鼠源性高亲和力H-Y噬菌体Fab抗体的筛选与早期胚胎性别鉴定
|
摘要
雄性特异性抗原,通常也称为雄性特异性弱组织相容性Y抗原,简称H-Y抗原。早期研究认为H-Y抗原是引起雌鼠对来自遗传性质相同的雄鼠皮肤移植片排斥反应的物质总称。血清学检测研究发现H-Y抗原位于雄性动物细胞膜,是一组雄性特异性基因的表达产物,检测哺乳动物早期胚胎细胞膜上是否存在H-Y抗原,即可鉴定胚胎的性别。H-Y抗原在异性间组织移植的排斥反应和性别控制等方面的研究中具有重要的理论和实践意义。由于H-Y抗原是一种弱抗原,导致常规免疫学方法无法获得高效价、高特异性的H-Y抗血清,而采用杂交瘤技术制备的H-Y单克隆抗体的效价不能得到明显的提高,限制了H-Y抗体在性别控制方面的应用。噬菌体抗体库技术也称抗体基因组合文库技术,它利用抗体基因噬菌体库代替B细胞克隆在体外表达抗体,具有多样性足够、生产周期短、技术灵活方便和筛选范围广等特点,是获得备种抗体的有效途径,而且噬菌体抗体的体外亲和力成熟可突破体内亲和力成熟的界限,能够使抗体的亲和力达到超自然的强度,噬菌体抗体库技术为H-Y抗体效价的提高提供了可能。
本研究以鼠源性抗雄性特异性抗原的初级噬菌体抗体库为基础,借助链替换技术对该库进行亲和力提高,并对小鼠早期胚胎进行性别鉴定的初步研究。主要研究内容如下：
1.抗血清学检测的H-Y抗原Fab抗体的表达和特异性鉴定
为研究抗血清学检测的H-Y抗原噬菌体Fab抗体的可溶性表达及其特异性,通过SpeⅠ和NheⅠ双酶切重组噬质粒,除去噬菌体gⅢ基因,经T4 DNA连接酶的作用后,构建Fab抗体的可溶性表达噬质粒。以IPTG诱导上述含重组质粒的阳性XL1-Blue菌液表达Fab抗体,利用免疫荧光FITC/DAPI共染技术和ELISA分析其特异性。结果表明,该抗体在约49 kD处有条带出现。在Fab抗体和H-Y抗血清的细胞免疫荧光比较分析试验中发现,从雌雄鼠脾细胞的阳性细胞数量和平均荧光强度的角度分析,Fab抗体的雄性特异性强于抗血清。石蜡切片免疫荧光定量分析显示Fab抗体与雄鼠肝脏的结合活性明显高于对应雌鼠,差异极显著(t=20.73,P=0.0023<0.01),而H-Y抗血清与雄鼠肝脏的结合活性略高于对应雌鼠,差异显著(t=7.11,P=0.0192<0.05)。以C57BL/6雄鼠脾细胞、睾丸细胞作为抗原的ELISA分析显示Fab抗体具有雄性特异性,但Fab抗体的OD值低于抗血清。结果提示,Fab抗体的雄性特异性高于H-Y抗血清,但其雄性特异性结合活性低于H-Y抗血清,Fab抗体具备雄性特异性的同时,仍有少量雌性非特异性结合,Fab抗体的体外亲和力成熟可作为后续研究工作以获得高亲和力、高特异性的可溶性Fab抗体分子。
2.利用链替换技术提高抗血清学检测的H-Y抗原Fab抗体的亲和力
提取雌性C57BL/6小鼠脾组织总RNA,反转录成cDNA,以其为模板扩增多样性小鼠Fab抗体基因,利用噬质粒pComb3,分别构建抗体轻链和重链基因文库。利用Sac I、Xba I双酶切轻链基因文库和原始克隆A8的重组噬质粒pComb3+K+fd,经T4 DNA连接酶的作用,构建了Fab抗体的轻链替换库,经过辅助噬菌体超感染,构建鼠源性次级噬菌体抗体库。通过雄、雌鼠脾细胞的“吸附-洗脱-扩增”筛选和ELISA分析,获得轻链替换后亲和力得到提高的克隆,将该克隆的轻链基因与重链库中的多样性fd段基因随机组合克隆到噬质粒pComb3,构建重链替换噬菌体抗体库,然后进行超感染、筛选和ELISA分析,挑选出高亲和力的抗血清检测的H-Y抗原噬菌体Fab抗体。研究发现,轻链替换的重组率约为80%,库容量7.6×107,重链替换库重组率约为70%,库容量6.4x107。抗体库筛选结果表明,噬菌体抗体亲和力及特异性逐渐得到提高。轻链或重链替换的噬菌体抗体库在第5轮筛选的回收率分别增加约35或15倍,从最后一轮挑取的25个单菌落制备噬菌体抗体,经ELISA检测,轻链替换的噬菌体抗体有8个(32%)显示较好雄性特异性结合活性,重链替换的噬菌体抗体有12个(48%)显示较好雄性特异性结合活性,重链替换后最佳克隆B9的OD值由原始克隆A8的0.505提高到0.961,约为1.90倍,从该角度分析,链替换后噬菌体抗体的亲和力有所提高。
3.高亲和力Fab抗体的特异性分析
采用pComb3载体和XL 1-Blue菌株对重链替换后的高亲和力Fab抗体基因进行表达,并利用已经建立的免疫荧光FITC/DAPI共染和ELISA方法分析其生物学活性,采用IMGT在线工具分析基因序列。在昆明系雌雄小鼠脾细胞与高亲和力B9 Fab抗体的细胞免疫荧光鉴定中发现,高亲和力B9 Fab抗体与雄鼠脾细胞亲和力强于雌鼠,从雄雌鼠脾细胞平均荧光强度对比的角度分析,重链替换的高亲和力B9 Fab抗体的雄性特异性(约3.62倍)强于原始克隆A8 Fab抗体(约2.07倍),而且筛选到的高亲和力B9 Fab抗体能够与睾丸组织特异结合,与卵巢反应不明显。原始克隆A8 (Fab-Kl, Fab-fd1)与高亲和力阳性克隆B9(Fab-κ2,Fab-fd2)的轻链和fd基因的CD3区比较分析发现,多个位点发生突变,提示CD3区位点的突变和长度的增加可能参与亲和力的提高。
4.利用高亲和力Fab抗体鉴定小鼠早期胚胎性别
利用间接免疫荧光法对70枚昆明系小白鼠桑椹期和囊胚期胚胎进行性别鉴定,参照已经建立的PCR法扩增SRY基因和常染色体113基因序列,验证性别鉴定准确率。28枚桑椹期和囊胚期胚胎有强荧光,判定为雄性胚胎,经PCR法验证,准确率(两种方法的符合率)为82.14%。36枚桑椹期和囊胚期胚胎呈灰绿荧光或透明带出现灰绿荧光环,判定为雌性胚胎,经PCR法验证,准确率为88.57%,从上述结果可判断B9 Fab抗体对血清学检测的H-Y抗原具有良好的特异性,但还需通过后续大量的动物实验来证明。
本研究开发了H-YFab抗体性别鉴定的实际应用潜能,高亲和力H-YFab抗体的筛选与制备将有助于揭示血清学检测的H-Y抗原和抗体的分子基础,为作用机理独特、具有复杂性的血清学检测的H-Y抗原的相关研究和生产应用提供理论依据。
Male specific antigen is often referred to as male-specific minor histocompati-bility Y antigen, also called H-Y antigen. In earlier studies, H-Y antigen was considered general material caused inbred female mice to reject skin grafts from male syngeneic mice. Serological findings showed that H-Y antigen was located at cell membranes of male animals, and were expressive products from a group of male specific genes, determining the existence of H-Y antigen at cell membrane of embryo is an approach for sexing embryo. H-Y antigen has an important theoretical and practical significance for the study of transplant rejection between different gender donor-recipient groups and sex control. Because H-Y antigen is a weak antigen, specific H-Y sera with high titer were not obtained by the conventional immunolog-ical methods, the titer of H-Y monoclonal antibody prepared by hybridoma technique could not be significantly improved, which limited the feasible application of sex control using H-Y antibody. The phage antibody library technique, also called combinatorial library technique of antibody genes, could express antibody fragments in vitro by using phage antibody library to replace B lymphocytes clones in vivo, which possess enough diversity, short production cycle, technology flexibility, wide screening range and other characteristics. So it is an effective way to prepare a variety of antibodies, and affinity maturation of phage antibody in vitro might break boundary of antibody affinity maturation in vivo, which could make antibody affinity reach supernatural intensity, phage antibody library technique provides a possibility to increase antibody titer.
This study based on primary phage antibody library against mouse male specific antigen constructed successfully, aimed to improve affinity of antibody from the phage library by chain shuffling, and was also a preliminary study of sexing mouse preimplantation embryo was also carried out. The main contents of our research are as following:
1. Expression and specific identification of Fab antibody against serologically detected H-Y antigen
To study soluble expression and specificity of Fab antibody against serologically detected H-Y antigen, the expressive plasmid was constructed by the ligation reaction system of T4DNA ligase, after the phage gⅢfragment was removed from the recombinant phagemid digested with Spe I and Nhe I. The soluble Fab antibody was expressed in positive XL1-Blue with recombinant phagemid by adding IPTG, the antibody specificity was determined by technique of immunofluorescence staining with FITC\DAPI and ELISA assay. The results showed that a band around 49 kD band appeared in the SDS-PAGE. Based on number of positive cells and mean fluorescence intensity of male or female spleen cells, comparison of cell immunofluorescence staining of Fab antibody and H-Y serum showed that male specificity and binding activity of Fab antibody were higher than those of antiserum. The quantitative analysis of immuno-fluorescence staining of paraffin sections showed that the binding activity of Fab antibody against male mouse liver was obviously higher than that of female mouse, there was high significant difference between them (t=20.73, P=0.0023<0.01), binding activity of H-Y serum against male mouse liver was slightly higher than that of female mouse, there was significant difference between them (t=7.11, P=0.0192<0.05). ELISA assay showed that the soluble Fab antibody had male specific activity with male mouse spleen and testicular cells as antigen, but OD values of Fab antibody were lower than that from antiserum. The results suggest that male specificity of Fab antibody was higher than that of H-Y serum, but its binding activity with male cells was lower than H-Y serum, Fab antibody with male specificity had non-specific binding to a small number of female tissue or cells, affinity maturation of Fab antibody in vitro would be used as follow-up study, in order to screen soluble Fab antibody with high affinity and specificity.
2. Improving affinity of Fab antibody against serologically detected male antigen by chain shuffling
The total RNA was isolated from spleen cells of C57BL/7 female mouse, then reverse transcribed into a single strand cDNA. Fab antibody genes with diversity were amplified using cDNA template, mouse combinatorial Fab gene libraries were constructed by inserting both light-chain genes and fd genes into pComb3 vector, respectively. The light-chain genes library and original clone A8 with pComb3+K+fd were digested by Sac I and Xba I, the light-chain shuffling library was constructed by the ligation reaction system of T4DNA ligase, then rescued by the infection with helper phage VCSM 13, the secondary phage antibody library was constructed. The panning cycles include specific absorption, elution and amplication with male or female spleen cells and ELISA were initiated, clones with the higher affinity were obtained after light-chain shuffling. The secondary phage antibody library was constructed again by light-chain gene of this clone and fd genes from high-chain genes library cloned randomly into pComb3 vector. The phage Fab antibody against serologically detected H-Y antigen with high affinity was finally selected by helper phage infection, screening and ELISA. The results showed that the recombination rates of light-chains shuffling library were 80%, the volume of the phage library was 7.6×107, and the recombination rates of high-chains shuffling library were 70%, the volume of the phage library was 6.4×107. The results from panning cycles of phage antibody library showed that the affinity and specificity of phage antibody were gradually improved, and the recovery rate of phage antibody library with light and high chain shuffling increased 35 and 25 times respectively in the fifth screening cycle. After the final panning, the phage antibody in 25 clones screened from final screening cycle were analysed with ELISA,8 (32%) phage antibodies from the library after light- chain shuffling had male specificity,12 (48%) phage antibodies from the library after high-chain shuffling had male specificity. After high chain shuffling, OD values of the best clone B9 increased to 0.961, compared that from the original clone A8 (0.505), which increased 1.90 times. From the point of view, the affinity of phage antibody was improved by chain shuffling.
