高效UV-致弱尾蚴单链抗体库的构建及Sj SCA66-68kDa ScFv的筛选
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摘要
研究目的
基于致弱尾蚴疫苗及天然分子疫苗高保护力的特点,利用噬菌体抗体库技术诸多的优点,构建高效日本和血吸虫UV-致弱尾蚴单链抗体(ScFv)表达文库,并以此作为高通量的库源筛选出针对有潜在保护力的天然分子抗原SjSCA66-68kDA的单链抗体,为进一步获取日本血吸虫候选疫苗天然分子SjSCA66-68kDA的相关编码基因奠定基础,加快抗日本血吸虫病高效天然分子基因疫苗研制的步伐。
研究方法
1.利用噬菌体展示技术构建高效日本血吸虫UV-照射致弱尾蚴单链抗体库,并从库容量、多样性等方面对文库进行鉴定。利用UV-照射日本血吸虫致弱尾蚴多次感染BALB/c小鼠,并将无菌UV-致弱尾蚴血清,在正常尾蚴攻击感染(尾蚴数为40±2条)前以及感染后一周,三周,共三次尾静脉注射转移至BALB/c小鼠体内,45天后处死小鼠,分别计算减虫率和减卵率,确保其致弱尾蚴血清的高效性。在成功构建致弱尾蚴小鼠模型的基础上,提取小鼠脾脏RNA,用RT-PCR法扩增得到全套VH和VL基因,经重叠PCR法扩增得到ScFv片段,将ScFv片段克隆至PCANTAB 5E载体,电转化感受态TG1菌,经辅助噬菌体M13K07拯救,获得的日本血吸虫UV-弱尾蚴单链抗体库。
2.利用建库时所获得的高效UV-致弱尾蚴血清与日本血吸虫不同发育阶段的抗原进行免疫反应,识别和选择免疫初筛的靶分子,并对候选抗原SCA66-68天然分子免疫生化特性和序列的初步研究:通过电泳切胶、电洗脱、超滤离心和冻干等方法分离和纯化SCA66-68KDa抗原,用含SCA66-68KDa抗原PAGE胶结合微量淋巴结注射法免疫新西兰兔制备出单特异性SCA66-68KDa免疫兔血清,用ELISA和Western blot检测和鉴定其效价和特异性;通过银染色和PAS染色确定SCA66-68KDa抗原的化学性质;用N—端测序的方法研究SCA66-68KDa抗原的蛋白质序列。
3.运用分离纯化的疫苗候选分子SCA66-68kDa抗原,通过抗原直接法和双抗体夹心法联合硝酸纤维膜富集法及菌落挖集法的组合策略筛选高通量的UV-照射致弱尾蚴单链抗体库,用Western blot对所获得的特异性SCA66-68kDa单链抗体进行鉴定。
研究结果:
1.UV-照射日本血吸虫致弱尾蚴血清被动转移BALB/c小鼠,可得到42.5%减虫率,显著高于感染血清转移组的减虫率28.0%。在成功构建UV-照射日本血吸虫致弱尾蚴小鼠模型的基础上,获得了高效日本血吸虫UV-弱尾蚴单链抗体库,其的库容量测定为1.9~*10~8,重组率100%,多样性好。
2.实验发现,建库时的高效致弱尾蚴血清可识别血吸虫不同阶段抗原的66-68kDa附近有共同的识别条带,加上本室先前的研究基础,选择尾蚴66-68kDa天然分子作为免疫初筛的靶分子抗原。成功分离纯化了电泳纯及免疫纯的SCA66-68kDa抗原,并成功制备出单特异性SCA66-68KDa免疫兔血清,抗体效价高达1:12800;硝酸银及PAS不同的染色方法来确定SCA66-68KD的化学性质为一种蛋白类组分且是一种非糖蛋白;用N—测序的方法研究SCA66-68KDa天然分子的蛋白序列,结果存在N-端肽段封闭现象,提出了SCA66-68KDa天然分子的蛋白质测序的优化策略。
3.通过高效UV-照射致弱尾蚴血清识别了SCA66-68kDa抗原,运用分离纯化的日本血吸虫病疫苗候选分子SCA66-68kDa对UV-照射致弱尾蚴单链抗体库进行筛选。运用抗原直接法和双抗夹心硝酸纤维膜法分别经过多轮淘洗富集,用集落挖掘法筛选致弱尾蚴单链抗体库:双抗夹心法第一轮假阳性太高,复筛没有得到阳性克隆;抗原直接法共获得6个SCA66-68kDa阳性克隆,将筛选获得阳性克隆转化至Ecoli HB2151菌,诱导可溶性scFv表达。经SDS-PAGE,Western blot分析,结果显示可溶性scFv获得了正确表达,且与相应抗原SCA66-68kDa特异性结合。结果表明通过抗原直接筛选法获得了特异性抗SCA66-68kDa单链抗体。
结论:
1.首次成功构建了高效日本血吸虫UV-致弱尾蚴单链抗体库,它的构建弥补了致弱疫苗应用的局限性,为开拓新的疫苗发展策略和新疫苗的设计提供借鉴,也为进一步筛选用于血吸虫病诊断和治疗的特异性单链抗体、进行抗原表位分析和疫苗研制提供了高通量的库源。
2.以候选疫苗SjSCA66-68kDa天然分子作为初筛靶抗原,成功分离纯化SjSCA66-68kDa天然分子,并对其免疫生化特性和序列进行初步研究,为进一步筛选致弱尾蚴单链抗体库提供了理论依据,也为天然分子编码基因工程疫苗的制备奠定基础。
3.成功地运用抗原直接法联合硝酸纤维膜富集法和集落挖掘法筛选获得了特异性抗SCA66-68kDa单链抗体。SCA66-68kDa特异性ScFv的获得在疫苗候选分子的筛选中体现优势,为成功构建有效的抗日本血吸虫病高效天然分子的基因工程疫苗及扩大现场应用奠定基础。另外,在利用单链抗体开展免疫诊断,免疫治疗和疾病致病分子机制研究等诸多方面,显示出广阔的应用前景。
Objective
To construct the highly efficient UV-attenuated Schistosoma japonicum cercariae single chain Fv phage display library and to obtain the SCA66-68kDa ScFv based on the advantages of UV-Attenuated Cercariae Vaccines,natural molecular vaccine and phage antibody library technology,inorder to get the new candidate vaccine coding genes of SjSCA66-68,which would advance the progress of vaccine for schistosoma japonicum.
Methods
1.Using the methods of phage display antibody library to construct the single-chain Fv(scFv)antibody library against UV-attenuated Schistosoma japonicum cercariae and characterize it from the way of capacity and diversity.BALB/c mice were immunized several times with UV-attenuated Schistosoma japonicum cercariae and In order to evaluate the protection potential of UV-attenuated cercariae serum, the serum was transferred passively to mice at 1 week before,1 week after and 3 weeks after challenged with Schistosoma japonicum cercariae.Once we get the protective serum,the spleens of these mice was used to prepare RNA.The variable heavy(VH)and variable light(VL)genes were amplified by RT-PCR and then the scFv was obtained through SOE-PCR.The scFv fragments were cloned into the vector PCANTAB 5E and the recombinant plasmids were electroporated into competent E.coli TG1 cells.UV-attenuated Schistosoma japonicum cercariae single chain Fv phage display library was obtained after the recombinant phagemids were rescued by infection of helper phage M13K07.
2.Confirm the target screening antigens with highly efficient UMS and the characterizations of immuno-biological chemistry of the new candidate vaccine antigens of SjSCA66-68:SjSCA66-68 were purifed from SjSCA as following procedures:SDS-PAGE,gel band secton, electroelution and ultrafiltraton,etc;The monospecific anti-sera against SCA66-68 was produced through immunization of New Zealand rabbit in the way of injection with microdosis SCA66-68 antigen gel into lymph node;Characterize the SCA66-68 antigen through silver and PAS staining;Sequence the SCA66-68 protein with N-terminal sequence analysis.
3.Screening of(scFv)antibody library against UV-attenuated Schistosoma japonicum cercariae with the purified antigen of SCA66-68 through the methods of NC meberance enrichment and colony lift assaydouble binded with the way of antibody sandwich method and antigen direct method respectively.The specificity of SCA66-68kDa ScFv was identified by western blot.
Results
1.BALB/c mice were transferred passively with UV-attenuated cercariae serum,the results showed the UV-attenuated cercariae serum conferred significant higher level of protection(42.5%)when transferred before challenge than that of natural infection(28.0%).As the base of the high protective animal model which induced by UV-attenuated cercariae,UV-attenuated Schistosoma japonicum cercariae single chain Fv phage display library were construct successfully with the capacity of 1.9*10~8 clones and the recombination rate of 100%and good diversity.
