抗生殖支原体MgPa’重组蛋白单克隆抗体的制备及鉴定

发布时间：2018-01-13 06:35

本文关键词：抗生殖支原体MgPa’重组蛋白单克隆抗体的制备及鉴定　出处：《南华大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的:将本试验室构建的含生殖支原体黏附素(Mycoplasma genitalium adhesin protein ,MgPa)蛋白优势表位基因(MgPa’3 223～4 092bp)的重组表达载体pET-30a(+)/MgPa’在表达宿主菌E.coliRosettaTM2(DE3)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MgPa’免疫的小鼠脾细胞进行融合,筛选抗重组蛋白MgPa’的单克隆抗体(mAb),并进行鉴定和纯化。由此初步确定该mAb的应用价值,并为研制Mg感染诊断试剂盒奠定基础。方法:(1)采用镍亲和层析法纯化重组蛋白MgPa’,并用SDS-PAGE分析和Western-blot鉴定表达产物。(2)以纯化复性后的MgPa’重组蛋白免疫BALB/c小鼠,取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,ELISA法检测抗体效价,采用竞争法ELISA和Western-blot分析mAb的特异性,用mAb亚类测定试剂盒鉴定mAb的类别,用硫酸铵沉淀法对腹水中的mAb进行纯化。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37kDa的目的蛋白,目的蛋白在菌体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;应用纯化复性后的重组蛋白MgPa’免疫6～8周龄的BALB/c小鼠,采用杂交瘤技术进行细胞融合;应用ELISA法筛选出了6株分泌抗MgPa’重组蛋白mAb的杂交瘤细胞株,分别命名为12E7、4B1、6E3、2B8、6E2和10E2;竞争法ELISA或Western-blot分析结果显示mAb均能与重组蛋白MgPa’很好的结合。用腹水诱生法大量制备mAb;ELISA检测6株杂交瘤细胞诱生腹水中的mAb效价分别为1:25 600、1:25 600、1:12 800、1:25 600、1:25 600和1:25 600;经Mouse Monoclonal Antibody Isotyping Kit测定,6株杂交瘤细胞中12E7、4B1分泌的mAb为IgG2b,其它4株分泌的mAb均为IgG1。结论: (1)筛选出6株稳定分泌抗MgPa’mAb的杂交瘤细胞株,经鉴定6株杂交瘤细胞分泌的mAb均能识别重组MgPa’。 (2)获得的抗重组MgPa’的mAb均为IgG类抗体,且能识别MgPa’抗原的天然表位,具有较好的特异性。
[Abstract]:Objective: the laboratory construct containing the adhesin of Mycoplasma genitalium (Mycoplasma genitalium adhesin protein, MgPa) protein epitope gene (MgPa 3223~4 092bp) recombinant expression vector pET-30a (+) /MgPa expression in E.coli E.coliRosettaTM2 (DE3) in the expression, purification and antigenicity analysis. The application of hybridoma technology, SP2/0 cells and the recombinant protein MgPa of immunized mice spleen cells were fused, screening monoclonal antibodies against recombinant protein MgPa '(mAb), was identified and purified. The first step to determine the application value of the mAb, and lay the foundation for the development of Mg infection diagnosis kit.
Methods: (1) using nickel affinity chromatography purification of recombinant protein MgPa, and analyzed by SDS-PAGE and Western-blot (2). The expressed product was identified by purification of refolded MgPa recombinant protein immunized BALB/c mice splenocytes from the immunized mice were fused with SP2/0 cells, using indirect enzyme-linked immunosorbent assay (ELISA) screening hybridoma cells secreting specific antibodies. (3) in ascites were induced to produce the antibody titers of ELISA method, the specific competition law ELISA and Western-blot analysis of mAb, mAb sub category determination kit for identification of mAb, of mAb in ascites were purified by ammonium sulfate precipitation method.
Results: the recombinant gene expression was induced by IPTG. The expression of the relative molecular weight of about 37kDa (Mr) protein, the protein in the bacteria mainly existed in the form of inclusion body; by Ni-NTA affinity chromatography. The purity of the recombinant protein was above 95%; should be purified after refolding of recombinant protein MgPa 'immune 6 ~ 8 week old BALB/c mice were fused by hybridoma technique; ELISA method was used to isolate 6 strains of hybridoma cell strains secreting anti MgPa recombinant protein mAb, named 12E7,4B1,6E3,2B8,6E2 and 10E2; competition law ELISA or Western-blot analysis showed that mAb can MgPa with recombinant protein good combination. The preparation of mAb ascites by induction; ELISA detection of 6 strains of hybridoma cells induced mAb titer in ascites were 1:25 600,1:25 600,1:12 800,1:25 600,1:25 600 and 1:25 600; Mouse Monoclonal Antibody Isotyping Kit determined that the mAb of 12E7,4B1 secreted in 6 hybridoma cells was IgG2b, and the other 4 secreted mAb were IgG1..
Conclusion:
(1) 6 hybridoma cells with stable secretion of anti MgPa 'mAb were screened, and the mAb secreted by 6 hybridoma cells could identify the recombinant MgPa'.
(2) the mAb anti recombinant MgPa 'is IgG antibody, and it can identify the natural epitopes of MgPa' antigen, which has good specificity.

【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392.1

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