3. The analysis of specificity of Fab antibody with high affinity
The phage Fab antibody gene with high affinity from the library after high-chain shuffling was expressed using pComb3 vector and XL1-Blue strain, the antibody specificity was determined by technique of immunofluorescence staining with FITC\DAPI and ELISA. The sequences of antibody genes were analysed with IMGT online tools. The quantitative analysis of immunofluorescence staining of Kunming mouse spleen cells showed that binding activity of B9 Fab antibody with high affinity against male mouse spleen cells was obviously higher than female mouse spleen cells, and male specificity of B9 Fab antibody (about 3.62 times) with high affinity after high chain shuffling was higher than that of Fab antibody (about 2.07 times) from the original clone. The B9 Fab antibody with high-affinity could specifically bind testis, its specific binding with ovarian was not obvious. The results from comparative analysis of CD3 regions of light or high chain genes between original clone A8 (Fab-Kl, Fab-fdl) and positive clone B9 with high affinity (Fab-K2, Fab-fd2) showed a few site mutations of CD3 regions occurred, which suggested that site mutations and increase of CD3 length may be related to improvement of antibody affinity.
4. Sexing mouse preimplantation embryos by Fab antibody with high affinity
The gender of 70 morulas and blastocysts from Kunming mice were identified using indirect immunofluorescence, then were evaluated by PCR amplification system of SRY and 113 genes established.28 morulas and blastocysts with strong fluorescence were determined as male embryos after PCR evaluation of genes from these embryos, the accuracy of embryos sexing (the coincidence rate of two methods) was 82.14%. 36 morulas and blastocysts with gray-green fluorescence or zona pellucida presenting gray-green fluorescence were determined as female embryo by the same PCR evaluation, the accuracy of embryos sexing was 88.57%. The results suggested this B9 Fab antibody had better specificity against serologically detected H-Y antigen, which need animal experiments to further determine antibody specificity.
This study developed a practical application of embryo sexing using H-Y Fab antibody, preparation of H-Y Fab antibody with high affinity will be helpful to understand the molecular basis of serologically detected H-Y antigen and antibody, and offer theoretical basis to study and application of serologically detected H-Y antigen with unique mechanism and complex characteristics.
本研究以鼠源性抗雄性特异性抗原的初级噬菌体抗体库为基础,借助链替换技术对该库进行亲和力提高,并对小鼠早期胚胎进行性别鉴定的初步研究。主要研究内容如下：
1.抗血清学检测的H-Y抗原Fab抗体的表达和特异性鉴定
为研究抗血清学检测的H-Y抗原噬菌体Fab抗体的可溶性表达及其特异性,通过SpeⅠ和NheⅠ双酶切重组噬质粒,除去噬菌体gⅢ基因,经T4 DNA连接酶的作用后,构建Fab抗体的可溶性表达噬质粒。以IPTG诱导上述含重组质粒的阳性XL1-Blue菌液表达Fab抗体,利用免疫荧光FITC/DAPI共染技术和ELISA分析其特异性。结果表明,该抗体在约49 kD处有条带出现。在Fab抗体和H-Y抗血清的细胞免疫荧光比较分析试验中发现,从雌雄鼠脾细胞的阳性细胞数量和平均荧光强度的角度分析,Fab抗体的雄性特异性强于抗血清。石蜡切片免疫荧光定量分析显示Fab抗体与雄鼠肝脏的结合活性明显高于对应雌鼠,差异极显著(t=20.73,P=0.0023<0.01),而H-Y抗血清与雄鼠肝脏的结合活性略高于对应雌鼠,差异显著(t=7.11,P=0.0192<0.05)。以C57BL/6雄鼠脾细胞、睾丸细胞作为抗原的ELISA分析显示Fab抗体具有雄性特异性,但Fab抗体的OD值低于抗血清。结果提示,Fab抗体的雄性特异性高于H-Y抗血清,但其雄性特异性结合活性低于H-Y抗血清,Fab抗体具备雄性特异性的同时,仍有少量雌性非特异性结合,Fab抗体的体外亲和力成熟可作为后续研究工作以获得高亲和力、高特异性的可溶性Fab抗体分子。
2.利用链替换技术提高抗血清学检测的H-Y抗原Fab抗体的亲和力
提取雌性C57BL/6小鼠脾组织总RNA,反转录成cDNA,以其为模板扩增多样性小鼠Fab抗体基因,利用噬质粒pComb3,分别构建抗体轻链和重链基因文库。利用Sac I、Xba I双酶切轻链基因文库和原始克隆A8的重组噬质粒pComb3+K+fd,经T4 DNA连接酶的作用,构建了Fab抗体的轻链替换库,经过辅助噬菌体超感染,构建鼠源性次级噬菌体抗体库。通过雄、雌鼠脾细胞的“吸附-洗脱-扩增”筛选和ELISA分析,获得轻链替换后亲和力得到提高的克隆,将该克隆的轻链基因与重链库中的多样性fd段基因随机组合克隆到噬质粒pComb3,构建重链替换噬菌体抗体库,然后进行超感染、筛选和ELISA分析,挑选出高亲和力的抗血清检测的H-Y抗原噬菌体Fab抗体。研究发现,轻链替换的重组率约为80%,库容量7.6×107,重链替换库重组率约为70%,库容量6.4x107。抗体库筛选结果表明,噬菌体抗体亲和力及特异性逐渐得到提高。轻链或重链替换的噬菌体抗体库在第5轮筛选的回收率分别增加约35或15倍,从最后一轮挑取的25个单菌落制备噬菌体抗体,经ELISA检测,轻链替换的噬菌体抗体有8个(32%)显示较好雄性特异性结合活性,重链替换的噬菌体抗体有12个(48%)显示较好雄性特异性结合活性,重链替换后最佳克隆B9的OD值由原始克隆A8的0.505提高到0.961,约为1.90倍,从该角度分析,链替换后噬菌体抗体的亲和力有所提高。
3.高亲和力Fab抗体的特异性分析
采用pComb3载体和XL 1-Blue菌株对重链替换后的高亲和力Fab抗体基因进行表达,并利用已经建立的免疫荧光FITC/DAPI共染和ELISA方法分析其生物学活性,采用IMGT在线工具分析基因序列。在昆明系雌雄小鼠脾细胞与高亲和力B9 Fab抗体的细胞免疫荧光鉴定中发现,高亲和力B9 Fab抗体与雄鼠脾细胞亲和力强于雌鼠,从雄雌鼠脾细胞平均荧光强度对比的角度分析,重链替换的高亲和力B9 Fab抗体的雄性特异性(约3.62倍)强于原始克隆A8 Fab抗体(约2.07倍),而且筛选到的高亲和力B9 Fab抗体能够与睾丸组织特异结合,与卵巢反应不明显。原始克隆A8 (Fab-Kl, Fab-fd1)与高亲和力阳性克隆B9(Fab-κ2,Fab-fd2)的轻链和fd基因的CD3区比较分析发现,多个位点发生突变,提示CD3区位点的突变和长度的增加可能参与亲和力的提高。
4.利用高亲和力Fab抗体鉴定小鼠早期胚胎性别
利用间接免疫荧光法对70枚昆明系小白鼠桑椹期和囊胚期胚胎进行性别鉴定,参照已经建立的PCR法扩增SRY基因和常染色体113基因序列,验证性别鉴定准确率。28枚桑椹期和囊胚期胚胎有强荧光,判定为雄性胚胎,经PCR法验证,准确率(两种方法的符合率)为82.14%。36枚桑椹期和囊胚期胚胎呈灰绿荧光或透明带出现灰绿荧光环,判定为雌性胚胎,经PCR法验证,准确率为88.57%,从上述结果可判断B9 Fab抗体对血清学检测的H-Y抗原具有良好的特异性,但还需通过后续大量的动物实验来证明。
本研究开发了H-YFab抗体性别鉴定的实际应用潜能,高亲和力H-YFab抗体的筛选与制备将有助于揭示血清学检测的H-Y抗原和抗体的分子基础,为作用机理独特、具有复杂性的血清学检测的H-Y抗原的相关研究和生产应用提供理论依据。
Male specific antigen is often referred to as male-specific minor histocompati-bility Y antigen, also called H-Y antigen. In earlier studies, H-Y antigen was considered general material caused inbred female mice to reject skin grafts from male syngeneic mice. Serological findings showed that H-Y antigen was located at cell membranes of male animals, and were expressive products from a group of male specific genes, determining the existence of H-Y antigen at cell membrane of embryo is an approach for sexing embryo. H-Y antigen has an important theoretical and practical significance for the study of transplant rejection between different gender donor-recipient groups and sex control. Because H-Y antigen is a weak antigen, specific H-Y sera with high titer were not obtained by the conventional immunolog-ical methods, the titer of H-Y monoclonal antibody prepared by hybridoma technique could not be significantly improved, which limited the feasible application of sex control using H-Y antibody. The phage antibody library technique, also called combinatorial library technique of antibody genes, could express antibody fragments in vitro by using phage antibody library to replace B lymphocytes clones in vivo, which possess enough diversity, short production cycle, technology flexibility, wide screening range and other characteristics. So it is an effective way to prepare a variety of antibodies, and affinity maturation of phage antibody in vitro might break boundary of antibody affinity maturation in vivo, which could make antibody affinity reach supernatural intensity, phage antibody library technique provides a possibility to increase antibody titer.
This study based on primary phage antibody library against mouse male specific antigen constructed successfully, aimed to improve affinity of antibody from the phage library by chain shuffling, and was also a preliminary study of sexing mouse preimplantation embryo was also carried out. The main contents of our research are as following:
1. Expression and specific identification of Fab antibody against serologically detected H-Y antigen
To study soluble expression and specificity of Fab antibody against serologically detected H-Y antigen, the expressive plasmid was constructed by the ligation reaction system of T4DNA ligase, after the phage gⅢfragment was removed from the recombinant phagemid digested with Spe I and Nhe I. The soluble Fab antibody was expressed in positive XL1-Blue with recombinant phagemid by adding IPTG, the antibody specificity was determined by technique of immunofluorescence staining with FITC\DAPI and ELISA assay. The results showed that a band around 49 kD band appeared in the SDS-PAGE. Based on number of positive cells and mean fluorescence intensity of male or female spleen cells, comparison of cell immunofluorescence staining of Fab antibody and H-Y serum showed that male specificity and binding activity of Fab antibody were higher than those of antiserum. The quantitative analysis of immuno-fluorescence staining of paraffin sections showed that the binding activity of Fab antibody against male mouse liver was obviously higher than that of female mouse, there was high significant difference between them (t=20.73, P=0.0023<0.01), binding activity of H-Y serum against male mouse liver was slightly higher than that of female mouse, there was significant difference between them (t=7.11, P=0.0192<0.05). ELISA assay showed that the soluble Fab antibody had male specific activity with male mouse spleen and testicular cells as antigen, but OD values of Fab antibody were lower than that from antiserum. The results suggest that male specificity of Fab antibody was higher than that of H-Y serum, but its binding activity with male cells was lower than H-Y serum, Fab antibody with male specificity had non-specific binding to a small number of female tissue or cells, affinity maturation of Fab antibody in vitro would be used as follow-up study, in order to screen soluble Fab antibody with high affinity and specificity.
