高效UV-致弱尾蚴单链抗体库的构建及Sj SCA66-68kDa ScFv的筛选

发布时间：2018-05-27 13:16

本文选题：日本血吸虫 + 致弱尾蚴　；参考：《中南大学》2008年硕士论文

【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。
[Abstract]:research objective
Based on the characteristics of the high protective ability of the weak cercariae vaccine and the natural molecular vaccine, using the advantages of the phage antibody library technology, the efficient expression library of the single chain antibody (ScFv) of the weak cercariae of Japanese and Schistosoma japonicum UV- was constructed and used as a high throughput library source to screen out the single chain of the natural molecular antigen SjSCA66-68kDA with potential protection. The antibody, which lays the foundation for further obtaining the related coding genes of the natural molecule SjSCA66-68kDA of the candidate vaccine for Schistosoma japonicum, speeds up the development of the high efficient natural molecular vaccine against schistosomiasis japonicum.
research method
1. using the phage display technique to construct the high efficient single chain antibody library of the weak cercariae caused by UV- irradiation of Schistosoma japonicum, and identify the library from the capacity and diversity of the library. UV- irradiated the weak cercariae caused by Schistosoma japonicum to infect BALB/c mice many times, and the aseptic UV- caused the weak cercariae in the normal cercariae (the number of cercariae is 40 + 2). Before and three weeks after infection, three caudal veins were injected into the BALB/c mice, and the mice were killed 45 days later to calculate the worm reduction rate and the egg reduction rate to ensure the high efficiency of the sera of the weak cercariae. On the basis of the successful construction of the mice model of the weak cercariae, the spleen RNA was extracted and the whole set of VH and VL genes were amplified by RT-PCR method. The ScFv fragment was amplified by overlapping PCR method, and the ScFv fragment was cloned to PCANTAB 5E vector, and TG1 bacteria were transformed by electricity, and the single chain antibody library of Schistosoma japonicum UV- weak cercariae was obtained by auxiliary phage M13K07.
2. the effective UV- induced weak cercariae and the antigen of different developmental stages of Schistosoma japonicum were immunized to identify and select the target molecules of the immunological screening, and the preliminary study on the immune biochemical characteristics and sequence of the candidate antigen SCA66-68 natural molecules: electrophoretic gel cutting, electric elution, ultrafiltration centrifugation and freeze drying. The SCA66-68KDa antigen was isolated and purified by the method of SCA66-68KDa antigen PAGE glue combined with microlymph node injection to immunize New Zealand rabbits with single specific SCA66-68KDa immunization. The titer and specificity were detected and identified by ELISA and Western blot, and the chemical properties of the SCA66-68KDa antigen were determined by silver staining and PAS staining; N - end was used to determine the chemical properties of the rabbit. The sequencing method was used to study the protein sequence of SCA66-68KDa antigen.
3. using the isolated and purified vaccine candidate SCA66-68kDa antigen, the single chain antibody library of high throughput UV- irradiation induced weak cercariae was screened through the combination of antigen direct method and double antibody sandwich method combined with nitric acid fibrous membrane enrichment and colony excavation method, and the specific SCA66-68kDa single chain antibody was identified by Western blot.
The results of the study:
1.UV- irradiated the weakly cercariae serum of Schistosoma japonicum to transfer BALB/c mice passively, and the 42.5% worm reduction rate was obtained, which was significantly higher than that of the infected sera group 28.0%.. On the basis of the successful construction of UV- irradiated Schistosoma japonicum induced weak cercariae, the high efficiency Japanese blood sucking worm single chain antibody library of UV- weak cercariae was obtained, and its library capacity was measured. For 1.9~*10~8, the recombination rate is 100%, and the diversity is good.
2. it was found that the high efficiency serum of cercariae in the construction of the library could identify the common bands near the 66-68kDa of different stages of Schistosoma, plus the previous research basis of this room, and selected the 66-68kDa natural molecules of cercariae as the target antigens of the immunization screening, and successfully isolated and purified the SCA66-68kDa antigens of pure electrophoresis and immuno pure. A single specific SCA66-68KDa immunized rabbit serum was successfully prepared. The antibody titer was up to 1:12800, and silver nitrate and PAS were used to determine the chemical properties of SCA66-68KD as a protein component and a non glycoprotein. The protein sequence of SCA66-68KDa natural fractions was studied by N sequencing. The results showed that the peptide segment of the N- end was closed now. Elephant, an optimization strategy for protein sequencing of SCA66-68KDa natural molecules.
3. the SCA66-68kDa antigen was identified by high efficiency UV- exposure to the weak cercariae serum, and the isolated and purified Japanese schistosomiasis vaccine candidate molecule SCA66-68kDa was used to screen the single chain antibody library of the weak cercariae induced by UV-. The single chain antibody library of the weak cercariae: the first round false positive of the double anti sandwich method was too high, and the resieve was not positive. 6 SCA66-68kDa positive clones were obtained by direct antigen method. The positive clones were screened and converted to Ecoli HB2151 bacteria to induce the expression of soluble scFv. The results showed that the soluble scFv was obtained by SDS-PAGE, Western blot analysis. The results showed that the specific anti SCA66-68kDa scFv was obtained by direct antigen screening.
Conclusion:
1. for the first time, a highly efficient single chain antibody library of Schistosoma japonicum UV- caused by Schistosoma japonicum was constructed. Its construction made up for the limitations of the application of weak vaccine. It provides reference for developing new vaccine development strategy and new vaccine design, and further screening specific single chain antibody for diagnosis and treatment of schistosomiasis, and analyzing antigen epitope analysis. And vaccine development provides a high throughput source.
2. the natural molecules of the candidate vaccine SjSCA66-68kDa were used as the primary screening target antigen, and the natural molecules of SjSCA66-68kDa were isolated and purified successfully. The preliminary study on its immune biochemical characteristics and sequences provided the theoretical basis for further screening of the single chain antibody library of the weak cercariae, and also laid the foundation for the preparation of the natural sub coding gene engineering vaccine.
3. the specificity of specific anti SCA66-68kDa single chain antibody.SCA66-68kDa specific ScFv was obtained by direct antigen direct method combined with nitric acid fibrous membrane enrichment and colony mining method to obtain the advantages of the vaccine candidate molecules, so as to successfully construct an effective gene engineering vaccine against the efficient natural molecules of schistosomiasis japonicum. In addition, the application of single chain antibody to immuno diagnosis, immunotherapy and the molecular mechanism of disease pathogenesis shows a broad application prospect.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

【参考文献】