2.66-68kDa antigen in different stage of schistosoma japonicum could be recognized by highly efficient UMS and SCA66-68kDa was taken as t the target screening antigens;SjSCA66-68 were purifed from SjSCA and the rabbit monospecific anti-sera with a titer as high as 1:12800 against SCA66-68 were produced successfully;The SCA66-68 antigen was characterize as a non-glycoprotein through silver and PAS staining;The result of N-terminal sequence analysis showed the N-terminally blocked proteins of SCA66-68,so we put forward some suggestions on the strategy of the sequencing of SCA66-68kDa.
3.Screening of(scFv)antibody library against UV-attenuated Schistosoma japonicum cercariae with the purified antigen of SCA66-68,after enriching and screening,we get six positive clones in the way of antigen direct method,and no positive clones with the antibody sandwich method.The Western blot analysis and SDS-PAGE showed that specific ScFv SCA66-68 was successfully abtained after Soluble expression of E-coli HB2151.
Conclusion
1.UV-attenuated Schistosoma japonicum cercariae single chain Fv phage display library was successfully constructed.The construction of this library may overcome the limitation of the application of radiate-attenuated vaccine,and benefit to develop the new strategies of prevention on Schistosoma japonicum.
2.SjSCA66-68kDa nature antigen could be recognized by UMS,the characterizations of immuno-biological chemistry of the new candidate vaccine antigens of SjSCA66-68 provides a theoretical basis for further screening of ScFv library,also lay a firm foundation for developing effective molecules of vaccine against schistosoma.
3.Successfully get the specific SCA66-68kDa ScFv with the methods of NC meberance enrichment and colony lift binded with antigen direct way,which has shown the wide application prospects in the field of the vaccine,immunodiagnosis and Immunotherapy.
基于致弱尾蚴疫苗及天然分子疫苗高保护力的特点,利用噬菌体抗体库技术诸多的优点,构建高效日本和血吸虫UV-致弱尾蚴单链抗体(ScFv)表达文库,并以此作为高通量的库源筛选出针对有潜在保护力的天然分子抗原SjSCA66-68kDA的单链抗体,为进一步获取日本血吸虫候选疫苗天然分子SjSCA66-68kDA的相关编码基因奠定基础,加快抗日本血吸虫病高效天然分子基因疫苗研制的步伐。
研究方法
1.利用噬菌体展示技术构建高效日本血吸虫UV-照射致弱尾蚴单链抗体库,并从库容量、多样性等方面对文库进行鉴定。利用UV-照射日本血吸虫致弱尾蚴多次感染BALB/c小鼠,并将无菌UV-致弱尾蚴血清,在正常尾蚴攻击感染(尾蚴数为40±2条)前以及感染后一周,三周,共三次尾静脉注射转移至BALB/c小鼠体内,45天后处死小鼠,分别计算减虫率和减卵率,确保其致弱尾蚴血清的高效性。在成功构建致弱尾蚴小鼠模型的基础上,提取小鼠脾脏RNA,用RT-PCR法扩增得到全套VH和VL基因,经重叠PCR法扩增得到ScFv片段,将ScFv片段克隆至PCANTAB 5E载体,电转化感受态TG1菌,经辅助噬菌体M13K07拯救,获得的日本血吸虫UV-弱尾蚴单链抗体库。
2.利用建库时所获得的高效UV-致弱尾蚴血清与日本血吸虫不同发育阶段的抗原进行免疫反应,识别和选择免疫初筛的靶分子,并对候选抗原SCA66-68天然分子免疫生化特性和序列的初步研究:通过电泳切胶、电洗脱、超滤离心和冻干等方法分离和纯化SCA66-68KDa抗原,用含SCA66-68KDa抗原PAGE胶结合微量淋巴结注射法免疫新西兰兔制备出单特异性SCA66-68KDa免疫兔血清,用ELISA和Western blot检测和鉴定其效价和特异性;通过银染色和PAS染色确定SCA66-68KDa抗原的化学性质;用N—端测序的方法研究SCA66-68KDa抗原的蛋白质序列。
3.运用分离纯化的疫苗候选分子SCA66-68kDa抗原,通过抗原直接法和双抗体夹心法联合硝酸纤维膜富集法及菌落挖集法的组合策略筛选高通量的UV-照射致弱尾蚴单链抗体库,用Western blot对所获得的特异性SCA66-68kDa单链抗体进行鉴定。
研究结果:
1.UV-照射日本血吸虫致弱尾蚴血清被动转移BALB/c小鼠,可得到42.5%减虫率,显著高于感染血清转移组的减虫率28.0%。在成功构建UV-照射日本血吸虫致弱尾蚴小鼠模型的基础上,获得了高效日本血吸虫UV-弱尾蚴单链抗体库,其的库容量测定为1.9~*10~8,重组率100%,多样性好。
2.实验发现,建库时的高效致弱尾蚴血清可识别血吸虫不同阶段抗原的66-68kDa附近有共同的识别条带,加上本室先前的研究基础,选择尾蚴66-68kDa天然分子作为免疫初筛的靶分子抗原。成功分离纯化了电泳纯及免疫纯的SCA66-68kDa抗原,并成功制备出单特异性SCA66-68KDa免疫兔血清,抗体效价高达1:12800;硝酸银及PAS不同的染色方法来确定SCA66-68KD的化学性质为一种蛋白类组分且是一种非糖蛋白;用N—测序的方法研究SCA66-68KDa天然分子的蛋白序列,结果存在N-端肽段封闭现象,提出了SCA66-68KDa天然分子的蛋白质测序的优化策略。
3.通过高效UV-照射致弱尾蚴血清识别了SCA66-68kDa抗原,运用分离纯化的日本血吸虫病疫苗候选分子SCA66-68kDa对UV-照射致弱尾蚴单链抗体库进行筛选。运用抗原直接法和双抗夹心硝酸纤维膜法分别经过多轮淘洗富集,用集落挖掘法筛选致弱尾蚴单链抗体库:双抗夹心法第一轮假阳性太高,复筛没有得到阳性克隆;抗原直接法共获得6个SCA66-68kDa阳性克隆,将筛选获得阳性克隆转化至Ecoli HB2151菌,诱导可溶性scFv表达。经SDS-PAGE,Western blot分析,结果显示可溶性scFv获得了正确表达,且与相应抗原SCA66-68kDa特异性结合。结果表明通过抗原直接筛选法获得了特异性抗SCA66-68kDa单链抗体。
结论:
1.首次成功构建了高效日本血吸虫UV-致弱尾蚴单链抗体库,它的构建弥补了致弱疫苗应用的局限性,为开拓新的疫苗发展策略和新疫苗的设计提供借鉴,也为进一步筛选用于血吸虫病诊断和治疗的特异性单链抗体、进行抗原表位分析和疫苗研制提供了高通量的库源。
2.以候选疫苗SjSCA66-68kDa天然分子作为初筛靶抗原,成功分离纯化SjSCA66-68kDa天然分子,并对其免疫生化特性和序列进行初步研究,为进一步筛选致弱尾蚴单链抗体库提供了理论依据,也为天然分子编码基因工程疫苗的制备奠定基础。
3.成功地运用抗原直接法联合硝酸纤维膜富集法和集落挖掘法筛选获得了特异性抗SCA66-68kDa单链抗体。SCA66-68kDa特异性ScFv的获得在疫苗候选分子的筛选中体现优势,为成功构建有效的抗日本血吸虫病高效天然分子的基因工程疫苗及扩大现场应用奠定基础。另外,在利用单链抗体开展免疫诊断,免疫治疗和疾病致病分子机制研究等诸多方面,显示出广阔的应用前景。
Objective
To construct the highly efficient UV-attenuated Schistosoma japonicum cercariae single chain Fv phage display library and to obtain the SCA66-68kDa ScFv based on the advantages of UV-Attenuated Cercariae Vaccines,natural molecular vaccine and phage antibody library technology,inorder to get the new candidate vaccine coding genes of SjSCA66-68,which would advance the progress of vaccine for schistosoma japonicum.