2. Improving affinity of Fab antibody against serologically detected male antigen by chain shuffling
The total RNA was isolated from spleen cells of C57BL/7 female mouse, then reverse transcribed into a single strand cDNA. Fab antibody genes with diversity were amplified using cDNA template, mouse combinatorial Fab gene libraries were constructed by inserting both light-chain genes and fd genes into pComb3 vector, respectively. The light-chain genes library and original clone A8 with pComb3+K+fd were digested by Sac I and Xba I, the light-chain shuffling library was constructed by the ligation reaction system of T4DNA ligase, then rescued by the infection with helper phage VCSM 13, the secondary phage antibody library was constructed. The panning cycles include specific absorption, elution and amplication with male or female spleen cells and ELISA were initiated, clones with the higher affinity were obtained after light-chain shuffling. The secondary phage antibody library was constructed again by light-chain gene of this clone and fd genes from high-chain genes library cloned randomly into pComb3 vector. The phage Fab antibody against serologically detected H-Y antigen with high affinity was finally selected by helper phage infection, screening and ELISA. The results showed that the recombination rates of light-chains shuffling library were 80%, the volume of the phage library was 7.6×107, and the recombination rates of high-chains shuffling library were 70%, the volume of the phage library was 6.4×107. The results from panning cycles of phage antibody library showed that the affinity and specificity of phage antibody were gradually improved, and the recovery rate of phage antibody library with light and high chain shuffling increased 35 and 25 times respectively in the fifth screening cycle. After the final panning, the phage antibody in 25 clones screened from final screening cycle were analysed with ELISA,8 (32%) phage antibodies from the library after light- chain shuffling had male specificity,12 (48%) phage antibodies from the library after high-chain shuffling had male specificity. After high chain shuffling, OD values of the best clone B9 increased to 0.961, compared that from the original clone A8 (0.505), which increased 1.90 times. From the point of view, the affinity of phage antibody was improved by chain shuffling.
3. The analysis of specificity of Fab antibody with high affinity
The phage Fab antibody gene with high affinity from the library after high-chain shuffling was expressed using pComb3 vector and XL1-Blue strain, the antibody specificity was determined by technique of immunofluorescence staining with FITC\DAPI and ELISA. The sequences of antibody genes were analysed with IMGT online tools. The quantitative analysis of immunofluorescence staining of Kunming mouse spleen cells showed that binding activity of B9 Fab antibody with high affinity against male mouse spleen cells was obviously higher than female mouse spleen cells, and male specificity of B9 Fab antibody (about 3.62 times) with high affinity after high chain shuffling was higher than that of Fab antibody (about 2.07 times) from the original clone. The B9 Fab antibody with high-affinity could specifically bind testis, its specific binding with ovarian was not obvious. The results from comparative analysis of CD3 regions of light or high chain genes between original clone A8 (Fab-Kl, Fab-fdl) and positive clone B9 with high affinity (Fab-K2, Fab-fd2) showed a few site mutations of CD3 regions occurred, which suggested that site mutations and increase of CD3 length may be related to improvement of antibody affinity.
4. Sexing mouse preimplantation embryos by Fab antibody with high affinity
The gender of 70 morulas and blastocysts from Kunming mice were identified using indirect immunofluorescence, then were evaluated by PCR amplification system of SRY and 113 genes established.28 morulas and blastocysts with strong fluorescence were determined as male embryos after PCR evaluation of genes from these embryos, the accuracy of embryos sexing (the coincidence rate of two methods) was 82.14%. 36 morulas and blastocysts with gray-green fluorescence or zona pellucida presenting gray-green fluorescence were determined as female embryo by the same PCR evaluation, the accuracy of embryos sexing was 88.57%. The results suggested this B9 Fab antibody had better specificity against serologically detected H-Y antigen, which need animal experiments to further determine antibody specificity.
This study developed a practical application of embryo sexing using H-Y Fab antibody, preparation of H-Y Fab antibody with high affinity will be helpful to understand the molecular basis of serologically detected H-Y antigen and antibody, and offer theoretical basis to study and application of serologically detected H-Y antigen with unique mechanism and complex characteristics.
引文
[1]Eichwald EJ SC. Untitled communication[J]. Transplant Bull 1955,2:148-149.
[2]Simpson E, Scott D, Chandler P. The male-specific histocompatibility antigen, H-Y: a history of transplantation, immune response genes, sex determination and expression cloning[J]. Annu Rev Immunol,1997,15:39-61.
[3]Wachtel S, Nakamura D, Wachtel G, et al. Sex selection with monoclonal H-Y anti-body[J]. Fertil Steril,1988,50(2):355-360.
[4]Goldberg EH. H-Y antigen and sex determination[J]. Philos Trans R Soc Lond B Biol Sci,1988,322(1208):73-81.
[5]Veerhuis R, Hendriksen PJ, Hengst AM, et al. The production of anti-H-Y monoclonal antibodies:their potential use in a sex test for bovine embryos[J]. Vet Immunol Immunopathol,1994,42(3-4):317-330.
[6]Gardon JC, Aguera S, Castejon F. Sexing in vitro produced bovine embryos, at different stages of development, using rat H-Y antiserum[J]. Theriogenology,2004, 62 (1-2):35-43.
[7]Muller U. H-Y antigens[J]. Hum Genet,1996,97(6):701-704.
[8]Wolf U. The serologically detected H-Y antigen revisited[J]. Cytogenet Cell Genet, 1998,80(1-4):232-235.
[9]Utsumi K, Hayashi M, Takakura R, et al. Embryo sex selection by a rat male-specific antibody and the cytogenetic and developmental confirmation in cattle embryos[J]. Mol Reprod Dev,1993,34(l):25-32.
[10]门红升,陈云鹤,周理良.间接免疫荧光法鉴别小鼠早期胚胎性别的研究[J].遗传学报,1992,19(2)：117-121.
[11]杨文胜,朱裕鼎.小鼠早期胚胎性别鉴定的研究[J].中国实验动物学报,1993,(2)：65-69.
[12]Scott DM, Ehrmann IE, Ellis PS, et al. Identification of a mouse male-specific transplantation antigen, H-Y[J]. Nature,1995,376(6542):695-698.
[13]Meadows L, Wang W, den Haan JM, et al. The HLA-A*0201-restricted H-Y antigen contains a posttranslationally modified cysteine that significantly affects T cell recognition[J]. Immunity,1997,6(3):273-281.
[14]Wang W, Meadows LR, den Haan JM, et al. Human H-Y:a male-specific his- tocompatibility antigen derived from the SMCY protein[J]. Science,1995,269 (5230):1588-1590.
[15]Warren EH, Gavin MA, Simpson E, et al. The human UTY gene encodes a novel HLA-B8-restricted H-Y antigen[J]. J Immunol,2000,164(5):2807-2814.
[16]Vogt MH, van den Muijsenberg JW, Goulmy E, et al. The DBY gene codes for an HLA-DQ5-restricted human male-specific minor histocompatibility antigen involved in graft-versus-host disease[J]. Blood,2002,99(8):3027-3032.
[17]Vogt MH, de Paus RA, Voogt PJ, et al. DFFRY codes for a new human male-specific minor transplantation antigen involved in bone marrow graft rejection[J]. Blood,2000,95(3):1100-1105.
[18]Pierce RA, Field ED, den Haan JM, et al. Cutting edge:the HLA-A*0101-restricted HY minor histocompatibility antigen originates from DFFRY and contains a cysteinylated cysteine residue as identified by a novel mass spectro-metric technique[J]. J Immunol,1999,163(12):6360-6364.
[19]Miklos DB, Kim HT, Miller KH, et al. Antibody responses to H-Y minor histocompatibility antigens correlate with chronic graft-versus-host disease and disease remission[J]. Blood,2005,105(7):2973-2978.
[20]Shapiro M, Erickson RP. Evidence that the serological determinant of H-Y antigen is carbohydrate[J]. Nature,1981,290(5806):503-505.
[21]Fellous M, Gunther E, Kemler R, et al. Association of the H-Y male antigen with beta2-microglobulin on human lymphoid and differentiated mouse teratocar-cinoma cell lines[J]. J Exp Med,1978,148(1):58-70.
[22]Wachtel SS, Koo GC, Boyse EA. Evolutionary conservation of H-Y ('male') antigen[J].Nature,1975,254(5497):270-272.
[23]Su H, Kozak CA, Veerhuis R, et al. Isolation of a phylogenetically conserved and testis-specific gene using a monoclonal antibody against the serological H-Y antigen[J]. J Reprod Immunol,1992,21(3):275-291.
[24]Lau YF, Chan KM, Kan YW, et al. Male-enhanced expression and genetic conservation of a gene isolated with an anti-H-Y antibody [J]. Trans Assoc Am Physicians,1987,100:45-53.
[25]Koo GC, Wachtel SS, Breg WR, et al. Mapping the locus of the H-Y antigen[J]. Cytogenet Cell Genet,1976,16(1-5):175-177.
[26]Wolf U, Fraccaro M, Mayerova A, et al. A gene controlling H-Y antigen on the X chromosome. Tentative assignment by deletion mapping to Xp223[J]. Hum Genet, 1980,54(2):149-154.
[27]Wolf U. X-linked genes and gonadal differentiation[J]. Differentiation,1983,23 Suppl:S 104-106.
[28]Wachtel SS. Where is the H-Y structural gene?[J]. Cell,1980,22(1):3-4.
[29]Ginalski K, Rychlewski L, Baker D, et al. Protein structure prediction for the male-specific region of the human Y chromosome [J]. Proc Natl Acad Sci U S A, 2004,101(8):2305-2310.
[30]Brunner M, Wachtel S, Chin S. Purification and analysis of a sex-specific antibody [J]. J Immunoassay,1988,9(2):105-124.
[31]White KL, Lindner GM, Anderson GB, et al. Cytolytic and fluorescent detection of H-Y antigen on preimplantation mouse embryos[J]. Theriogenology,1983,19 (5): 701-705.
[32]Wang N, Xue L, Yuan A, et al. Identification of Mouse 8-Cell Embryo Stage-Specific Genes by Digital Differential Display [J]. Experimental animals,2009,58 (5):547-556.
[33]Bonney EA, Onyekwuluje J. Maternal tolerance to H-Y is independent of IL-10[J]. Immunol Invest,2004,33(4):385-395.
[34]Bonney EA,Onyekwuluje J.The H-Y response in midgestation and long after delivery in mice primed before pregnancy[J].Immunol Invest,2003,32(1):71-81.
[35]Bonney EA. Maternal tolerance is not critically dependent on interleukin-4[J]. Immunology,2001,103(3):382-389.
[36]Vacchio MS, Hodes RJ. Fetal expression of Fas ligand is necessary and sufficient for induction of CD8 T cell tolerance to the fetal antigen H-Y during pregnancy[J]. J Immunol,2005,174(8):4657-4661.
