抗牛精子膜蛋白单链抗体基因的克隆与表达
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摘要
由于精子膜蛋白结构上的多形性、功能上的复杂性，加之缺乏有效的分离纯化手段，有关精子膜蛋白的研究一直是一个难点。sp18是由本实验室首次发现的一组牛精子膜蛋白，由于尚未纯化得到sp18，有关sp18的研究难以突破。为此，本研究从分泌抗sp18单抗的杂交瘤细胞入手，欲克隆其抗体基因并分析其序列特征，以此推测其针对的抗原性质。
应用重组噬菌体抗体库技术，从分泌小鼠抗牛精子sp18抗体的杂交瘤细胞系中分离总RNA，克隆抗体重链和轻链可变区基因，加入连接肽引物(Linker primer)组装成单链抗体ScFv(single chain fragment variable)基因并用RS引物进行扩增，Sfi Ⅰ、Not Ⅰ酶切，回收后与pCANTAB5E载体相连，转化E.coli TG1宿主菌，构建单链抗体文库。经辅助噬菌体M13K07超感染，将ScFv展示于噬菌体表面。以牛精子作为包被抗原，经过三轮“亲和吸附-洗脱-扩增”的淘选后，进行phage-ELISA筛选和鉴定，在波长450nm处测定吸光值，吸光值在阴性对照两倍以上的视为阳性克隆。取阳性重组噬菌体抗体克隆株pCSA1，PCR扩增其ScFv基因，筛选重组子进行序列测定，发现其序列符合小鼠抗体基因的一般特征，并且与几株抗磷酸胆碱的抗体重链和轻链可变区序列的同源性达80％以上；推测pCSA1 ScFv针对的抗原是磷酸胆碱类物质。将pCSA1 ScFv基因克隆到pET-30a(+)载体上，用IPTG进行诱导表达，SDS-PAGE蛋白电泳分析，发现重组蛋白表达量占菌体总蛋白含量的30％。这些结果表明，本研究成功获得了杂交瘤细胞的单链抗体基因，并且在大肠杆菌细胞中得到了高表达；推测抗sp18单抗针对的抗原可能是磷酸胆碱类物质。
精子蛋白种类繁多，为了进一步研究牛精子蛋白功能，本研究欲构建一个针对牛精子的噬菌体抗体文库。首先用牛精子免疫雌性四周龄BALB／c小鼠，从其脾脏组织分离总RNA，应用重组噬菌体抗体库技术，构建了一个针对牛精子的噬菌体抗体文库。经PCR检测，计算文库的有效库容为1.2×10~4TFU。用phage-ELISA从文库中筛选并鉴定出一个针对牛精子的阳性克隆，序列分析发现，该单链抗体克隆的序列符合小鼠抗体可变区的一般特征，这说明构建的针对牛精子的噬菌体抗体文库是成功的。
本研究成功获得了杂交瘤细胞的单链抗体基因，并且推测抗sp18单抗针对的抗原可能是磷酸胆碱类物质；成功构建了针对牛精子的噬菌体抗体文库，为日后牛精子蛋白的功能研究与应用研究奠定了一定的基础。
It was difficult to confirm the role of sperm membrane protein during mammalian fertilization and embryogenesis for the multiple configuration and lack of purified protein of sperm membrane proteins. In our previous studies, the primary role of sp18 family proteins, located on the post head of bull sperm plasma was described. In the present study, the express library of monoclonal anti-sp18 ScFv (Single Chain Fragment Variable) gene is constructed and selected for further study of sp18 antigen on mammalian fertilization and embryogenesis.
Total RNA were firstly isolated from these growing hybridoma cells which secretes monoclonal anti-sp18 antibodies. After obtained using RPAS system, VH and VL genes were used to assemble ScFv gene fragment with a Linker primer. The ScFv fragment was then amplified with RS primer, followed by Sfi I and Not I degistion together and ligation into pCANTAB5E vector. ScFv antibody library was constructed after transform of pCANTAB5E into E.coli TGI. The library was rescued with phage M13K07 in order to display ScFv on the surface of the phage and to form the recombinant phage antibody library. One of positive ScFv clones, named pCSAl, was selected with phage-ELISA after panning and screening by bull sperm three times. ScFv fragment, amplified from pCSA1, was ligated to pMD18-T vector for sequencing analysis. Analysis on sequence indicated that the ScFv shown more than 80% homogolous sequence with anti-phosphorylcholine mAbs. These results suggested that ScFv CDR of pCSAl, and sp18 antigens, might be phosphorylchol
ine. The recombinant protein of pET30a/ScFv was expressed by E.coli BL21 (DE3) after ligation ScFv fragment into pET-30a (+) vector followed by transform and induction of IPTG. The patterns of SDS-PAGE indicated more than 30 % of recombinant proteins could be obtained from the extract of E.coli BL21.