Methods
1.Using the methods of phage display antibody library to construct the single-chain Fv(scFv)antibody library against UV-attenuated Schistosoma japonicum cercariae and characterize it from the way of capacity and diversity.BALB/c mice were immunized several times with UV-attenuated Schistosoma japonicum cercariae and In order to evaluate the protection potential of UV-attenuated cercariae serum, the serum was transferred passively to mice at 1 week before,1 week after and 3 weeks after challenged with Schistosoma japonicum cercariae.Once we get the protective serum,the spleens of these mice was used to prepare RNA.The variable heavy(VH)and variable light(VL)genes were amplified by RT-PCR and then the scFv was obtained through SOE-PCR.The scFv fragments were cloned into the vector PCANTAB 5E and the recombinant plasmids were electroporated into competent E.coli TG1 cells.UV-attenuated Schistosoma japonicum cercariae single chain Fv phage display library was obtained after the recombinant phagemids were rescued by infection of helper phage M13K07.
2.Confirm the target screening antigens with highly efficient UMS and the characterizations of immuno-biological chemistry of the new candidate vaccine antigens of SjSCA66-68:SjSCA66-68 were purifed from SjSCA as following procedures:SDS-PAGE,gel band secton, electroelution and ultrafiltraton,etc;The monospecific anti-sera against SCA66-68 was produced through immunization of New Zealand rabbit in the way of injection with microdosis SCA66-68 antigen gel into lymph node;Characterize the SCA66-68 antigen through silver and PAS staining;Sequence the SCA66-68 protein with N-terminal sequence analysis.
3.Screening of(scFv)antibody library against UV-attenuated Schistosoma japonicum cercariae with the purified antigen of SCA66-68 through the methods of NC meberance enrichment and colony lift assaydouble binded with the way of antibody sandwich method and antigen direct method respectively.The specificity of SCA66-68kDa ScFv was identified by western blot.
Results
1.BALB/c mice were transferred passively with UV-attenuated cercariae serum,the results showed the UV-attenuated cercariae serum conferred significant higher level of protection(42.5%)when transferred before challenge than that of natural infection(28.0%).As the base of the high protective animal model which induced by UV-attenuated cercariae,UV-attenuated Schistosoma japonicum cercariae single chain Fv phage display library were construct successfully with the capacity of 1.9*10~8 clones and the recombination rate of 100%and good diversity.
2.66-68kDa antigen in different stage of schistosoma japonicum could be recognized by highly efficient UMS and SCA66-68kDa was taken as t the target screening antigens;SjSCA66-68 were purifed from SjSCA and the rabbit monospecific anti-sera with a titer as high as 1:12800 against SCA66-68 were produced successfully;The SCA66-68 antigen was characterize as a non-glycoprotein through silver and PAS staining;The result of N-terminal sequence analysis showed the N-terminally blocked proteins of SCA66-68,so we put forward some suggestions on the strategy of the sequencing of SCA66-68kDa.
3.Screening of(scFv)antibody library against UV-attenuated Schistosoma japonicum cercariae with the purified antigen of SCA66-68,after enriching and screening,we get six positive clones in the way of antigen direct method,and no positive clones with the antibody sandwich method.The Western blot analysis and SDS-PAGE showed that specific ScFv SCA66-68 was successfully abtained after Soluble expression of E-coli HB2151.
Conclusion
1.UV-attenuated Schistosoma japonicum cercariae single chain Fv phage display library was successfully constructed.The construction of this library may overcome the limitation of the application of radiate-attenuated vaccine,and benefit to develop the new strategies of prevention on Schistosoma japonicum.
2.SjSCA66-68kDa nature antigen could be recognized by UMS,the characterizations of immuno-biological chemistry of the new candidate vaccine antigens of SjSCA66-68 provides a theoretical basis for further screening of ScFv library,also lay a firm foundation for developing effective molecules of vaccine against schistosoma.
3.Successfully get the specific SCA66-68kDa ScFv with the methods of NC meberance enrichment and colony lift binded with antigen direct way,which has shown the wide application prospects in the field of the vaccine,immunodiagnosis and Immunotherapy.
引文
[1]朱晓华,佑恩.血吸虫病全球流行病学状况及其控制的新进展.国外医学寄生虫病分册,2004,31(3):116-119
[2]肖东楼,余晴,党辉,等2003年全国血吸虫病疫情通报.中国血吸虫病防治杂志,2004,16(6):401-405
[3]汪世平,周泪波,曾宪芳,等.抗日本血吸虫生殖及卵胚发育免疫的研究.中国血吸虫病防治杂志.1998,10(7):7-12
[4]汪世平,周汨波,沈国励,等.日本血吸虫未成熟虫卵26/28kDa抗原诱导抗雌虫生殖和抗卵胚发育免疫的研究.中国寄生虫学与寄生虫病杂志,1997,15(2):79-83
[5]Shi YE.Jiang CF.Han JJ.et al.Schistosoma japonicum:An ultravilet-attenuated cercarial vaccine applicable in the field for water buffaloes[J].XP Parasitol,1990,71:100-104.
[6]Rashika ER,Toshihiro O.Hiroshi S,et al.Immunization of mice with ultraviolet-attenuated cercariae of Schistosoma mansoni transiently reduces the fecundity of challenge worm[J].International Journal for Parasitology.1997.37(5):581-586.
[7]Sheets MD,Amersdorfer P,Firmem R,et al.Efficient construction of a large nonimmune phage antibody library:the production of high-affinity human single-chain antibodies to protein antigens.Proc Natl Acad Sci U S A,1998,95(11):6157-6163.
[8]俞小淙,蒋欣,等.抗日本血吸虫膜蛋白特异性单链抗体的构建及表达[J].中国寄生虫学与寄生虫病杂志,2001,(3):9-14
[9]陈代雄,詹希美,何蔼,等.抗日本血吸虫噬菌体抗体库的构建和初步应用.热带医学杂志,2003,3(4):391-395.
[10]徐劲,吴忠道,等.抗日本血吸虫卵单抗33F5可变区轻、重链基因的串联及克隆[J].中国血吸虫病防治杂志,2002,14(3):173-175.
[11]宋晓彤,冯振卿,等.日本血吸虫单克隆抗独特型抗体NP30单链抗体基因的构建和表达[J].中国寄生虫学与寄生虫病杂志,2002,(3):26-28
[12]朱毅,等.日本血吸虫单克隆抗抗独特型抗体NP48单特异性双链抗体的构建表达与初步鉴定[J].中国血吸虫病防治杂志,2005,(4):241-245
[13]岳国峰,等.人源日本血吸虫Fab抗体库的构建及抗独特型抗体的筛选、鉴定[J]. 南京医科大学学报,2006,3 6(11):997-1000.
[14]雷黎,赵志荣等.人源性抗日本血吸虫噬菌体抗体库的构建及初步鉴定[J].中国人兽共患病学报,2007,23(4):386-389.
[15]李小红.血吸虫致弱疫苗的研究进展[J].国外医学.寄生虫病分册,2003,(3):97-102
[16]芳丽.等紫外线减毒日本血吸虫尾蚴免痉小鼠的体液免痉和NK细胞括性的动志变化[J]中国寄生虫病防治杂志,1996.9:94
[17]谢闻悦,周晓红,等.紫外线减毒血吸虫尾蚴免疫兔血清IgG水平动态及被动转移至小鼠诱导保护力研究[J].中国人兽共患病杂志,2001,(1):42-44
[18]胡姝颖,张美娟,等.UV-致弱日本血吸虫尾蚴诱导C57BL/6小鼠免疫保护作用的研究[J].中国病原生物学杂志,2006,(3):181-185
[19]Zhang YB,Taylor MG,Bickle QD,et al.Vaccination of mice with 7-irradiated cercariae[J].Parasitlmmunol,1999,21(1):111-117.
[20]Gui M,Wales A,Jones JT,et al.Vaccination of mice with radiation-attenuated larvae of Schitosomajaponicum or S.mansoni[J].Mitt Osterr Ges Tropenmed,Parasitol,1993,15:43-50
[21]陈家旭,刘述先,曹建平,等.日本血吸虫97kDa DNA疫苗与致弱尾蚴疫苗诱导免疫应答特征的比较研究[J].中国寄生虫学与寄生虫病杂志,2002,20:257-261.
[22]Villella J,Gomberg H J,Gould SE.Immunization to Schistosoma mansoni in mice inoculated with radiated cercariae.Science,1961,134(3):1073-1075
[23]Perlowagara-Szumlewicz A.Schistoma mansoni:humoral transfer of protective factors produced by irradiated cercariae.Nature,1964,20;4(5):1009-1010
[24]Perlowagara-Szumlewicz A.The role of attenuated cercariae in effective immunization against Schistosoma mansoni.Rev Bras Malariol Doeneas Trop,1964,16(4):505-525
[25]Taylor MG.Schistosomiasis vaccines:farewell to the God of plague[J]Trop Med Hyg,1994,97(5):257-268
[26]Craig Gerard.purification of glycoprotein.Methods in enzyme.1990,Vol.182.529-39.