[37]Barjaktarevic I, Vukmanovic S. Paternal cell immunization raises autoantibodies and improves pregnancy success in mice[J]. Am J Reprod Immunol,2008,60 (6): 497-500.
[38]Goldberg EH, Boyse EA, Bennett D, et al. Serological demonstration of H-Y (male) antigen on mouse sperm[J]. Nature,1971,232(5311):478-480.
[39]Koo GC, Tada N, Chaganti R, et al. Application of monoclonal anti-HY antibody for human H-Y typing[J]. Hum Genet,1981,57(1):64-67.
[40]Shapiro M, Goldberg EH. Analysis of a serological determinant of H-Y antigen: evidence for carbohydrate specificity using an H-Y specific monoclonal antibody [J]. J Immunogenet,1984,11(3-4):209-218.
[41]Meck JM, Goldberg EH. Serological detection of H-Y antigen in humans with a cellular radioimmunobinding assay and monoclonal antibody[J].J Immunol Methods,1984,73(2):293-299.
[42]Ramalho MF, Garcia JM, Esper CR, et al. Sexing of murine and bovine embryos by developmental arrest induced by high-titer H-Y antisera[J]. Theriogenology, 2004,62(9):1569-1576.
[43]Bradley MP, Ebensperger C, Wiberg UH. Determination of the serological sex-specific (Sxs) antigen ("H-Y antigen") in birds and mammals using high-titer antisera and a sensitive urease ELISA[J]. Hum Genet,1987,76(4):352-356.
[44]Heslop BF, Bradley MP, Baird MA. The H-Y antigen[J]. Asian Pac J Allergy Immunol,1987,5(1):1-4.
[45]Bradley MP, Forrester IT, Heslop BF. Identification of a male-specific (H-Y) antigen on the flagellar plasma membrane of ram epididymal spermatozoa[J]. Hum Genet,1987,75(4):362-367.
[46]Koo GC, Varano A. Inhibition of H-Y cell-mediated cytolysis by monoclonal H-Y specific antibody[J]. Immunogenetics,1981,14(3-4):183-188.
[47]Moreira-Filho CA, Wachtel SS. Study of H-Y antigen in abnormal sex determin-ation with monoclonal antibody and an ELISA[J]. Am J Med Genet,1985,20(3): 525-534.
[48]Brunner M, Jaswaney V, Wachtel S. Reaction of monoclonal H-Y antibody in the ELISA[J]. J Reprod Immunol,1987,11(3):181-191.
[49]Brunner M, Wachtel SS. Two new ELISAs using monoclonal H-Y antibody[J]. J Immunol Methods,1988,106(1):49-55.
[50]Wang N, Xu D, Yuan A, et al. Construction and characterization of phage display library:Recognition of mouse serologically detected male (SDM) antigen[J]. Animal reproduction science,2008,104(1):93-110.
[51]Naidong W, Liqun X, Daojun X. Construction of phage display library of Fab antibodies repertoies of mouse male specific antigen; Fab[J]. Chinese Journal of Animal and Veterinary Sciences,2007,38(4):344-351.
[52]王乃东,薛立群,许道军,等.鼠源性抗雄性特异性抗原噬菌体Fab抗体的制备及分析[J].生物工程学报,2006,22(5)：727-732.
[53]Whitfield L, Lovell-Badge R, Goodfellow P. Rapid sequence evolution of the mammalian sex-determining gene SRY[J]. Nature,1993,364:713-715.
[54]Berta P, Hawkins J, Sinclair A, et al. Genetic evidence equating SRY and the testis-determining factor[J]. Nature,1990,348(6300):448-450.
[55]Swain A, Narvaez V, Burgoyne P, et al. Daxl antagonizes Sry action in mamma-lian sex determination[J]. Nature,1998,391(6669):761-767.
[56]Utsumi K, Hayashi M, Takakura R, et al. Embryo sex selection by a rat male-specific antibody and the cytogenetic and developmental confirmation in cattle embryos[J]. Molecular reproduction and development,1993,34(l):25-32.
[57]Williams T. A technique for sexing mouse embryos by a visual colorimetric assay of the X-linked enzyme, glucose 6-phosphate dehydrogenase[J]. Theriogenology, 1986,25(5):733-739.
[58]Tiffin G, Rieger D, Betteridge K, et al. Glucose and glutamine metabolism in pre-attachment cattle embryos in relation to sex and stage of development [J]. Reproduction,1991,93(1):125.
[59]Van Vliet R, Verrinder Gibbins A, Walton J. Livestock embryo sexing:a review of current methods, with emphasis on Y-specific DNA probes [J]. Theriogenology, 1989,32(3):421-438.
[60]Fajfar-Whetstone C, Rayburn A, Schook L, et al. Sex determination of porcine preimplantation embryos via Y-chromosome specific DNA sequences [J]. Animal Biotechnology,1993,4(2):183-193.
[61]Leonard M, Kirszenbaum M, Cotinot C, et al. Sexing bovine embryos using Y chromosome specific DNA probe[J]. Theriogenology,1987,27(1):248.
[62]Hacker A, Capel B, Goodfellow P, et al. Expression of Sry, the mouse sex determining gene[J]. Development,1995,121(6):1603.
[63]Zwingman T, Erickson R, Boyer T, et al. Transcription of the sex-determining region genes Sry and Zfy in the mouse preimplantation embryo [J]. Proceedings of the National Academy of Sciences of the United States of America,1993,90(3): 814.
[64]McClive P, Sinclair A. Rapid DNA extraction and PCR-sexing of mouse embryos[J]. Molecular reproduction and development,2001,60(2):225-226.
[65]李晓红,庄广伦.单细胞PCR对种植前胚胎的性别鉴定[J].中山医科大学学报,1998,19(1)：11-13.
[66]White K, Lindner G, Anderson G, et al. Cytolytic and fluorescent detection of HY antigen on preimplantation mouse embryos[J]. Theriogenology,1983,19(5):701-705.
[67]Hossepian de Lima V, Moreira-Filho C, De Bem A, et al. Sex determination of murine and bovine embryos using cytotoxicity and immunofluorescence assays[J]. Theriogenology,1993,39(6):1343-1352.
[68]周颂成,龚振明.用H-Y抗血清对大鼠胚胎进行性别鉴定的研究[J].上海农学院学报,1993,11(003)：177-181.
[69]Avery B, Schmidt M. Sex determination of bovine embryos using H-Y antibodies [J].Acta Vet Scand,1989,30(2):155-164.
[70]Goldbard SB, Warner CM. Genes affect the timing of early mouse embryo development[J]. Biol Reprod,1982,27(2):419-424.
[71]Avery B, Jorgensen CB, Madison V, et al. Morphological development and sex of bovine in vitro-fertilized embryos[J]. Mol Reprod Dev,1992,32(3):265-270.
[72]许晓霁,马向东.用H-Y抗原方法鉴定小鼠胚胎性别的研究[J].东北农学院学报,1990,21(2)：186-191.
[73]White KL, Anderson GB, Bondurant RH. Expression of a male-specific factor on various stages of preimplantation bovine embryos[J]. Biol Reprod,1987,37(4): 867-873.
[74]White KL, Anderson GB, Berger TJ, et al. Identification of a male-specific histocompatibility protein on preimplantation porcine embryos[J]. Gamete Res, 1987,17(2):107-113.
[75]White KL, Anderson GB, Pashen RL, et al. Detection of histocompatibility-Y antigen:identification of sex of pre-implantation ovine embryos [J]. J Reprod Immunol,1987,10(1):27-32.
[76]Utsumi K, Iritani A. Embryo sexing by male specific antibody and by PCR using male specific (SRY) primer[J]. Mol Reprod Dev,1993,36(2):238-241.
[77]Utsumi K, Satoh E, Iritani A. Sexing of rat embryos with antisera specific for male rats[J]. J Exp Zool,1991,260(1):99-105.
[78]马云生,郭彩霞.用抗雄性特异性单克隆抗体鉴别小鼠胚胎性别[J].天津农业科学,1999,5(4)：9-12.
[79]曹文广,董伟.应用H-Y单克隆抗体鉴定小鼠和家兔胚胎性别[J].畜牧兽医学报,1993,24(6)：506-511.
[80]Ali JI, Eldridge FE, Koo GC, et al. Enrichment of bovine X-and Y- chromosome-bearing sperm with monoclonal H-Y antibody-fluorescence-activated cell sorter [J].Arch Androl,1990,24(3):235-245.
[81]Hendriksen P. Do X and Y spermatozoa differ in proteins?[J]. Theriogenology, 1999,52(8):1295-1307.
[82]Hendriksen P, Tieman M, Van Der Lende T, et al. Binding of anti H Y monoclonal antibodies to separated X and Y chromosome bearing porcine and bovine sperm[J]. Molecular reproduction and development,1993,35(2):189-196.
[83]Barbas CF,3rd. Recent advances in phage display[J]. Curr Opin Biotechnol,1993, 4(5):526-530.
[84]Barbas CF,3rd, Kang AS, Lerner RA, et al. Assembly of combinatorial antibody libraries on phage surfaces:the gene Ⅲ site[J]. Proc Natl Acad Sci U S A,1991, 88(18):7978-7982.
[85]Willats WG. Phage display:practicalities and prospects[J]. Plant Mol Biol,2002, 50(6):837-854.
[86]Winter G, Griffiths AD, Hawkins RE, et al. Making antibodies by phage display technology [J]. Annu Rev Immunol,1994,12:433-455.
[87]Hoogenboom HR, de Bruine AP, Hufton SE, et al. Antibody phage display technology and its applications [J]. Immunotechnology,1998,4(1):1-20.
[88]Koehler M, Mielzynska I, Bubala H, et al. [Use of an immunoenzyme method with monoclonal antibodies in cytologic studies for the optimal diagnosis of non-Hodgkin's lymphoma in children][J]. Patol Pol,1989,40(3):311-323.
[89]Smith G. Filamentous fusion phage:novel expression vectors that display cloned antigens on the virion surface[J]. Science,1985,228(4705):1315.
[90]吴雯峰,王双,孙志伟.噬菌体展示抗体库筛选技术研究进展[J].生物技术通讯,2007,18(3)：476-479.
[91]Kretzschmar T, von Ruden T. Antibody discovery:phage display[J]. Current opinion in biotechnology,2002,13(6):598-602.
[92]Schmitz U, Versmold A, Kaufmann P, et al. Phage display:a molecular tool for the generation of antibodies--a review[J], Placenta,2000,21:106-112.
[93]Rodi DJ, Makowski L, Kay BK. One from column A and two from column B:the benefits of phage display in molecular-recognition studies[J]. Curr Opin Chem Biol,2002,6(1):92-96.
[94]Whaley SR, English DS, Hu EL, et al. Selection of peptides with semiconductor binding specificity for directed nanocrystal assembly[J].Nature,2000,405(6787): 665-668.
[95]Marks JD, Griffiths AD, Malmqvist M, et al. By-passing immunization:building high affinity human antibodies by chain shuffling[J]. Biotechnology (N Y),1992, 10(7):779-783.
[96]Kang AS, Jones TM, Burton DR. Antibody redesign by chain shuffling from random combinatorial immunoglobulin libraries[J]. Proc Natl Acad Sci U S A, 1991,88 (24):11120-11123.
[97]Bowen AJ, Corcoran AE. How chromatin remodelling allows shuffling of immunoglobulin heavy chain genes[J]. Mol Biosyst,2008,4(8):790-798.
