抗梅毒螺旋体Tp0453基因重组蛋白单克隆抗体的制备、鉴定及初步应用

发布时间：2018-04-19 02:19

本文选题：梅毒螺旋体 + 单克隆抗体　；参考：《南华大学》2007年硕士论文

【摘要】： 目的:将本室构建的含梅毒螺旋体(Treponema pallidum,Tp)外膜蛋白Tp0453的优势表位(28～288aa)基因的重组表达体在E.coli M15中进行诱导表达,纯化表达产物并进行抗原性分析。应用杂交瘤技术,将SP2/0细胞与经重组蛋白Tp0453免疫的小鼠脾细胞进行融合,筛选抗重组蛋白Tp0453的单克隆抗体(mAb) ,并进行鉴定和纯化。用此mAb检测Tp感染疑似患者的分泌物标本,由此确定该mAb的应用价值,并为Tp感染诊断的研究奠定基础。方法:(1)使用镍亲和层析法纯化重组蛋白Tp0453。SDS-PAGE和Western- blot鉴定表达产物。(2)以复性后纯化的Tp0453重组蛋白免疫BALB/c小鼠,将免疫小鼠的脾细胞与SP2/0细胞融合,用间接酶联免疫吸附试验(ELISA)筛选分泌特异性抗体的杂交瘤细胞。(3)体内诱生腹水法制备抗体,用硫酸铵沉淀法对腹水中的mAb进行纯化,ELISA法检测抗体效价,mAb亚类测定试剂盒鉴定mAb的类别,通过细胞涂片染色镜检计数杂交瘤细胞株染色体数目。(4)间接ELISA和间接荧光免疫试验(IIF)检测临床标本及mAb的特异性,并进行统计学分析。结果:重组目的基因表达菌在IPTG的诱导下,表达了相对分子量(Mr)约为32kDa的目的蛋白,目的蛋白在菌体细胞内主要以包涵体形式存在;经Ni-NTA亲和层析法纯化获得了纯度在95%以上的重组蛋白;Western-blot检测其能与Anti-His mAb发生特异性反应;应用纯化复性后的重组Tp0453免疫8周龄的BALB/c小鼠,经过杂交瘤技术进行细胞融合;应用ELISA法筛选出了4株分泌抗Tp0453重组蛋白mAb的杂交瘤细胞株,分别命名为2F8、8B9、9C6和15B2;Western-blot结果显示mAb均能与重组Tp0453很好的结合。用腹水诱生法大量制备mAb;ELISA检测4株杂交瘤细胞诱生腹水中的mAb效价分别为1:25 000、1:8 000、1:50 000和1:10 000;根据mAb亲和常数公式计算4株杂交瘤细胞分泌的mAb亲和常数分别为4.12×107M-1,2.73×108M-1,4.71×108M-1和3.64×107M-1;经Mouse Monoclonal Antibody Isotyping Kit测定,4株杂交瘤细胞分泌的mAb,2F8为IgG2b,其它3株均为IgG1。4株mAb腹水经SDS-PAGE均显示出一条分子量约为50kDa的重链条带和一条分子量为26kDa的轻链条带。4株杂交瘤细胞染色体数在90～108范围内变化。IIF和间接ELISA对85份临床标本进行检测结果表明自制mAb能与天然抗原结合,并与镀银染色的检测结果基本符合。结论: (1)筛选出4株稳定分泌抗Tp0453 mAb的杂交瘤细胞株,经鉴定4株杂交瘤细胞分泌的mAb均能识别重组Tp0453。 (2)获得的抗重组Tp0453的mAb均为IgG类抗体,且能识别Tp0453抗原的天然表位,具有较好的特异性。 (3)自制mAb与镀银染色的检测结果比较具有较好的一致性,有望应用于Tp抗原的检测。
[Abstract]:Aim: to induce the recombinant expression of the outer membrane protein Tp0453 containing Treponema pallidum Tp1 in our laboratory into E.coli M15, and to purify the expressed product and analyze its antigenicity.SP2/0 cells were fused with murine spleen cells immunized with recombinant protein Tp0453 by hybridoma technique. Monoclonal antibodies against recombinant protein Tp0453 were screened, identified and purified.The mAb was used to detect the secretions of suspected patients with TP infection. The application value of the mAb was determined and the foundation for the diagnosis of TP infection was established.Methods the recombinant protein Tp0453.SDS-PAGE and Western blot were purified by nickel affinity chromatography. The purified recombinant Tp0453 protein was used to immunize BALB/c mice, and the spleen cells of the immunized mice were fused with SP2/0 cells.Indirect enzyme-linked immunosorbent assay (Elisa) was used to screen hybridoma cells secreting specific antibodies.MAb in ascites was purified by ammonium sulfate precipitation method. Elisa was used to detect antibody titer and mAb subclass test kit was used to identify the type of mAb.Indirect ELISA and indirect fluorescence immunoassay (IIFI) were used to detect the specificity of clinical specimens and mAb.Results: under the induction of IPTG, the recombinant target gene expressed the target protein of 32kDa, and the target protein existed mainly in the form of inclusion body.The recombinant protein with purity of more than 95% was purified by Ni-NTA affinity chromatography and its specific reaction with Anti-His mAb was detected by Western-blot. The purified recombinant Tp0453 was used to immunize the 8-week-old BALB/c mice, and the cells were fused by hybridoma technique.Four hybridoma cell lines secreting mAb against Tp0453 recombinant protein were screened by ELISA method and named as 2F8B9C6 and 15B2 Western-blot, respectively. The results showed that mAb could bind well with recombinant Tp0453.The mAb titers in ascites of four hybridoma cell lines were measured by Elisa with the method of ascites induction. The mAb affinity constants of the four hybridoma cells were calculated by mAb affinity constant formula as 4.12 脳 107M-12.73 脳 108M-1 and 4.71 脳 108M-1, respectively.The mAb2F8 secreted by Mouse Monoclonal Antibody Isotyping Kit was IgG2b, and the other three IgG1.4 strains mAb ascites showed a heavy chain band with a molecular weight of 50kDa and a light chain band with a molecular weight of 26kDa.The changes of chromosome number in 90 ~ (10 ~ 8) range. IIF and indirect ELISA were used to detect 85 clinical specimens. The results showed that self-made mAb could bind to natural antigen.The results are in good agreement with the results of silver-plated staining.Conclusion:1) four hybridoma cell lines secreting stable anti- mAb were screened, and all the mAb secreted by the four hybridoma cells could recognize recombinant Tp0453.(2) the mAb of the recombinant Tp0453 were all IgG class antibodies, and could recognize the natural epitopes of Tp0453 antigen, and had good specificity.(3) the results of self-made mAb and silver staining are in good agreement with each other, and it is expected to be applied to the detection of TP antigen.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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