[27]Tarrab-Hazdai R,SchechtmanD.LoweLlG.etal.Protesome delivery of a protective 9B-antigen against Schistosoma mansoni[J].Intemational Journal of Immunopharmacology.1999.21:205-218.
[28]Quentin DB.Joanne O.Characterization of a stage-specific Mr16000 schistosomular surface glycoprotein antigen of Schistosoma mansoni [J].Molecular and Biochemical Parasitology.1999,100:85-94.
[29]King,C.H.,R.R.Lett,J.Nanduri etal.Isolation and characterization of a protective antigen for adjuvant-free immunization against Schistosoma mansoni.Journal of Immunology.,139(1987):4218-4224.
[30]King,C.H.,S.Ellbiary,I.Navawi etal.Intensity of Schistosoma mansoni infection in a human population is inversely correlated with antibody response to Sm68,a protective parasite antigen.J.Infect.Dis.,160(1989):686-691
[31]Blanton RE,MatsumotoY,KingCH,etal Ultrastructural localization of a protective 68,000 molecular weight antigen in Schistosoma mansoni.Am J Trop Med Hvg.1991,45(1):112-120
[32]李华,易新元,曾宪芳,等.日本血吸虫67kDa分子抗原的纯化及其对血吸虫病的诊断和疗效考核冲国热带医学,2001,1(2):93-95
[33]李华,汪世平,曾宪芳,等.日本血吸虫未成熟虫卵67kDa分子抗原诊断血吸虫病的研究.中国血吸虫病防治杂志,1998,10(5):271-274
[34]袁仕善,易新元,曾宪芳,等日本血吸虫成虫组分抗原的分离及小鼠免疫试验.中国人兽共患病杂志,2003,19(5):85-88
[35]汪世平,朱平安,刘立鹏,等.日本血吸虫未成熟虫卵糖蛋白抗原生物学特性的初步分析.中国血吸虫病防治杂志,2002,14(2):92-9
[36]吕志跃,汪世平,彭先楚,等..致弱尾蚴免疫血清与日本血吸虫不同发育阶段抗原免疫反应性的研究.中国人兽共患病杂志.2005,21(3):197-201
[37]姜孝新.sjAWA38kDa和SIEA66-68kDa天然分子抗原的分离纯化及其免疫保护性效果的观察.中南大学硕士学位论文,2006,16-17.
[38]Horowitz,S.,M.Smolaraky,R.Amon.Protection against Schistosoma mansoni achieved by immunization with sonicated parasite.Eur.J.Immunol.1982,12(4):327-332
[39]刘雪琴,汪世平,曾少华,等.高效天然分子疫苗免疫猪血清筛选尾蚴抗原的结果分析[J].寄生虫与医学昆虫学报,2007,(03),141-144
[40]夏其昌.蛋白质化学研究技术与进展。科学出版社.1997
[41]Scott DI.Immunoblotting and dot-blotting.J Imrnunol Methods,1989;119:153.
[42]Renner SW.Immunoblottting and immunobinding.Arch Pathol Lab Med,1988:112:780.
[43]Radossevic K,Voerman JS,Heroines A,et al.Coloy lift as2 say using cellcoated filters:a fast and efficient method to screen phage libraries for cell- bindig clones[J].J Immunol Methods,2003,272(1-2):219-233.
[44]Ingram DT,Lamichhane CM,Rollins DM,et al.Development of a colony lift immunoassay to facilitate rapid detection and quantification of Escherichia coli O157:H7 from agar plates and filter monitor membranes.Clin Diagn Lab Immunol,1998(5):567-573.
[45]Carroll SA,Carr LE,Mallinson ET,et al.A colony lift immunoassay for the specific identification and quantification of Listeria monocytogenes,J.Microbiol.Methods,2000(41):145-153.
[46]Radossevic K,Voerman JS,Hemmes A,et al.Colony lift assay using cell-coated filters:a fast and efficient method to screen phage libraries for cell-binding clones.J.Immunol Methods.2003.272(1-2):219-233.
[47]周旻,杨冬华,汤绍辉,等.集落挖掘法在抗肝癌噬菌体单链抗体库筛选中的应用.中国病理生理杂志,2005,21(6):1246-1248.
[48]Tanaka T,Ito T,Furuta M,Eguchi C,IL oda H,Wakabayashi-Takai E,Kaneko K.In situ phage screening.A method for identification of subnanogram tissue components in situ.)Biol Chem.2002,277(33):30382-7
[49]de Wildt RM,Tomlinson IM,Ong JL,Holliger P.Related Articles,Isolation of reeeptor-ligand pairs by capture of long-lived multivalent interaction complexes.Proc Nat,Acad Sci U S A.2002,99(13):8530-5.
[50]Mutuberria R,Hoogenboom HR,van der Linden E,de Bruine AP,Roovers RC.Model systems to study the parameters determining the success of phage antibody selections on complex antigens.)Immunol Methods.1999,231(1-2):65-81.
[51]Johns M,George A J,Ritter MA.In vivo selection of sFv from phage display libraries.)Immunol Methods.2000,239(1-2):137-51
[52]Trepel M,Arap W,Pasqualini R.In vivo phage display and vascular heterogeneity:implications for targeted medicine.Curr Opin Chem Biol.2002,6(3):399-404.
[53]Hombach A,Pohl C,Heuser C Sircar R,Diehl V,Abken H.Isolation of single chain antibody fragments with specificity for cell surface antigens by phage display utilizing internal image anti-idiotypic antibodies.J Immunol Methods.1998,218(1-2):53-61
[54]王勇,苏川,张兆松,等.日本血吸虫特异性IgE相关抗原编码基因的克隆和鉴定.中国寄生虫学与寄生病杂志,2001:19(5):272-275
[55]张桂药,张兆松,沈一平.血吸虫基因工程免疫研制策略及进展.国外医学寄 生虫病分册,1997:24(1):6-10
[56]何卓,周帅锋,汪世平,等。日本血吸虫未成熟卵单链抗体库的构建、筛选及初步应用.生物化学与生物物理进展(待发表)。
[57]陈欲晓,易新元,曾宪芳,等日本血吸虫雄虫免疫血清对成虫cDNA文库的筛选及克隆分析.中国人畜共患病杂志,2003,19(1):51-54
1.刘尚林,古锦泰,张仁利等.日本血吸虫Clpain蛋白免疫原性研究[J].中国热带医学杂志,2004;4(4):493-496.
2.Katok,MiuraM.On the comlrison of some stool examinationmethods[J].Japan.J Parasitology,1954,3(1):35.
3.KatzN.Et al.A simple device for quantitative thick Tlef.rtechniquein schistosomiasis mansoni[J].RevtalnstMedTropsP ub,1972,14:397.
4.Peters PA.Quick Kato smear for field quantification of schimosorna mansoni eggs[J].Am J Trop Med Hyg,1980,29:217.
5.WHO.The Control of schistosomiasis[R].Report of a WHO Expert Committee.Geneva,World Health Organization,WHO Technical Report Series No.728,1985.
6.刘晓明,尹晓梅,周利,等.新型定量集卵直检技术现场诊断血吸虫病的效果评价[J].中国寄生虫病防治杂志.2002,15(4):237-238.
7.CheererAW.et al..Countingof schistommamansuni eggs infaeces.Comlmrisonof afiltratkntechnique 8n adilutiontechnique[J].JParasitol,1968,54:632.
8.余森海.尼龙袋集卵法诊断血吸虫病的研究[J].中华医学检验杂志,1981,(4):210.
9.张经文,方爱珍.h形顶管孵化法诊断血吸虫病的研究[J].中国血吸虫病防治杂志,1994,6(1):44.
10.王槐震.粪便检查促孵法诊断血吸虫病的初步观察[J].江苏医药,1982,(8):4
11.陈争.血吸虫病粪检球击集卵机[J].中国农村医学,1982,(4):17.
12.毛守白.血吸虫生物学与血吸虫病的防治[M].北京:人民卫生出版社,1990.456-461.
13.徐国余.新的血吸虫病查病工具-直肠显微镜的研制和使用[J].中华医学杂志,1982,62.112。
14.吴观陵.我国血吸虫病免疫诊断发展的回顾与展望[J].中国寄生虫学与寄生虫病杂志,2005,23(5)增刊:323-328.