[98]Marks JD. Antibody affinity maturation by chain shuffling[J]. Methods Mol Biol, 2004,248:327-343.
[99]Lantto J, Jirholt P, Barrios Y, et al. Chain shuffling to modify properties of recombinant immunoglobulins[J]. Methods Mol Biol,2002,178:303-316.
[100]Park SG, Lee JS, Je EY, et al. Affinity maturation of natural antibody using a chain shuffling technique and the expression of recombinant antibodies in Escheri- chia coli[J]. Biochem Biophys Res Commun,2000,275(2):553-557.
[101]Figini M, Marks JD, Winter G, et al. In vitro assembly of repertoires of antibody chains on the surface of phage by renaturation[J]. J Mol Biol,1994,239(1):68-78.
[102]Guo-Qiang B, Xian-Li H. Guided selection methods through chain shuffling[J]. Methods Mol Biol,2009,562:133-142.
[103]Kwong KY, Baskar S, Zhang H, et al. Generation, affinity maturation, and characterization of a human anti-human NKG2D monoclonal antibody with dual antagonistic and agonistic activity[J]. J Mol Biol,2008,384(5):1143-1156.
[104]Yoshinaga K, Matsumoto M, Torikai M, et al. Ig L-chain shuffling for affinity maturation of phage library-derived human anti-human MCP-1 antibody blocking its chemotactic activity[J]. J Biochem,2008,143(5):593-601.
[105]Chodorge M, Fourage L, Ravot G, et al. In vitro DNA recombination by L-Shuffling during ribosome display affinity maturation of an anti-Fas antibody increases the population of improved variants [J]. Protein Eng Des Sel,2008,21(5): 343-351.
[106]Jia L, Yu J, Song H, et al. Screening of human antibody Fab fragment against HBsAg and the construction of its dsFv form[J]. Int J Biol Sci,2008,4(2):103-110.
[107]刘雪林,唐晓敏,李申龙,等.轻链替换文库对抗HBs基因工程Fab抗体亲和力的影响[J].解放军医学杂志,2006,31(8)：770-772.
[108]Luginbuhl B, Kanyo Z, Jones RM, et al. Directed evolution of an anti-prion protein scFv fragment to an affinity of 1 pM and its structural interpretation [J]. J Mol Biol,2006,363(1):75-97.
[109]Watkins NA, Dafforn TR, Kuijpers M, et al. Molecular studies of anti-HLA-A2 using light-chain shuffling:a structural model for HLA antibody binding[J]. Tissue Antigens,2004,63(4):345-354.
[110]汪保安.抗TNFa抗体的体外亲和力成熟[D].中国人民解放军军事医学科学院博士论文,2004.
[111]林周.人TNF-α嵌合抗体体外亲和力成熟作用研究[D].中国人民解放军军事医学科学院博士论文,2004.
[112]王刚,王琰,刘玉峰.高亲和力抗角蛋白人抗体的基因工程制备[J].中华皮肤科杂志,2003,36(10)：559-561.
[113]Schier R, Bye J, Apell G, et al. Isolation of high-affinity monomeric human anti-c-erbB-2 single chain Fv using affinity-driven selection[J]. J Mol Biol,1996, 255(1):28-43.
[114]Proulx C, Boyer L, St-Amour I, et al. Higher affinity human D MoAb prepared by light-chain shuffling and selected by phage display[J].Transfusion,2002,42(1):59-65.
[115]Suzuki M, Takemura H, Suzuki H, et al. Light chain determines the binding property of human anti-dsDNA IgG autoantibodies[J]. Biochem Biophys Res Commun,2000,271(1):240-243.
[116]Kumar S, Nagl S, Kalsi JK, et al. Beta-2-glycoprotein specificity of human anti-phospholipid antibody resides on the light chain:a novel mechanism for acquisition of cross-reactivity by an autoantibody[J]. Mol Immunol,2005,42(1): 39-48.
[117]Kim SJ, Hong HJ. Guided selection of human antibody light chains against TAG-72 using a phage display chain shuffling approach[J]. J Microbiol,2007, 45(6):572-577.
[118]Kuepper MB, Huhn M, Spiegel H, et al. Generation of human antibody fragments against Streptococcus mutans using a phage display chain shuffling approach[J]. BMC Biotechnol,2005,5:4.
[119]包国强,马庆久,邢金良,等.用导向选择链更替技术建立抗肝癌抗原HAb18G的人源性Fab基因库[J].细胞与分子免疫学杂志,2004,20(4)：437-440.
[120]Rojas G, Talavera A, Munoz Y, et al. Light-chain shuffling results in successful phage display selection of functional prokaryotic-expressed antibody fragments to N-glycolyl GM3 ganglioside[J]. J Immunol Methods,2004,293(1-2):71-83.
[121]Christensen PA, Danielczyk A, Ravn P, et al. Modifying antibody specificity by chain shuffling of V/V between antibodies with related specificities [J]. Scand J Immunol,2009,69(1):1-10.
[122]Lau Y, Chan K, Sparkes R. Male-enhanced antigen gene is phylogenetically conserved and expressed at late stages of spermatogenesis[J]. Proceedings of the National Academy of Sciences of the United States of America,1989,86(21):8462.
[123]Kondo M, Sutou S. Cloning and molecular characterization of cDNA encoding a mouse male-enhanced antigen-2 (Mea-2):a putative family of the Golgi autoanti-gen[J]. Mitochondrial DNA,1997,7(2):71-82.
[124]王乃东,薛立群,许道军,等.鼠源性H-Y抗血清制备及特异性分析[J].中国比较医学杂志,2006,16(8)：485-488.
[125]Wang ND, Xue LQ, Xu DJ, et al. [Cloning and analysis of phage Fab antibodies of mouse male specific antigen][J]. Sheng Wu Gong Cheng Xue Bao,2006,22(5): 727-732.
[126]McCafferty J, Griffiths A, Winter G, et al. Phage antibodies:filamentous phage displaying antibody variable domains[J].1990.348(6301):552-554.
[127]Hudson P, Souriau C. Engineered antibodies [J]. Nature medicine,2003,9(l):129-134.
[128]王群,刘军莉,高飞,等.人源性TAT-Fab抗体的表达优化及生物活性测定[J].浙江大学学报,2007,33(6)：651-655.
[129]Hust M, Dubel S. Phage display vectors for the in vitro generation of human antibody fragments [J]. Immunochemical protocols,2005,295:71-95.
[130]Barbas C, Kang A, Lerner R, et al. Assembly of combinatorial antibody libraries on phage surfaces:the gene Ⅲ site[J]. Proceedings of the National Academy of Sciences of the United States of America,1991,88(18):7978.
[131]Barbas S, Barbas C. Filamentous phage display[J]. Fibrinolysis,1994,8:245-252.
[132]陆慧琦,韩焕兴,叶伟民,等.PBAD表达系统对抗-HBs Fab抗体表达的影 响[J].免疫学杂志,2005,21(1)：66-68.
[133]Corisdeo S, Wang B. Functional expression and display of an antibody Fab fragment in Escherichia coli:study of vector designs and culture conditions[J]. Protein expression and purification,2004,34(2):270-279.
[134]Backovic M, Johansson D, Klupp B, et al Efficient method for production of high yields of Fab fragments in Drosophila S2 cells[J]. Protein Engineering Design and Selection,2010,23(4):169.
[135]Mao C, Song H, Li J. [Improvement in affinity of phage antibodies against HBsAg by light chain shuffling][J].Zhonghua Gan Zang Bing Za Zhi,1999,7(1): 36-38.
[136]Ohlin M, Owman H, Mach M, et al. Light chain shuffling of a high affinity anti-body results in a drift in epitope recognition[J]. Mol Immunol,1996,33(1):47-56.
[137]O'Brien P, Aitken R. Antibody phage display:Methods and protocols[M].Humana Pr Inc,2002.
[138]Wysocki L, Gefter M. Gene conversion and the generation of antibody diversity [J]. Annual review of biochemistry,1989,58(1):509-527.
[139]Honjo T, Alt F. Immunoglobulin genes[M]. Elsevier,1995.
[140]Palmer DB, Crompton T, Marandi MB, et al. Intrathymic function of the human cortical epithelial cell surface antigen gp200-MR6:single-chain antibodies to evolutionarily conserved determinants disrupt mouse thymus development J]. Immunology,1999,96(2):236-245.
[141]Pereira S, Maruyama H, Siegel D, et al. A model system for detection and isola-tion of a tumor cell surface antigen using antibody phage display[J]. J Immunol Methods,1997,203(1):11-24.
[142]McWhirter JR, Kretz-Rommel A, Saven A, et al. Antibodies selected from com-binatorial libraries block a tumor antigen that plays a key role in immunomo dula-tion[J]. Proc Natl Acad Sci U S A,2006,103(4):1041-1046.
[143]Valadon P, Garnett JD, Testa JE, et al. Screening phage display libraries for organ-specific vascular immunotargeting in vivo [J]. Proc Natl Acad Sci U S A,2006, 103(2):407-412.
[144]Williams B, Atkins A, Zhang H, et al. Cell-based selection of internalizing fully human antagonistic antibodies directed against FLT3 for suppression of leukemia cell growth[J]. Leukemia,2005,19(8):1432-1438.
[145]Piazza T, Cha E, Bongarzone I, et al. Internalization and recycling of ALCAM/ CD 166 detected by a fully human single-chain recombinant antibody[J]. J Cell Sci,2005,118(7):1515-1525.
[146]Geuijen CA, Bijl N, Smit RC, et al. A proteomic approach to tumour target identi-fication using phage display, affinity purification and mass spectrometry[J]. Eur J Cancer,2005,41(1):178-187.
[147]Li Y, Moysey R, Molloy PE, et al. Directed evolution of human T-cell receptors with picomolar affinities by phage display[J]. Nat Biotechnol,2005,23(3):349-354.
[148]罗弋,庞华,李少林,等.人源肺腺癌噬菌体抗体库的构建及筛选[J].第二军医大学学报,2009,30(1)：87-90.
[149]谢平丽,李官成,李艳东,等.全人源抗结肠癌噬菌体单链抗体库的构建及筛选鉴定[J].生命科学研究,2010(1)：57-61.
[150]Siegel RW, Coleman JR, Miller KD, et al. High efficiency recovery and epitope-specific sorting of an scFv yeast display library[J]. J Immunol Methods,2004,286 (1-2):141-153.
[151]Shen Y, Yang X, Dong N, et al. Generation, screening and preliminary identifica-tion of specific Fab phage antibody library against Daudi cell strain[J]. Xi bao yu fen zi mian yi xue za zhi=Chinese journal of cellular and molecular immunology, 2009,25(2):150.
[152]Zhu Z, Bossart KN, Bishop KA, et al. Exceptionally potent cross-reactive neutr-alization of Nipah and Hendra viruses by a human monoclonal antibody [J]. J Infect Dis,2008,197(6):846-853.
[153]Sui J, Aird DR, Tamin A, et al. Broadening of neutralization activity to directly block a dominant antibody-driven SARS-coronavirus evolution pathway [J]. PLoS Pathog,2008,4(11):e1000197.
[154]Huls G, Gestel D, van der Linden J, et al. Tumor cell killing by in vitro affinity-matured recombinant human monoclonal antibodies[J]. Cancer Immunol Immun-other,2001,50(3):163-171.
[155]焦永军,周镇先,曾晓燕,等.人源Fab抗体库的构建和抗HBsAg抗体的筛选和鉴定[J].免疫学杂志,2008(1)：61-64.
