【作者】 周帅锋；

【导师】 汪世平；

【作者基本信息】 中南大学 ， 微生物学， 2008， 硕士

【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。更多还原

【Abstract】 ObjectiveTo 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.Methods1.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.Results1.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.Conclusion1.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.更多还原