15.Zhu YC,He W,Liang YS,et al.Development of a rapid,simple dipstick dye immunoassay for schistosomiasis diagnosis[J].Immunol Methods,2002,266(102):1-5.
16.李允鹤.我国血吸虫病诊断工作面临的问题及展望.中国寄生虫学与寄生虫病志,1994,12(3):227-230.
17.袁有赉.血吸虫病环卵沉淀反应方法的改进[J].安徽医学院学报,1985,20:24.
18.Kamiya H.,Technique of the intraoval precipitin reaction by using formalin fixed tissue section for the diagnosis of schistosomiasis[J].Jon J Parasitol,1981,30:161.
19.郑思民,汤淡云,龚玲妹,等.酶联环卵沉淀试验诊断日本血吸虫病的研究[J].寄生虫学与寄生虫杂志,1983,(1):147.
20.中华人民共和国卫生部疾病控制司编.血吸虫病防治手册[M].上海:上海科技出版社,2000,88-91.
21.吴海玮,罗建平,吴观陵,等.用ELISA法研究血吸虫抗体水平时OD值标准化的方法[J].寄生虫学与医学昆虫学报,1995,2(3):140-145.
22.梁幼生,朱荫昌,宁安,等.胶体染料试纸条免疫诊断试剂盒诊断血吸虫病的现场应用研究[J].中国寄生虫病防治杂志,2001a,14:12-14.
23.李允鹤主编.寄生虫病免疫学及免疫诊断.南京:江苏科学技术出版社,第1版1991:215.
24.王秀珍,黎世涛.快速ELISA诊断日本血吸虫病的研究及应用[J].中国血吸虫病防治杂志1990;2:31.
25.陶如华,仇镇宁.抗独特型抗体作为日本血吸虫病诊断抗原的研究.[J].中国人兽共患病杂志1996:12(6):8.10.
26.黄佩君,仇镇宁,冯振卿,等.用单克隆抗体NP30检测血吸虫病短程抗体的研究[J]..南京医科大学学报1999;19:4.
27.朱荫昌,华万全,刘韵娟,等.日本血吸虫虫卵组分抗原疗效考核价值的研究.中国血吸虫病防治杂志1996;8(6):321.
28.刘韵娟,朱荫昌.应用日本血吸虫卵组分抗原检测病人短程抗体的研究.实用寄生虫病杂志1997:5:59.
29.Yogore MG.I.ewert RM,Bias BI- Schistosomiasis japonica in Barrio San Antonio,Basey,Sam ar in the Philippines:V.Theenzyme linked inmm nosorbent assay(EIlSA)tom pared with quantitative stool examination and the eircumoval precipitin[J].Am J Trop Mcd Hyg.1 981.30(6):1252 1 262.
30.苏正明,胡敏,何汇,等:微波酶联免疫吸附试验诊断日本血吸虫病[J].中国血吸虫病防治杂志2004;16(3)178-180.
31.何伟,朱荫昌,华万全,等.血吸虫病快速免疫诊断.腹体染料试纸条法的研究.[J]中国血吸虫病防治杂志2000;12:18-20.
32.杨忠,殷关麟,范崇正.金标免疫法诊断血吸虫病效果观察[J].寄生虫病感染疾病.2007:5(2):97-98.
33.Xiao X,Wang TP,Tian ZG.Development of a rapid,sensitive,dye immunoassay for schistosomiasis diagnosis:a colloidal dye immunofiltration assay[J].Journal of Immunological Methods 2003;280:49-57.
34.Xiang Xiao,Tianping Wang,Hongzhuan Ye et al.Field evaluation of a rapid,visually-read colloidal dye immunofiltration assay for Schistosoma japonicum for screening in areas of low transmission[J].Bulletin of the World Health Organization,2005;83(7):526-533.
35.Hua Wang,Yun Zhang,Bani Yan et al.Rapid,Simple,and Sensitive Immunoagglutination Assay with SiO2 Particles and Quartz Crystal Microbalance for Quantifying Schistosoma japonicum Antibodies[J].Clinical Chemistry,2006;52(11):2065-2072.
36.陆萍,钱宗立,Van EL,等.试剂条夹心酶联免疫吸附试验检测13本血吸虫 感染者经尿液排泄的循环趋阴极抗原[J].中国寄生虫学与中寄生虫病杂志,1996;14(4):253.
37.Van L,De Jonge N,Um y NA,et al.Improved diagnostic performance of time circulating antigen assay in human sehistosomiasis by paraleU testing for circulating anodic and Pilhodic antigens and urine[J].Am J Med Hyg,1992;47:463.
38.Yi XinYuan,Fie YongKang,Li Yue sheng.Co llaborativestudy on detection of circulating antigens in schistosomiasis japonica.Chinese J Parasitology &Parosific Diseases.1995:13:277.
39.娄文娴.九种抗13本血吸虫单克隆抗体特性鉴定及其组合试验[J].中国血吸虫病防治杂志,1995:7。
40.管小红,石佑恩.评估13本血吸虫病免疫学诊断方法疗效考核价值的合作研究[J].中国血吸虫病防治杂志,1996;8:72.
41.余传信,朱荫昌,殷旭仁,等.日本血吸虫(中国大陆株)磷酸丙糖异构酶(Sie-TPI)基因克隆与序列分析[J].中国血吸虫病防治杂志,1997;9:321.
42.余传信,朱荫昌,殷旭仁,等.重组13本血吸虫病中国大陆株磷酸丙糖异构酶蛋白质表达、纯化及特性分析[J].中国人兽共患病杂志,1999:15:31.
43.朱荫昌,管晓虹,杨小红,等.抗rSiCnn单克隆抗体检测血吸虫循环抗原的研究[J].中国寄生虫病防治.
44.吴锦雅,杨培良,周晓红,等.应用抗SjP38的单克隆抗体建立血吸虫感染的胶体金免疫层析检测体系[J].第一军医大学学报,2005;25(5):538-540.
45.DedderAN,DeJongeN,OttoC,et.Sensitive determination anodic antigen in schisto6oma mansoni infectedindividuals by enzyme - linked inmmno6orbent assay usingmonoelonal antibody[J].Am J Trop meal Hyg,1989;40(3):268.
46.Mohamed SA,Nourel Din,Dieuwke Komelis,et 81.In,lmunologic characterization oftow monoelonal antibodies reactive tllrepetitve carbohydrate epitopes of circulating Schisto6oma mansoni egg antigeia[J].Am J Trop led Hyg,1994:50(4):487.
47.Van Etteia L,Folman CC,Eggelte TA,et 81.Rapid diagnosesof schisto6omiasis by antigen detection in urine with a reagentstrip[J].clin.Microbiol,1994:32(10):2404.
48.田海生,朱昌亮,管晓虹,等.日本血吸虫感染者尿液诊断标志物的电泳筛选及单克隆抗体的制备和初步应用[J].中国人兽共患病杂志,2000;16(1):39.
49.王兆军,薜纯良,吴维铎,等.慢性日本血吸虫病患者唾液中特异性抗体的检测[J].中国寄生虫学与寄生虫病杂志,2000;18(3):152.
50.马立人,蒋中华.生物芯片[M].北京:化学工业出版社,2000,2-3.
51.周钧,钱万红,李越希,等日本血吸虫检测基因芯片的研制及初步应用[J].江苏医药杂志,2004;30(6):455-457.
52.Grothaus MC,Srivastava N,Smithson SL,et al.Selection of an immunogenic peptide mimic of the capsular polysaccharide of Neisserie meningitides serogroup A using a peptide display library[J].Vaccine,2000,18(13):1253-1263.
53.Willis AE,Perham RN,Wraith D.Immunological properties of foreign peptides in multiple display on a filamentous bacteriophage[J].Gene,1993,128(1):79 -83.
54.Folgori A.Taft R,Meola A,et al.A general strategy to identify mimotopes of pathological antigens using only random peptide libraries and human sera[J].EMBO J,1994,13(9):2236-2243.
55.Iniguez P,Zientara S,Marauh M,et al.Screening of horse polyclonal antibodies with a random peptide library displayed onphage:identification of ligands used as antigens in an El ISA test to detect the presence Of antibodies tO equine artedtis virus[J].J Virol Methods,1998,73(2):175-183.
56.王林纤,蔡春,易新元.日本血吸虫雌虫抗原模拟表位的筛选及免疫保护性.中国寄生虫病防治杂志,2004;17(2):110-112.
57.余传信,朱荫昌,殷旭仁,等,模拟抗原表位基因测定及合成多肽的血吸虫病诊断价值的研究[J].中国血吸虫病防治杂志,2004;16(2):95-99.