[156]韩焕兴,陆慧琦,郑大勇,等.人源抗-HBs Fab表达系统的转换与效果[J].第二军医大学学报,2004,25(12)：1338-1340.
[157]王勤,李金,廖贵清.黑色素瘤抗原基因-A在头颈部肿瘤中的研究进展[J].国际口腔医学杂志,2008(S1)：35-39.
[158]丁春艳,桑梅香.黑色素瘤抗原A家族与肿瘤的关系[J].国际肿瘤学杂志2010,37(5)：121-126.
[159]Simpson E. Review lecture. Immunology of H-Y antigen and its role in sex deter-mination[J]. Proc R Soc Lond B Biol Sci,1983,220(1218):31-46.
[160]Men H, Chen Y, Zhou X, et al. Identification of sex in mouse preimplantation embryos by indirect immunofluorescent assay[J]. Yi Chuan Xue Bao,1992,19(2): 117-121.
[161]Ross DG, Bowles J, Hope M, et al. Profiles of gonadal gene expression in the developing bovine embryo[J]. Sex Dev,2009,3(5):273-283.
[162]Lambert J, Benoit B, Colvin G, et al. Quick sex determination of mouse fetuses[J]. Journal of neuroscience methods,2000,95(2):127-132.
[163]张丽,杜卫华,陈宏,等.生殖激素对小鼠胚胎性别影响的研究[J].分子细胞生物学报,2006,39(6)：573-577.
[164]Fu Q, Zhang M, Qin WS, et al. Cloning the swamp buffalo SRY gene for embryo sexing with multiplex-nested PCR[J]. Theriogenology,2007,68(9):1211-1218.
[165]Mulder LC, Sacco MG, Mangiarini L, et al. Preimplantation embryo sexing by polymerase chain reaction amplification of the sry gene on single mouse blasto-meres[J]. Genet Anal Tech Appl,1993,10(6):147-149.
[166]谭国,杨文胜.H-Y抗血清的制备及其在小鼠胚胎性别鉴定上的应用[J].高技术通讯,1993,3(5)：9-12.
[167]杭苏琴,刘为敏,牛树理,等.H-Y抗体对小鼠胚胎H-Y抗原表达的研究[J].畜牧与兽医,2003,35(12)：3-4.
[168]Piedrahita J, Anderson G. Investigation of sperm cytotoxicity as an indicator of ability of antisera to detect male-specific antigen on preimplantation mouse embryos[J]. Reproduction,1985,74(2):637.
[2]Simpson E, Scott D, Chandler P. The male-specific histocompatibility antigen, H-Y: a history of transplantation, immune response genes, sex determination and expression cloning[J]. Annu Rev Immunol,1997,15:39-61.
[3]Wachtel S, Nakamura D, Wachtel G, et al. Sex selection with monoclonal H-Y anti-body[J]. Fertil Steril,1988,50(2):355-360.
[4]Goldberg EH. H-Y antigen and sex determination[J]. Philos Trans R Soc Lond B Biol Sci,1988,322(1208):73-81.
[5]Veerhuis R, Hendriksen PJ, Hengst AM, et al. The production of anti-H-Y monoclonal antibodies:their potential use in a sex test for bovine embryos[J]. Vet Immunol Immunopathol,1994,42(3-4):317-330.
[6]Gardon JC, Aguera S, Castejon F. Sexing in vitro produced bovine embryos, at different stages of development, using rat H-Y antiserum[J]. Theriogenology,2004, 62 (1-2):35-43.
[7]Muller U. H-Y antigens[J]. Hum Genet,1996,97(6):701-704.
[8]Wolf U. The serologically detected H-Y antigen revisited[J]. Cytogenet Cell Genet, 1998,80(1-4):232-235.
[9]Utsumi K, Hayashi M, Takakura R, et al. Embryo sex selection by a rat male-specific antibody and the cytogenetic and developmental confirmation in cattle embryos[J]. Mol Reprod Dev,1993,34(l):25-32.
[10]门红升,陈云鹤,周理良.间接免疫荧光法鉴别小鼠早期胚胎性别的研究[J].遗传学报,1992,19(2)：117-121.
[11]杨文胜,朱裕鼎.小鼠早期胚胎性别鉴定的研究[J].中国实验动物学报,1993,(2)：65-69.
[12]Scott DM, Ehrmann IE, Ellis PS, et al. Identification of a mouse male-specific transplantation antigen, H-Y[J]. Nature,1995,376(6542):695-698.
[13]Meadows L, Wang W, den Haan JM, et al. The HLA-A*0201-restricted H-Y antigen contains a posttranslationally modified cysteine that significantly affects T cell recognition[J]. Immunity,1997,6(3):273-281.
[14]Wang W, Meadows LR, den Haan JM, et al. Human H-Y:a male-specific his- tocompatibility antigen derived from the SMCY protein[J]. Science,1995,269 (5230):1588-1590.
[15]Warren EH, Gavin MA, Simpson E, et al. The human UTY gene encodes a novel HLA-B8-restricted H-Y antigen[J]. J Immunol,2000,164(5):2807-2814.
[16]Vogt MH, van den Muijsenberg JW, Goulmy E, et al. The DBY gene codes for an HLA-DQ5-restricted human male-specific minor histocompatibility antigen involved in graft-versus-host disease[J]. Blood,2002,99(8):3027-3032.
[17]Vogt MH, de Paus RA, Voogt PJ, et al. DFFRY codes for a new human male-specific minor transplantation antigen involved in bone marrow graft rejection[J]. Blood,2000,95(3):1100-1105.
[18]Pierce RA, Field ED, den Haan JM, et al. Cutting edge:the HLA-A*0101-restricted HY minor histocompatibility antigen originates from DFFRY and contains a cysteinylated cysteine residue as identified by a novel mass spectro-metric technique[J]. J Immunol,1999,163(12):6360-6364.
[19]Miklos DB, Kim HT, Miller KH, et al. Antibody responses to H-Y minor histocompatibility antigens correlate with chronic graft-versus-host disease and disease remission[J]. Blood,2005,105(7):2973-2978.
[20]Shapiro M, Erickson RP. Evidence that the serological determinant of H-Y antigen is carbohydrate[J]. Nature,1981,290(5806):503-505.
[21]Fellous M, Gunther E, Kemler R, et al. Association of the H-Y male antigen with beta2-microglobulin on human lymphoid and differentiated mouse teratocar-cinoma cell lines[J]. J Exp Med,1978,148(1):58-70.
[22]Wachtel SS, Koo GC, Boyse EA. Evolutionary conservation of H-Y ('male') antigen[J].Nature,1975,254(5497):270-272.
[23]Su H, Kozak CA, Veerhuis R, et al. Isolation of a phylogenetically conserved and testis-specific gene using a monoclonal antibody against the serological H-Y antigen[J]. J Reprod Immunol,1992,21(3):275-291.
[24]Lau YF, Chan KM, Kan YW, et al. Male-enhanced expression and genetic conservation of a gene isolated with an anti-H-Y antibody [J]. Trans Assoc Am Physicians,1987,100:45-53.
[25]Koo GC, Wachtel SS, Breg WR, et al. Mapping the locus of the H-Y antigen[J]. Cytogenet Cell Genet,1976,16(1-5):175-177.
[26]Wolf U, Fraccaro M, Mayerova A, et al. A gene controlling H-Y antigen on the X chromosome. Tentative assignment by deletion mapping to Xp223[J]. Hum Genet, 1980,54(2):149-154.
[27]Wolf U. X-linked genes and gonadal differentiation[J]. Differentiation,1983,23 Suppl:S 104-106.
[28]Wachtel SS. Where is the H-Y structural gene?[J]. Cell,1980,22(1):3-4.
[29]Ginalski K, Rychlewski L, Baker D, et al. Protein structure prediction for the male-specific region of the human Y chromosome [J]. Proc Natl Acad Sci U S A, 2004,101(8):2305-2310.
[30]Brunner M, Wachtel S, Chin S. Purification and analysis of a sex-specific antibody [J]. J Immunoassay,1988,9(2):105-124.
[31]White KL, Lindner GM, Anderson GB, et al. Cytolytic and fluorescent detection of H-Y antigen on preimplantation mouse embryos[J]. Theriogenology,1983,19 (5): 701-705.
[32]Wang N, Xue L, Yuan A, et al. Identification of Mouse 8-Cell Embryo Stage-Specific Genes by Digital Differential Display [J]. Experimental animals,2009,58 (5):547-556.
[33]Bonney EA, Onyekwuluje J. Maternal tolerance to H-Y is independent of IL-10[J]. Immunol Invest,2004,33(4):385-395.
[34]Bonney EA,Onyekwuluje J.The H-Y response in midgestation and long after delivery in mice primed before pregnancy[J].Immunol Invest,2003,32(1):71-81.
[35]Bonney EA. Maternal tolerance is not critically dependent on interleukin-4[J]. Immunology,2001,103(3):382-389.
[36]Vacchio MS, Hodes RJ. Fetal expression of Fas ligand is necessary and sufficient for induction of CD8 T cell tolerance to the fetal antigen H-Y during pregnancy[J]. J Immunol,2005,174(8):4657-4661.
[37]Barjaktarevic I, Vukmanovic S. Paternal cell immunization raises autoantibodies and improves pregnancy success in mice[J]. Am J Reprod Immunol,2008,60 (6): 497-500.
[38]Goldberg EH, Boyse EA, Bennett D, et al. Serological demonstration of H-Y (male) antigen on mouse sperm[J]. Nature,1971,232(5311):478-480.
[39]Koo GC, Tada N, Chaganti R, et al. Application of monoclonal anti-HY antibody for human H-Y typing[J]. Hum Genet,1981,57(1):64-67.
[40]Shapiro M, Goldberg EH. Analysis of a serological determinant of H-Y antigen: evidence for carbohydrate specificity using an H-Y specific monoclonal antibody [J]. J Immunogenet,1984,11(3-4):209-218.
[41]Meck JM, Goldberg EH. Serological detection of H-Y antigen in humans with a cellular radioimmunobinding assay and monoclonal antibody[J].J Immunol Methods,1984,73(2):293-299.
[42]Ramalho MF, Garcia JM, Esper CR, et al. Sexing of murine and bovine embryos by developmental arrest induced by high-titer H-Y antisera[J]. Theriogenology, 2004,62(9):1569-1576.
[43]Bradley MP, Ebensperger C, Wiberg UH. Determination of the serological sex-specific (Sxs) antigen ("H-Y antigen") in birds and mammals using high-titer antisera and a sensitive urease ELISA[J]. Hum Genet,1987,76(4):352-356.
[44]Heslop BF, Bradley MP, Baird MA. The H-Y antigen[J]. Asian Pac J Allergy Immunol,1987,5(1):1-4.
[45]Bradley MP, Forrester IT, Heslop BF. Identification of a male-specific (H-Y) antigen on the flagellar plasma membrane of ram epididymal spermatozoa[J]. Hum Genet,1987,75(4):362-367.
[46]Koo GC, Varano A. Inhibition of H-Y cell-mediated cytolysis by monoclonal H-Y specific antibody[J]. Immunogenetics,1981,14(3-4):183-188.
[47]Moreira-Filho CA, Wachtel SS. Study of H-Y antigen in abnormal sex determin-ation with monoclonal antibody and an ELISA[J]. Am J Med Genet,1985,20(3): 525-534.
[48]Brunner M, Jaswaney V, Wachtel S. Reaction of monoclonal H-Y antibody in the ELISA[J]. J Reprod Immunol,1987,11(3):181-191.