Furthermore, library of recombinant phage antibodies was constructed from total RNA isolated
from spleen of the female BALB/c mice immunized by bull sperm. PCR detection found there were
1.2x104 TFU in the library. One of colonies of ScFv antibody, which binds to bull sperm, was selected
and sequenced. Sequence analysis indicates that the ScFv colony resembles the mouse antibody.
应用重组噬菌体抗体库技术，从分泌小鼠抗牛精子sp18抗体的杂交瘤细胞系中分离总RNA，克隆抗体重链和轻链可变区基因，加入连接肽引物(Linker primer)组装成单链抗体ScFv(single chain fragment variable)基因并用RS引物进行扩增，Sfi Ⅰ、Not Ⅰ酶切，回收后与pCANTAB5E载体相连，转化E.coli TG1宿主菌，构建单链抗体文库。经辅助噬菌体M13K07超感染，将ScFv展示于噬菌体表面。以牛精子作为包被抗原，经过三轮“亲和吸附-洗脱-扩增”的淘选后，进行phage-ELISA筛选和鉴定，在波长450nm处测定吸光值，吸光值在阴性对照两倍以上的视为阳性克隆。取阳性重组噬菌体抗体克隆株pCSA1，PCR扩增其ScFv基因，筛选重组子进行序列测定，发现其序列符合小鼠抗体基因的一般特征，并且与几株抗磷酸胆碱的抗体重链和轻链可变区序列的同源性达80％以上；推测pCSA1 ScFv针对的抗原是磷酸胆碱类物质。将pCSA1 ScFv基因克隆到pET-30a(+)载体上，用IPTG进行诱导表达，SDS-PAGE蛋白电泳分析，发现重组蛋白表达量占菌体总蛋白含量的30％。这些结果表明，本研究成功获得了杂交瘤细胞的单链抗体基因，并且在大肠杆菌细胞中得到了高表达；推测抗sp18单抗针对的抗原可能是磷酸胆碱类物质。
精子蛋白种类繁多，为了进一步研究牛精子蛋白功能，本研究欲构建一个针对牛精子的噬菌体抗体文库。首先用牛精子免疫雌性四周龄BALB／c小鼠，从其脾脏组织分离总RNA，应用重组噬菌体抗体库技术，构建了一个针对牛精子的噬菌体抗体文库。经PCR检测，计算文库的有效库容为1.2×10~4TFU。用phage-ELISA从文库中筛选并鉴定出一个针对牛精子的阳性克隆，序列分析发现，该单链抗体克隆的序列符合小鼠抗体可变区的一般特征，这说明构建的针对牛精子的噬菌体抗体文库是成功的。
本研究成功获得了杂交瘤细胞的单链抗体基因，并且推测抗sp18单抗针对的抗原可能是磷酸胆碱类物质；成功构建了针对牛精子的噬菌体抗体文库，为日后牛精子蛋白的功能研究与应用研究奠定了一定的基础。
It was difficult to confirm the role of sperm membrane protein during mammalian fertilization and embryogenesis for the multiple configuration and lack of purified protein of sperm membrane proteins. In our previous studies, the primary role of sp18 family proteins, located on the post head of bull sperm plasma was described. In the present study, the express library of monoclonal anti-sp18 ScFv (Single Chain Fragment Variable) gene is constructed and selected for further study of sp18 antigen on mammalian fertilization and embryogenesis.
Total RNA were firstly isolated from these growing hybridoma cells which secretes monoclonal anti-sp18 antibodies. After obtained using RPAS system, VH and VL genes were used to assemble ScFv gene fragment with a Linker primer. The ScFv fragment was then amplified with RS primer, followed by Sfi I and Not I degistion together and ligation into pCANTAB5E vector. ScFv antibody library was constructed after transform of pCANTAB5E into E.coli TGI. The library was rescued with phage M13K07 in order to display ScFv on the surface of the phage and to form the recombinant phage antibody library. One of positive ScFv clones, named pCSAl, was selected with phage-ELISA after panning and screening by bull sperm three times. ScFv fragment, amplified from pCSA1, was ligated to pMD18-T vector for sequencing analysis. Analysis on sequence indicated that the ScFv shown more than 80% homogolous sequence with anti-phosphorylcholine mAbs. These results suggested that ScFv CDR of pCSAl, and sp18 antigens, might be phosphorylchol
ine. The recombinant protein of pET30a/ScFv was expressed by E.coli BL21 (DE3) after ligation ScFv fragment into pET-30a (+) vector followed by transform and induction of IPTG. The patterns of SDS-PAGE indicated more than 30 % of recombinant proteins could be obtained from the extract of E.coli BL21.
Furthermore, library of recombinant phage antibodies was constructed from total RNA isolated
from spleen of the female BALB/c mice immunized by bull sperm. PCR detection found there were
1.2x104 TFU in the library. One of colonies of ScFv antibody, which binds to bull sperm, was selected
and sequenced. Sequence analysis indicates that the ScFv colony resembles the mouse antibody.
引文
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