58.陆正贤,许静,龚唯.聚合酶链式反应检测日本血吸虫DNA的实验研究[J].中国人兽共患病学报,2007;23(5):479-481.
59.汪世平,吴朝阳,沈国励等,日本血吸虫分子诊断抗原压电免疫传感器的研究,中国人兽共患病杂志2000,16(1):15-17.
60.石佑恩,Dell R.日本血吸虫成虫31/32kDa 诊断蛋白:与血吸虫单克隆抗体和病人血清的免疫印斑反应[J]中国寄生虫学与寄生虫病杂志,1988,6(4):2451
61.汪世平,曾宪芳,易新元,等.日本血吸虫31/32kDa抗原纯化及诊断应用的研究(英文)[J]中国寄生虫学与寄生虫病杂志,1995,13(1):251-253.
62.Ya-Min Zhou,Guo-Dong Liu,Zho-Yang Wu,et al.An Amperometric Immunosensor Based on a Conducting Immunocomposite Electrode for the Determination of Schistosoma Japonieum Antigen[J].ANALYTICAL SCIENCES,2002;18:155-159.
63.蔡卫民,郑敏.日本血吸虫病的超声诊断[J].中国血吸虫病防治杂志,2003;15(4):245-246.
64.易松涛,黄维恒,彭亿新.血吸虫病肝损害超声分级与门静脉及脾静脉血流动力学分析[J].中国血吸虫病防治杂志,2007;19(4):293-295.
65.孙晓枫.血吸虫病肝硬化变患者肝段下腔静脉内径的超声研究[J].中国病原生物学杂志,2007;2(3):221-223.
66.何伟,朱荫昌,梁幼生.粪检与免疫诊断方法检测日本血吸虫感染效果比较[J].中国血吸虫病防治杂志,2007;19(2):107-109.
67.秦志强,曾庆仁.日本血吸虫病免疫诊断的研究进展与问题[J].中国寄生虫病防治杂志,2001;14(4)306-308.
[2]肖东楼,余晴,党辉,等2003年全国血吸虫病疫情通报.中国血吸虫病防治杂志,2004,16(6):401-405
[3]汪世平,周泪波,曾宪芳,等.抗日本血吸虫生殖及卵胚发育免疫的研究.中国血吸虫病防治杂志.1998,10(7):7-12
[4]汪世平,周汨波,沈国励,等.日本血吸虫未成熟虫卵26/28kDa抗原诱导抗雌虫生殖和抗卵胚发育免疫的研究.中国寄生虫学与寄生虫病杂志,1997,15(2):79-83
[5]Shi YE.Jiang CF.Han JJ.et al.Schistosoma japonicum:An ultravilet-attenuated cercarial vaccine applicable in the field for water buffaloes[J].XP Parasitol,1990,71:100-104.
[6]Rashika ER,Toshihiro O.Hiroshi S,et al.Immunization of mice with ultraviolet-attenuated cercariae of Schistosoma mansoni transiently reduces the fecundity of challenge worm[J].International Journal for Parasitology.1997.37(5):581-586.
[7]Sheets MD,Amersdorfer P,Firmem R,et al.Efficient construction of a large nonimmune phage antibody library:the production of high-affinity human single-chain antibodies to protein antigens.Proc Natl Acad Sci U S A,1998,95(11):6157-6163.
[8]俞小淙,蒋欣,等.抗日本血吸虫膜蛋白特异性单链抗体的构建及表达[J].中国寄生虫学与寄生虫病杂志,2001,(3):9-14
[9]陈代雄,詹希美,何蔼,等.抗日本血吸虫噬菌体抗体库的构建和初步应用.热带医学杂志,2003,3(4):391-395.
[10]徐劲,吴忠道,等.抗日本血吸虫卵单抗33F5可变区轻、重链基因的串联及克隆[J].中国血吸虫病防治杂志,2002,14(3):173-175.
[11]宋晓彤,冯振卿,等.日本血吸虫单克隆抗独特型抗体NP30单链抗体基因的构建和表达[J].中国寄生虫学与寄生虫病杂志,2002,(3):26-28
[12]朱毅,等.日本血吸虫单克隆抗抗独特型抗体NP48单特异性双链抗体的构建表达与初步鉴定[J].中国血吸虫病防治杂志,2005,(4):241-245
[13]岳国峰,等.人源日本血吸虫Fab抗体库的构建及抗独特型抗体的筛选、鉴定[J]. 南京医科大学学报,2006,3 6(11):997-1000.
[14]雷黎,赵志荣等.人源性抗日本血吸虫噬菌体抗体库的构建及初步鉴定[J].中国人兽共患病学报,2007,23(4):386-389.
[15]李小红.血吸虫致弱疫苗的研究进展[J].国外医学.寄生虫病分册,2003,(3):97-102
[16]芳丽.等紫外线减毒日本血吸虫尾蚴免痉小鼠的体液免痉和NK细胞括性的动志变化[J]中国寄生虫病防治杂志,1996.9:94
[17]谢闻悦,周晓红,等.紫外线减毒血吸虫尾蚴免疫兔血清IgG水平动态及被动转移至小鼠诱导保护力研究[J].中国人兽共患病杂志,2001,(1):42-44
[18]胡姝颖,张美娟,等.UV-致弱日本血吸虫尾蚴诱导C57BL/6小鼠免疫保护作用的研究[J].中国病原生物学杂志,2006,(3):181-185
[19]Zhang YB,Taylor MG,Bickle QD,et al.Vaccination of mice with 7-irradiated cercariae[J].Parasitlmmunol,1999,21(1):111-117.
[20]Gui M,Wales A,Jones JT,et al.Vaccination of mice with radiation-attenuated larvae of Schitosomajaponicum or S.mansoni[J].Mitt Osterr Ges Tropenmed,Parasitol,1993,15:43-50
[21]陈家旭,刘述先,曹建平,等.日本血吸虫97kDa DNA疫苗与致弱尾蚴疫苗诱导免疫应答特征的比较研究[J].中国寄生虫学与寄生虫病杂志,2002,20:257-261.
[22]Villella J,Gomberg H J,Gould SE.Immunization to Schistosoma mansoni in mice inoculated with radiated cercariae.Science,1961,134(3):1073-1075
[23]Perlowagara-Szumlewicz A.Schistoma mansoni:humoral transfer of protective factors produced by irradiated cercariae.Nature,1964,20;4(5):1009-1010
[24]Perlowagara-Szumlewicz A.The role of attenuated cercariae in effective immunization against Schistosoma mansoni.Rev Bras Malariol Doeneas Trop,1964,16(4):505-525
[25]Taylor MG.Schistosomiasis vaccines:farewell to the God of plague[J]Trop Med Hyg,1994,97(5):257-268
[26]Craig Gerard.purification of glycoprotein.Methods in enzyme.1990,Vol.182.529-39.
[27]Tarrab-Hazdai R,SchechtmanD.LoweLlG.etal.Protesome delivery of a protective 9B-antigen against Schistosoma mansoni[J].Intemational Journal of Immunopharmacology.1999.21:205-218.
[28]Quentin DB.Joanne O.Characterization of a stage-specific Mr16000 schistosomular surface glycoprotein antigen of Schistosoma mansoni [J].Molecular and Biochemical Parasitology.1999,100:85-94.
[29]King,C.H.,R.R.Lett,J.Nanduri etal.Isolation and characterization of a protective antigen for adjuvant-free immunization against Schistosoma mansoni.Journal of Immunology.,139(1987):4218-4224.
[30]King,C.H.,S.Ellbiary,I.Navawi etal.Intensity of Schistosoma mansoni infection in a human population is inversely correlated with antibody response to Sm68,a protective parasite antigen.J.Infect.Dis.,160(1989):686-691
[31]Blanton RE,MatsumotoY,KingCH,etal Ultrastructural localization of a protective 68,000 molecular weight antigen in Schistosoma mansoni.Am J Trop Med Hvg.1991,45(1):112-120
[32]李华,易新元,曾宪芳,等.日本血吸虫67kDa分子抗原的纯化及其对血吸虫病的诊断和疗效考核冲国热带医学,2001,1(2):93-95
[33]李华,汪世平,曾宪芳,等.日本血吸虫未成熟虫卵67kDa分子抗原诊断血吸虫病的研究.中国血吸虫病防治杂志,1998,10(5):271-274
[34]袁仕善,易新元,曾宪芳,等日本血吸虫成虫组分抗原的分离及小鼠免疫试验.中国人兽共患病杂志,2003,19(5):85-88
[35]汪世平,朱平安,刘立鹏,等.日本血吸虫未成熟虫卵糖蛋白抗原生物学特性的初步分析.中国血吸虫病防治杂志,2002,14(2):92-9
[36]吕志跃,汪世平,彭先楚,等..致弱尾蚴免疫血清与日本血吸虫不同发育阶段抗原免疫反应性的研究.中国人兽共患病杂志.2005,21(3):197-201
[37]姜孝新.sjAWA38kDa和SIEA66-68kDa天然分子抗原的分离纯化及其免疫保护性效果的观察.中南大学硕士学位论文,2006,16-17.