[49]Brunner M, Wachtel SS. Two new ELISAs using monoclonal H-Y antibody[J]. J Immunol Methods,1988,106(1):49-55.
[50]Wang N, Xu D, Yuan A, et al. Construction and characterization of phage display library:Recognition of mouse serologically detected male (SDM) antigen[J]. Animal reproduction science,2008,104(1):93-110.
[51]Naidong W, Liqun X, Daojun X. Construction of phage display library of Fab antibodies repertoies of mouse male specific antigen; Fab[J]. Chinese Journal of Animal and Veterinary Sciences,2007,38(4):344-351.
[52]王乃东,薛立群,许道军,等.鼠源性抗雄性特异性抗原噬菌体Fab抗体的制备及分析[J].生物工程学报,2006,22(5)：727-732.
[53]Whitfield L, Lovell-Badge R, Goodfellow P. Rapid sequence evolution of the mammalian sex-determining gene SRY[J]. Nature,1993,364:713-715.
[54]Berta P, Hawkins J, Sinclair A, et al. Genetic evidence equating SRY and the testis-determining factor[J]. Nature,1990,348(6300):448-450.
[55]Swain A, Narvaez V, Burgoyne P, et al. Daxl antagonizes Sry action in mamma-lian sex determination[J]. Nature,1998,391(6669):761-767.
[56]Utsumi K, Hayashi M, Takakura R, et al. Embryo sex selection by a rat male-specific antibody and the cytogenetic and developmental confirmation in cattle embryos[J]. Molecular reproduction and development,1993,34(l):25-32.
[57]Williams T. A technique for sexing mouse embryos by a visual colorimetric assay of the X-linked enzyme, glucose 6-phosphate dehydrogenase[J]. Theriogenology, 1986,25(5):733-739.
[58]Tiffin G, Rieger D, Betteridge K, et al. Glucose and glutamine metabolism in pre-attachment cattle embryos in relation to sex and stage of development [J]. Reproduction,1991,93(1):125.
[59]Van Vliet R, Verrinder Gibbins A, Walton J. Livestock embryo sexing:a review of current methods, with emphasis on Y-specific DNA probes [J]. Theriogenology, 1989,32(3):421-438.
[60]Fajfar-Whetstone C, Rayburn A, Schook L, et al. Sex determination of porcine preimplantation embryos via Y-chromosome specific DNA sequences [J]. Animal Biotechnology,1993,4(2):183-193.
[61]Leonard M, Kirszenbaum M, Cotinot C, et al. Sexing bovine embryos using Y chromosome specific DNA probe[J]. Theriogenology,1987,27(1):248.
[62]Hacker A, Capel B, Goodfellow P, et al. Expression of Sry, the mouse sex determining gene[J]. Development,1995,121(6):1603.
[63]Zwingman T, Erickson R, Boyer T, et al. Transcription of the sex-determining region genes Sry and Zfy in the mouse preimplantation embryo [J]. Proceedings of the National Academy of Sciences of the United States of America,1993,90(3): 814.
[64]McClive P, Sinclair A. Rapid DNA extraction and PCR-sexing of mouse embryos[J]. Molecular reproduction and development,2001,60(2):225-226.
[65]李晓红,庄广伦.单细胞PCR对种植前胚胎的性别鉴定[J].中山医科大学学报,1998,19(1)：11-13.
[66]White K, Lindner G, Anderson G, et al. Cytolytic and fluorescent detection of HY antigen on preimplantation mouse embryos[J]. Theriogenology,1983,19(5):701-705.
[67]Hossepian de Lima V, Moreira-Filho C, De Bem A, et al. Sex determination of murine and bovine embryos using cytotoxicity and immunofluorescence assays[J]. Theriogenology,1993,39(6):1343-1352.
[68]周颂成,龚振明.用H-Y抗血清对大鼠胚胎进行性别鉴定的研究[J].上海农学院学报,1993,11(003)：177-181.
[69]Avery B, Schmidt M. Sex determination of bovine embryos using H-Y antibodies [J].Acta Vet Scand,1989,30(2):155-164.
[70]Goldbard SB, Warner CM. Genes affect the timing of early mouse embryo development[J]. Biol Reprod,1982,27(2):419-424.
[71]Avery B, Jorgensen CB, Madison V, et al. Morphological development and sex of bovine in vitro-fertilized embryos[J]. Mol Reprod Dev,1992,32(3):265-270.
[72]许晓霁,马向东.用H-Y抗原方法鉴定小鼠胚胎性别的研究[J].东北农学院学报,1990,21(2)：186-191.
[73]White KL, Anderson GB, Bondurant RH. Expression of a male-specific factor on various stages of preimplantation bovine embryos[J]. Biol Reprod,1987,37(4): 867-873.
[74]White KL, Anderson GB, Berger TJ, et al. Identification of a male-specific histocompatibility protein on preimplantation porcine embryos[J]. Gamete Res, 1987,17(2):107-113.
[75]White KL, Anderson GB, Pashen RL, et al. Detection of histocompatibility-Y antigen:identification of sex of pre-implantation ovine embryos [J]. J Reprod Immunol,1987,10(1):27-32.
[76]Utsumi K, Iritani A. Embryo sexing by male specific antibody and by PCR using male specific (SRY) primer[J]. Mol Reprod Dev,1993,36(2):238-241.
[77]Utsumi K, Satoh E, Iritani A. Sexing of rat embryos with antisera specific for male rats[J]. J Exp Zool,1991,260(1):99-105.
[78]马云生,郭彩霞.用抗雄性特异性单克隆抗体鉴别小鼠胚胎性别[J].天津农业科学,1999,5(4)：9-12.
[79]曹文广,董伟.应用H-Y单克隆抗体鉴定小鼠和家兔胚胎性别[J].畜牧兽医学报,1993,24(6)：506-511.
[80]Ali JI, Eldridge FE, Koo GC, et al. Enrichment of bovine X-and Y- chromosome-bearing sperm with monoclonal H-Y antibody-fluorescence-activated cell sorter [J].Arch Androl,1990,24(3):235-245.
[81]Hendriksen P. Do X and Y spermatozoa differ in proteins?[J]. Theriogenology, 1999,52(8):1295-1307.
[82]Hendriksen P, Tieman M, Van Der Lende T, et al. Binding of anti H Y monoclonal antibodies to separated X and Y chromosome bearing porcine and bovine sperm[J]. Molecular reproduction and development,1993,35(2):189-196.
[83]Barbas CF,3rd. Recent advances in phage display[J]. Curr Opin Biotechnol,1993, 4(5):526-530.
[84]Barbas CF,3rd, Kang AS, Lerner RA, et al. Assembly of combinatorial antibody libraries on phage surfaces:the gene Ⅲ site[J]. Proc Natl Acad Sci U S A,1991, 88(18):7978-7982.
[85]Willats WG. Phage display:practicalities and prospects[J]. Plant Mol Biol,2002, 50(6):837-854.
[86]Winter G, Griffiths AD, Hawkins RE, et al. Making antibodies by phage display technology [J]. Annu Rev Immunol,1994,12:433-455.
[87]Hoogenboom HR, de Bruine AP, Hufton SE, et al. Antibody phage display technology and its applications [J]. Immunotechnology,1998,4(1):1-20.
[88]Koehler M, Mielzynska I, Bubala H, et al. [Use of an immunoenzyme method with monoclonal antibodies in cytologic studies for the optimal diagnosis of non-Hodgkin's lymphoma in children][J]. Patol Pol,1989,40(3):311-323.
[89]Smith G. Filamentous fusion phage:novel expression vectors that display cloned antigens on the virion surface[J]. Science,1985,228(4705):1315.
[90]吴雯峰,王双,孙志伟.噬菌体展示抗体库筛选技术研究进展[J].生物技术通讯,2007,18(3)：476-479.
[91]Kretzschmar T, von Ruden T. Antibody discovery:phage display[J]. Current opinion in biotechnology,2002,13(6):598-602.
[92]Schmitz U, Versmold A, Kaufmann P, et al. Phage display:a molecular tool for the generation of antibodies--a review[J], Placenta,2000,21:106-112.
[93]Rodi DJ, Makowski L, Kay BK. One from column A and two from column B:the benefits of phage display in molecular-recognition studies[J]. Curr Opin Chem Biol,2002,6(1):92-96.
[94]Whaley SR, English DS, Hu EL, et al. Selection of peptides with semiconductor binding specificity for directed nanocrystal assembly[J].Nature,2000,405(6787): 665-668.
[95]Marks JD, Griffiths AD, Malmqvist M, et al. By-passing immunization:building high affinity human antibodies by chain shuffling[J]. Biotechnology (N Y),1992, 10(7):779-783.
[96]Kang AS, Jones TM, Burton DR. Antibody redesign by chain shuffling from random combinatorial immunoglobulin libraries[J]. Proc Natl Acad Sci U S A, 1991,88 (24):11120-11123.
[97]Bowen AJ, Corcoran AE. How chromatin remodelling allows shuffling of immunoglobulin heavy chain genes[J]. Mol Biosyst,2008,4(8):790-798.
[98]Marks JD. Antibody affinity maturation by chain shuffling[J]. Methods Mol Biol, 2004,248:327-343.
[99]Lantto J, Jirholt P, Barrios Y, et al. Chain shuffling to modify properties of recombinant immunoglobulins[J]. Methods Mol Biol,2002,178:303-316.
[100]Park SG, Lee JS, Je EY, et al. Affinity maturation of natural antibody using a chain shuffling technique and the expression of recombinant antibodies in Escheri- chia coli[J]. Biochem Biophys Res Commun,2000,275(2):553-557.
[101]Figini M, Marks JD, Winter G, et al. In vitro assembly of repertoires of antibody chains on the surface of phage by renaturation[J]. J Mol Biol,1994,239(1):68-78.
[102]Guo-Qiang B, Xian-Li H. Guided selection methods through chain shuffling[J]. Methods Mol Biol,2009,562:133-142.
[103]Kwong KY, Baskar S, Zhang H, et al. Generation, affinity maturation, and characterization of a human anti-human NKG2D monoclonal antibody with dual antagonistic and agonistic activity[J]. J Mol Biol,2008,384(5):1143-1156.
[104]Yoshinaga K, Matsumoto M, Torikai M, et al. Ig L-chain shuffling for affinity maturation of phage library-derived human anti-human MCP-1 antibody blocking its chemotactic activity[J]. J Biochem,2008,143(5):593-601.
[105]Chodorge M, Fourage L, Ravot G, et al. In vitro DNA recombination by L-Shuffling during ribosome display affinity maturation of an anti-Fas antibody increases the population of improved variants [J]. Protein Eng Des Sel,2008,21(5): 343-351.
[106]Jia L, Yu J, Song H, et al. Screening of human antibody Fab fragment against HBsAg and the construction of its dsFv form[J]. Int J Biol Sci,2008,4(2):103-110.
[107]刘雪林,唐晓敏,李申龙,等.轻链替换文库对抗HBs基因工程Fab抗体亲和力的影响[J].解放军医学杂志,2006,31(8)：770-772.
[108]Luginbuhl B, Kanyo Z, Jones RM, et al. Directed evolution of an anti-prion protein scFv fragment to an affinity of 1 pM and its structural interpretation [J]. J Mol Biol,2006,363(1):75-97.
[109]Watkins NA, Dafforn TR, Kuijpers M, et al. Molecular studies of anti-HLA-A2 using light-chain shuffling:a structural model for HLA antibody binding[J]. Tissue Antigens,2004,63(4):345-354.