[38]Horowitz,S.,M.Smolaraky,R.Amon.Protection against Schistosoma mansoni achieved by immunization with sonicated parasite.Eur.J.Immunol.1982,12(4):327-332
[39]刘雪琴,汪世平,曾少华,等.高效天然分子疫苗免疫猪血清筛选尾蚴抗原的结果分析[J].寄生虫与医学昆虫学报,2007,(03),141-144
[40]夏其昌.蛋白质化学研究技术与进展。科学出版社.1997
[41]Scott DI.Immunoblotting and dot-blotting.J Imrnunol Methods,1989;119:153.
[42]Renner SW.Immunoblottting and immunobinding.Arch Pathol Lab Med,1988:112:780.
[43]Radossevic K,Voerman JS,Heroines A,et al.Coloy lift as2 say using cellcoated filters:a fast and efficient method to screen phage libraries for cell- bindig clones[J].J Immunol Methods,2003,272(1-2):219-233.
[44]Ingram DT,Lamichhane CM,Rollins DM,et al.Development of a colony lift immunoassay to facilitate rapid detection and quantification of Escherichia coli O157:H7 from agar plates and filter monitor membranes.Clin Diagn Lab Immunol,1998(5):567-573.
[45]Carroll SA,Carr LE,Mallinson ET,et al.A colony lift immunoassay for the specific identification and quantification of Listeria monocytogenes,J.Microbiol.Methods,2000(41):145-153.
[46]Radossevic K,Voerman JS,Hemmes A,et al.Colony lift assay using cell-coated filters:a fast and efficient method to screen phage libraries for cell-binding clones.J.Immunol Methods.2003.272(1-2):219-233.
[47]周旻,杨冬华,汤绍辉,等.集落挖掘法在抗肝癌噬菌体单链抗体库筛选中的应用.中国病理生理杂志,2005,21(6):1246-1248.
[48]Tanaka T,Ito T,Furuta M,Eguchi C,IL oda H,Wakabayashi-Takai E,Kaneko K.In situ phage screening.A method for identification of subnanogram tissue components in situ.)Biol Chem.2002,277(33):30382-7
[49]de Wildt RM,Tomlinson IM,Ong JL,Holliger P.Related Articles,Isolation of reeeptor-ligand pairs by capture of long-lived multivalent interaction complexes.Proc Nat,Acad Sci U S A.2002,99(13):8530-5.
[50]Mutuberria R,Hoogenboom HR,van der Linden E,de Bruine AP,Roovers RC.Model systems to study the parameters determining the success of phage antibody selections on complex antigens.)Immunol Methods.1999,231(1-2):65-81.
[51]Johns M,George A J,Ritter MA.In vivo selection of sFv from phage display libraries.)Immunol Methods.2000,239(1-2):137-51
[52]Trepel M,Arap W,Pasqualini R.In vivo phage display and vascular heterogeneity:implications for targeted medicine.Curr Opin Chem Biol.2002,6(3):399-404.
[53]Hombach A,Pohl C,Heuser C Sircar R,Diehl V,Abken H.Isolation of single chain antibody fragments with specificity for cell surface antigens by phage display utilizing internal image anti-idiotypic antibodies.J Immunol Methods.1998,218(1-2):53-61
[54]王勇,苏川,张兆松,等.日本血吸虫特异性IgE相关抗原编码基因的克隆和鉴定.中国寄生虫学与寄生病杂志,2001:19(5):272-275
[55]张桂药,张兆松,沈一平.血吸虫基因工程免疫研制策略及进展.国外医学寄 生虫病分册,1997:24(1):6-10
[56]何卓,周帅锋,汪世平,等。日本血吸虫未成熟卵单链抗体库的构建、筛选及初步应用.生物化学与生物物理进展(待发表)。
[57]陈欲晓,易新元,曾宪芳,等日本血吸虫雄虫免疫血清对成虫cDNA文库的筛选及克隆分析.中国人畜共患病杂志,2003,19(1):51-54
1.刘尚林,古锦泰,张仁利等.日本血吸虫Clpain蛋白免疫原性研究[J].中国热带医学杂志,2004;4(4):493-496.
2.Katok,MiuraM.On the comlrison of some stool examinationmethods[J].Japan.J Parasitology,1954,3(1):35.
3.KatzN.Et al.A simple device for quantitative thick Tlef.rtechniquein schistosomiasis mansoni[J].RevtalnstMedTropsP ub,1972,14:397.
4.Peters PA.Quick Kato smear for field quantification of schimosorna mansoni eggs[J].Am J Trop Med Hyg,1980,29:217.
5.WHO.The Control of schistosomiasis[R].Report of a WHO Expert Committee.Geneva,World Health Organization,WHO Technical Report Series No.728,1985.
6.刘晓明,尹晓梅,周利,等.新型定量集卵直检技术现场诊断血吸虫病的效果评价[J].中国寄生虫病防治杂志.2002,15(4):237-238.
7.CheererAW.et al..Countingof schistommamansuni eggs infaeces.Comlmrisonof afiltratkntechnique 8n adilutiontechnique[J].JParasitol,1968,54:632.
8.余森海.尼龙袋集卵法诊断血吸虫病的研究[J].中华医学检验杂志,1981,(4):210.
9.张经文,方爱珍.h形顶管孵化法诊断血吸虫病的研究[J].中国血吸虫病防治杂志,1994,6(1):44.
10.王槐震.粪便检查促孵法诊断血吸虫病的初步观察[J].江苏医药,1982,(8):4
11.陈争.血吸虫病粪检球击集卵机[J].中国农村医学,1982,(4):17.
12.毛守白.血吸虫生物学与血吸虫病的防治[M].北京:人民卫生出版社,1990.456-461.
13.徐国余.新的血吸虫病查病工具-直肠显微镜的研制和使用[J].中华医学杂志,1982,62.112。
14.吴观陵.我国血吸虫病免疫诊断发展的回顾与展望[J].中国寄生虫学与寄生虫病杂志,2005,23(5)增刊:323-328.
15.Zhu YC,He W,Liang YS,et al.Development of a rapid,simple dipstick dye immunoassay for schistosomiasis diagnosis[J].Immunol Methods,2002,266(102):1-5.
16.李允鹤.我国血吸虫病诊断工作面临的问题及展望.中国寄生虫学与寄生虫病志,1994,12(3):227-230.
17.袁有赉.血吸虫病环卵沉淀反应方法的改进[J].安徽医学院学报,1985,20:24.
18.Kamiya H.,Technique of the intraoval precipitin reaction by using formalin fixed tissue section for the diagnosis of schistosomiasis[J].Jon J Parasitol,1981,30:161.
19.郑思民,汤淡云,龚玲妹,等.酶联环卵沉淀试验诊断日本血吸虫病的研究[J].寄生虫学与寄生虫杂志,1983,(1):147.
20.中华人民共和国卫生部疾病控制司编.血吸虫病防治手册[M].上海:上海科技出版社,2000,88-91.
21.吴海玮,罗建平,吴观陵,等.用ELISA法研究血吸虫抗体水平时OD值标准化的方法[J].寄生虫学与医学昆虫学报,1995,2(3):140-145.
22.梁幼生,朱荫昌,宁安,等.胶体染料试纸条免疫诊断试剂盒诊断血吸虫病的现场应用研究[J].中国寄生虫病防治杂志,2001a,14:12-14.
23.李允鹤主编.寄生虫病免疫学及免疫诊断.南京:江苏科学技术出版社,第1版1991:215.
24.王秀珍,黎世涛.快速ELISA诊断日本血吸虫病的研究及应用[J].中国血吸虫病防治杂志1990;2:31.
25.陶如华,仇镇宁.抗独特型抗体作为日本血吸虫病诊断抗原的研究.[J].中国人兽共患病杂志1996:12(6):8.10.
26.黄佩君,仇镇宁,冯振卿,等.用单克隆抗体NP30检测血吸虫病短程抗体的研究[J]..南京医科大学学报1999;19:4.
27.朱荫昌,华万全,刘韵娟,等.日本血吸虫虫卵组分抗原疗效考核价值的研究.中国血吸虫病防治杂志1996;8(6):321.
28.刘韵娟,朱荫昌.应用日本血吸虫卵组分抗原检测病人短程抗体的研究.实用寄生虫病杂志1997:5:59.
29.Yogore MG.I.ewert RM,Bias BI- Schistosomiasis japonica in Barrio San Antonio,Basey,Sam ar in the Philippines:V.Theenzyme linked inmm nosorbent assay(EIlSA)tom pared with quantitative stool examination and the eircumoval precipitin[J].Am J Trop Mcd Hyg.1 981.30(6):1252 1 262.
30.苏正明,胡敏,何汇,等:微波酶联免疫吸附试验诊断日本血吸虫病[J].中国血吸虫病防治杂志2004;16(3)178-180.
31.何伟,朱荫昌,华万全,等.血吸虫病快速免疫诊断.腹体染料试纸条法的研究.[J]中国血吸虫病防治杂志2000;12:18-20.
32.杨忠,殷关麟,范崇正.金标免疫法诊断血吸虫病效果观察[J].寄生虫病感染疾病.2007:5(2):97-98.
33.Xiao X,Wang TP,Tian ZG.Development of a rapid,sensitive,dye immunoassay for schistosomiasis diagnosis:a colloidal dye immunofiltration assay[J].Journal of Immunological Methods 2003;280:49-57.
34.Xiang Xiao,Tianping Wang,Hongzhuan Ye et al.Field evaluation of a rapid,visually-read colloidal dye immunofiltration assay for Schistosoma japonicum for screening in areas of low transmission[J].Bulletin of the World Health Organization,2005;83(7):526-533.
35.Hua Wang,Yun Zhang,Bani Yan et al.Rapid,Simple,and Sensitive Immunoagglutination Assay with SiO2 Particles and Quartz Crystal Microbalance for Quantifying Schistosoma japonicum Antibodies[J].Clinical Chemistry,2006;52(11):2065-2072.
36.陆萍,钱宗立,Van EL,等.试剂条夹心酶联免疫吸附试验检测13本血吸虫 感染者经尿液排泄的循环趋阴极抗原[J].中国寄生虫学与中寄生虫病杂志,1996;14(4):253.
37.Van L,De Jonge N,Um y NA,et al.Improved diagnostic performance of time circulating antigen assay in human sehistosomiasis by paraleU testing for circulating anodic and Pilhodic antigens and urine[J].Am J Med Hyg,1992;47:463.
38.Yi XinYuan,Fie YongKang,Li Yue sheng.Co llaborativestudy on detection of circulating antigens in schistosomiasis japonica.Chinese J Parasitology &Parosific Diseases.1995:13:277.
39.娄文娴.九种抗13本血吸虫单克隆抗体特性鉴定及其组合试验[J].中国血吸虫病防治杂志,1995:7。
40.管小红,石佑恩.评估13本血吸虫病免疫学诊断方法疗效考核价值的合作研究[J].中国血吸虫病防治杂志,1996;8:72.
41.余传信,朱荫昌,殷旭仁,等.日本血吸虫(中国大陆株)磷酸丙糖异构酶(Sie-TPI)基因克隆与序列分析[J].中国血吸虫病防治杂志,1997;9:321.
42.余传信,朱荫昌,殷旭仁,等.重组13本血吸虫病中国大陆株磷酸丙糖异构酶蛋白质表达、纯化及特性分析[J].中国人兽共患病杂志,1999:15:31.
43.朱荫昌,管晓虹,杨小红,等.抗rSiCnn单克隆抗体检测血吸虫循环抗原的研究[J].中国寄生虫病防治.
44.吴锦雅,杨培良,周晓红,等.应用抗SjP38的单克隆抗体建立血吸虫感染的胶体金免疫层析检测体系[J].第一军医大学学报,2005;25(5):538-540.
45.DedderAN,DeJongeN,OttoC,et.Sensitive determination anodic antigen in schisto6oma mansoni infectedindividuals by enzyme - linked inmmno6orbent assay usingmonoelonal antibody[J].Am J Trop meal Hyg,1989;40(3):268.
46.Mohamed SA,Nourel Din,Dieuwke Komelis,et 81.In,lmunologic characterization oftow monoelonal antibodies reactive tllrepetitve carbohydrate epitopes of circulating Schisto6oma mansoni egg antigeia[J].Am J Trop led Hyg,1994:50(4):487.
47.Van Etteia L,Folman CC,Eggelte TA,et 81.Rapid diagnosesof schisto6omiasis by antigen detection in urine with a reagentstrip[J].clin.Microbiol,1994:32(10):2404.
48.田海生,朱昌亮,管晓虹,等.日本血吸虫感染者尿液诊断标志物的电泳筛选及单克隆抗体的制备和初步应用[J].中国人兽共患病杂志,2000;16(1):39.
49.王兆军,薜纯良,吴维铎,等.慢性日本血吸虫病患者唾液中特异性抗体的检测[J].中国寄生虫学与寄生虫病杂志,2000;18(3):152.
50.马立人,蒋中华.生物芯片[M].北京:化学工业出版社,2000,2-3.
51.周钧,钱万红,李越希,等日本血吸虫检测基因芯片的研制及初步应用[J].江苏医药杂志,2004;30(6):455-457.
52.Grothaus MC,Srivastava N,Smithson SL,et al.Selection of an immunogenic peptide mimic of the capsular polysaccharide of Neisserie meningitides serogroup A using a peptide display library[J].Vaccine,2000,18(13):1253-1263.
53.Willis AE,Perham RN,Wraith D.Immunological properties of foreign peptides in multiple display on a filamentous bacteriophage[J].Gene,1993,128(1):79 -83.
54.Folgori A.Taft R,Meola A,et al.A general strategy to identify mimotopes of pathological antigens using only random peptide libraries and human sera[J].EMBO J,1994,13(9):2236-2243.
55.Iniguez P,Zientara S,Marauh M,et al.Screening of horse polyclonal antibodies with a random peptide library displayed onphage:identification of ligands used as antigens in an El ISA test to detect the presence Of antibodies tO equine artedtis virus[J].J Virol Methods,1998,73(2):175-183.
56.王林纤,蔡春,易新元.日本血吸虫雌虫抗原模拟表位的筛选及免疫保护性.中国寄生虫病防治杂志,2004;17(2):110-112.
57.余传信,朱荫昌,殷旭仁,等,模拟抗原表位基因测定及合成多肽的血吸虫病诊断价值的研究[J].中国血吸虫病防治杂志,2004;16(2):95-99.
58.陆正贤,许静,龚唯.聚合酶链式反应检测日本血吸虫DNA的实验研究[J].中国人兽共患病学报,2007;23(5):479-481.
59.汪世平,吴朝阳,沈国励等,日本血吸虫分子诊断抗原压电免疫传感器的研究,中国人兽共患病杂志2000,16(1):15-17.
60.石佑恩,Dell R.日本血吸虫成虫31/32kDa 诊断蛋白:与血吸虫单克隆抗体和病人血清的免疫印斑反应[J]中国寄生虫学与寄生虫病杂志,1988,6(4):2451
61.汪世平,曾宪芳,易新元,等.日本血吸虫31/32kDa抗原纯化及诊断应用的研究(英文)[J]中国寄生虫学与寄生虫病杂志,1995,13(1):251-253.
62.Ya-Min Zhou,Guo-Dong Liu,Zho-Yang Wu,et al.An Amperometric Immunosensor Based on a Conducting Immunocomposite Electrode for the Determination of Schistosoma Japonieum Antigen[J].ANALYTICAL SCIENCES,2002;18:155-159.
63.蔡卫民,郑敏.日本血吸虫病的超声诊断[J].中国血吸虫病防治杂志,2003;15(4):245-246.
64.易松涛,黄维恒,彭亿新.血吸虫病肝损害超声分级与门静脉及脾静脉血流动力学分析[J].中国血吸虫病防治杂志,2007;19(4):293-295.
65.孙晓枫.血吸虫病肝硬化变患者肝段下腔静脉内径的超声研究[J].中国病原生物学杂志,2007;2(3):221-223.
66.何伟,朱荫昌,梁幼生.粪检与免疫诊断方法检测日本血吸虫感染效果比较[J].中国血吸虫病防治杂志,2007;19(2):107-109.
67.秦志强,曾庆仁.日本血吸虫病免疫诊断的研究进展与问题[J].中国寄生虫病防治杂志,2001;14(4)306-308.