[110]汪保安.抗TNFa抗体的体外亲和力成熟[D].中国人民解放军军事医学科学院博士论文,2004.
[111]林周.人TNF-α嵌合抗体体外亲和力成熟作用研究[D].中国人民解放军军事医学科学院博士论文,2004.
[112]王刚,王琰,刘玉峰.高亲和力抗角蛋白人抗体的基因工程制备[J].中华皮肤科杂志,2003,36(10)：559-561.
[113]Schier R, Bye J, Apell G, et al. Isolation of high-affinity monomeric human anti-c-erbB-2 single chain Fv using affinity-driven selection[J]. J Mol Biol,1996, 255(1):28-43.
[114]Proulx C, Boyer L, St-Amour I, et al. Higher affinity human D MoAb prepared by light-chain shuffling and selected by phage display[J].Transfusion,2002,42(1):59-65.
[115]Suzuki M, Takemura H, Suzuki H, et al. Light chain determines the binding property of human anti-dsDNA IgG autoantibodies[J]. Biochem Biophys Res Commun,2000,271(1):240-243.
[116]Kumar S, Nagl S, Kalsi JK, et al. Beta-2-glycoprotein specificity of human anti-phospholipid antibody resides on the light chain:a novel mechanism for acquisition of cross-reactivity by an autoantibody[J]. Mol Immunol,2005,42(1): 39-48.
[117]Kim SJ, Hong HJ. Guided selection of human antibody light chains against TAG-72 using a phage display chain shuffling approach[J]. J Microbiol,2007, 45(6):572-577.
[118]Kuepper MB, Huhn M, Spiegel H, et al. Generation of human antibody fragments against Streptococcus mutans using a phage display chain shuffling approach[J]. BMC Biotechnol,2005,5:4.
[119]包国强,马庆久,邢金良,等.用导向选择链更替技术建立抗肝癌抗原HAb18G的人源性Fab基因库[J].细胞与分子免疫学杂志,2004,20(4)：437-440.
[120]Rojas G, Talavera A, Munoz Y, et al. Light-chain shuffling results in successful phage display selection of functional prokaryotic-expressed antibody fragments to N-glycolyl GM3 ganglioside[J]. J Immunol Methods,2004,293(1-2):71-83.
[121]Christensen PA, Danielczyk A, Ravn P, et al. Modifying antibody specificity by chain shuffling of V/V between antibodies with related specificities [J]. Scand J Immunol,2009,69(1):1-10.
[122]Lau Y, Chan K, Sparkes R. Male-enhanced antigen gene is phylogenetically conserved and expressed at late stages of spermatogenesis[J]. Proceedings of the National Academy of Sciences of the United States of America,1989,86(21):8462.
[123]Kondo M, Sutou S. Cloning and molecular characterization of cDNA encoding a mouse male-enhanced antigen-2 (Mea-2):a putative family of the Golgi autoanti-gen[J]. Mitochondrial DNA,1997,7(2):71-82.
[124]王乃东,薛立群,许道军,等.鼠源性H-Y抗血清制备及特异性分析[J].中国比较医学杂志,2006,16(8)：485-488.
[125]Wang ND, Xue LQ, Xu DJ, et al. [Cloning and analysis of phage Fab antibodies of mouse male specific antigen][J]. Sheng Wu Gong Cheng Xue Bao,2006,22(5): 727-732.
[126]McCafferty J, Griffiths A, Winter G, et al. Phage antibodies:filamentous phage displaying antibody variable domains[J].1990.348(6301):552-554.
[127]Hudson P, Souriau C. Engineered antibodies [J]. Nature medicine,2003,9(l):129-134.
[128]王群,刘军莉,高飞,等.人源性TAT-Fab抗体的表达优化及生物活性测定[J].浙江大学学报,2007,33(6)：651-655.
[129]Hust M, Dubel S. Phage display vectors for the in vitro generation of human antibody fragments [J]. Immunochemical protocols,2005,295:71-95.
[130]Barbas C, Kang A, Lerner R, et al. Assembly of combinatorial antibody libraries on phage surfaces:the gene Ⅲ site[J]. Proceedings of the National Academy of Sciences of the United States of America,1991,88(18):7978.
[131]Barbas S, Barbas C. Filamentous phage display[J]. Fibrinolysis,1994,8:245-252.
[132]陆慧琦,韩焕兴,叶伟民,等.PBAD表达系统对抗-HBs Fab抗体表达的影 响[J].免疫学杂志,2005,21(1)：66-68.
[133]Corisdeo S, Wang B. Functional expression and display of an antibody Fab fragment in Escherichia coli:study of vector designs and culture conditions[J]. Protein expression and purification,2004,34(2):270-279.
[134]Backovic M, Johansson D, Klupp B, et al Efficient method for production of high yields of Fab fragments in Drosophila S2 cells[J]. Protein Engineering Design and Selection,2010,23(4):169.
[135]Mao C, Song H, Li J. [Improvement in affinity of phage antibodies against HBsAg by light chain shuffling][J].Zhonghua Gan Zang Bing Za Zhi,1999,7(1): 36-38.
[136]Ohlin M, Owman H, Mach M, et al. Light chain shuffling of a high affinity anti-body results in a drift in epitope recognition[J]. Mol Immunol,1996,33(1):47-56.
[137]O'Brien P, Aitken R. Antibody phage display:Methods and protocols[M].Humana Pr Inc,2002.
[138]Wysocki L, Gefter M. Gene conversion and the generation of antibody diversity [J]. Annual review of biochemistry,1989,58(1):509-527.
[139]Honjo T, Alt F. Immunoglobulin genes[M]. Elsevier,1995.
[140]Palmer DB, Crompton T, Marandi MB, et al. Intrathymic function of the human cortical epithelial cell surface antigen gp200-MR6:single-chain antibodies to evolutionarily conserved determinants disrupt mouse thymus development J]. Immunology,1999,96(2):236-245.
[141]Pereira S, Maruyama H, Siegel D, et al. A model system for detection and isola-tion of a tumor cell surface antigen using antibody phage display[J]. J Immunol Methods,1997,203(1):11-24.
[142]McWhirter JR, Kretz-Rommel A, Saven A, et al. Antibodies selected from com-binatorial libraries block a tumor antigen that plays a key role in immunomo dula-tion[J]. Proc Natl Acad Sci U S A,2006,103(4):1041-1046.
[143]Valadon P, Garnett JD, Testa JE, et al. Screening phage display libraries for organ-specific vascular immunotargeting in vivo [J]. Proc Natl Acad Sci U S A,2006, 103(2):407-412.
[144]Williams B, Atkins A, Zhang H, et al. Cell-based selection of internalizing fully human antagonistic antibodies directed against FLT3 for suppression of leukemia cell growth[J]. Leukemia,2005,19(8):1432-1438.
[145]Piazza T, Cha E, Bongarzone I, et al. Internalization and recycling of ALCAM/ CD 166 detected by a fully human single-chain recombinant antibody[J]. J Cell Sci,2005,118(7):1515-1525.
[146]Geuijen CA, Bijl N, Smit RC, et al. A proteomic approach to tumour target identi-fication using phage display, affinity purification and mass spectrometry[J]. Eur J Cancer,2005,41(1):178-187.
[147]Li Y, Moysey R, Molloy PE, et al. Directed evolution of human T-cell receptors with picomolar affinities by phage display[J]. Nat Biotechnol,2005,23(3):349-354.
[148]罗弋,庞华,李少林,等.人源肺腺癌噬菌体抗体库的构建及筛选[J].第二军医大学学报,2009,30(1)：87-90.
[149]谢平丽,李官成,李艳东,等.全人源抗结肠癌噬菌体单链抗体库的构建及筛选鉴定[J].生命科学研究,2010(1)：57-61.
[150]Siegel RW, Coleman JR, Miller KD, et al. High efficiency recovery and epitope-specific sorting of an scFv yeast display library[J]. J Immunol Methods,2004,286 (1-2):141-153.
[151]Shen Y, Yang X, Dong N, et al. Generation, screening and preliminary identifica-tion of specific Fab phage antibody library against Daudi cell strain[J]. Xi bao yu fen zi mian yi xue za zhi=Chinese journal of cellular and molecular immunology, 2009,25(2):150.
[152]Zhu Z, Bossart KN, Bishop KA, et al. Exceptionally potent cross-reactive neutr-alization of Nipah and Hendra viruses by a human monoclonal antibody [J]. J Infect Dis,2008,197(6):846-853.
[153]Sui J, Aird DR, Tamin A, et al. Broadening of neutralization activity to directly block a dominant antibody-driven SARS-coronavirus evolution pathway [J]. PLoS Pathog,2008,4(11):e1000197.
[154]Huls G, Gestel D, van der Linden J, et al. Tumor cell killing by in vitro affinity-matured recombinant human monoclonal antibodies[J]. Cancer Immunol Immun-other,2001,50(3):163-171.
[155]焦永军,周镇先,曾晓燕,等.人源Fab抗体库的构建和抗HBsAg抗体的筛选和鉴定[J].免疫学杂志,2008(1)：61-64.
[156]韩焕兴,陆慧琦,郑大勇,等.人源抗-HBs Fab表达系统的转换与效果[J].第二军医大学学报,2004,25(12)：1338-1340.
[157]王勤,李金,廖贵清.黑色素瘤抗原基因-A在头颈部肿瘤中的研究进展[J].国际口腔医学杂志,2008(S1)：35-39.
[158]丁春艳,桑梅香.黑色素瘤抗原A家族与肿瘤的关系[J].国际肿瘤学杂志2010,37(5)：121-126.
[159]Simpson E. Review lecture. Immunology of H-Y antigen and its role in sex deter-mination[J]. Proc R Soc Lond B Biol Sci,1983,220(1218):31-46.
[160]Men H, Chen Y, Zhou X, et al. Identification of sex in mouse preimplantation embryos by indirect immunofluorescent assay[J]. Yi Chuan Xue Bao,1992,19(2): 117-121.
[161]Ross DG, Bowles J, Hope M, et al. Profiles of gonadal gene expression in the developing bovine embryo[J]. Sex Dev,2009,3(5):273-283.
[162]Lambert J, Benoit B, Colvin G, et al. Quick sex determination of mouse fetuses[J]. Journal of neuroscience methods,2000,95(2):127-132.
[163]张丽,杜卫华,陈宏,等.生殖激素对小鼠胚胎性别影响的研究[J].分子细胞生物学报,2006,39(6)：573-577.
[164]Fu Q, Zhang M, Qin WS, et al. Cloning the swamp buffalo SRY gene for embryo sexing with multiplex-nested PCR[J]. Theriogenology,2007,68(9):1211-1218.
[165]Mulder LC, Sacco MG, Mangiarini L, et al. Preimplantation embryo sexing by polymerase chain reaction amplification of the sry gene on single mouse blasto-meres[J]. Genet Anal Tech Appl,1993,10(6):147-149.
[166]谭国,杨文胜.H-Y抗血清的制备及其在小鼠胚胎性别鉴定上的应用[J].高技术通讯,1993,3(5)：9-12.
[167]杭苏琴,刘为敏,牛树理,等.H-Y抗体对小鼠胚胎H-Y抗原表达的研究[J].畜牧与兽医,2003,35(12)：3-4.
[168]Piedrahita J, Anderson G. Investigation of sperm cytotoxicity as an indicator of ability of antisera to detect male-specific antigen on preimplantation mouse embryos[J]. Reproduction,1985,74(2):637.