淋球菌IgA蛋白酶中和表位表达载体的构建及其诱导小鼠的粘膜免疫
发布时间：2018-01-12 09:00
本文关键词：淋球菌IgA蛋白酶中和表位表达载体的构建及其诱导小鼠的粘膜免疫 出处：《南华大学》2007年硕士论文 论文类型：学位论文
更多相关文章： 淋球菌 IgA蛋白酶 中和表位 表达 粘膜免疫
【摘要】： 【目的】: (1)构建淋球菌IgA蛋白酶中和表位原核表达载体,并在大肠杆菌中诱导表达,纯化表达产物6His-IgA蛋白酶中和表位融合蛋白,检测融合蛋白的免疫原性。 (2)构建淋球菌IgA蛋白酶中和表位真核表达载体,通过生殖道粘膜途径接种,在动物体内表达相应产物,诱导粘膜免疫,为研制人用高效抗淋病核酸疫苗提供实验依据。 【方法】: (1)IgA蛋白酶中和表位原核表达载体的构建、表达、纯化及其免疫原性鉴定通过PCR扩增淋球菌MS11株IgA蛋白酶编码基因1 601～2 722位序列,构建pQE30-IgA蛋白酶中和表位原核表达载体;测序正确后在大肠杆菌M15中诱导表达,包涵体重组蛋白经8M尿素变性后通过镍琼脂凝胶FF分离纯化,SDS-PAGE和Western-Blot分析及鉴定该纯化蛋白;后者经尿素浓度梯度透析复性、SDS-PAGE鉴定正确后,与佐剂混合于皮内多点注射免疫新西兰兔,诱导产生抗淋球菌IgA蛋白酶中和表位的多克隆抗体;以复性后的重组蛋白抗原包板,建立间接ELISA法,检测兔免疫血清中IgA蛋白酶中和表位的抗体效价。 (2)构建IgA蛋白酶中和表位真核表达载体,诱导小鼠产生粘膜免疫通过PCR扩增淋球菌MS11株IgA蛋白酶编码基因1 601-2 722位序列,构建pcDNA3.1(-)-IgA蛋白酶中和表位真核表达载体;经鉴定后,制备重组质粒壳聚糖混合颗粒,通过阴道灌注法免疫小鼠,诱导其产生粘膜免疫,对照组免疫不含重组质粒的壳聚糖溶液和pcDNA3.1(-)壳聚糖混合颗粒溶液;第3周和第5周加强免疫一次,间接免疫荧光法检测目的抗原在小鼠阴道组织内的表达,ELISA法检测小鼠血清阴道冲洗液中抗IgA蛋白酶中和表位的IgA和IgG类抗体。 【结果】: (1)成功构建了淋球菌IgA蛋白酶中和表位的原核表达载体。通过PCR扩增获得的淋球菌IgA蛋白酶中和表位序列与GenBank报道的一致。所克隆的基因在大肠杆菌中获得高效表达,目的蛋白在菌体细胞内以包涵体形式存在,Western-Blot结果显示该纯化蛋白能被抗6-His单抗识别,复性后得到溶解状态的复性蛋白。免疫组兔血清中IgA蛋白酶中和表位抗体效价达1:6 400。 (2)成功构建了淋球菌IgA蛋白酶中和表位真核表达载体。以壳聚糖为释放系统经阴道免疫后第4天开始,通过间接免疫荧光法检测,在免疫组小鼠阴道上皮细胞中可以观察到一定亮度的绿色荧光,提示IgA蛋白酶中和表位抗原能在小鼠阴道组织内表达。免疫组小鼠阴道冲洗液中检测到的抗IgA蛋白酶中和表位的sIgA水平明显高于对照组。 【结论】: (1)成功构建了淋球菌pQE30-IgA蛋白酶中和表位原核表达载体,免疫动物后获得了具有较好免疫原性的纯化复性IgA蛋白酶中和表位蛋白。 (2)成功构建了淋球菌pcDNA3.1(-)-IgA蛋白酶中和表位真核表达载体,其壳聚糖混合颗粒经阴道免疫小鼠,能诱导产生有效的生殖道粘膜免疫。 (3)pcDNA3.1(-)-IgA蛋白酶中和表位蛋白能在小鼠阴道上皮组织内表达。
[Abstract]:[Objective]:
(1) construct prokaryotic expression vector of Neisseria gonorrhoeae IgA protease neutralization epitope, and express it in Escherichia coli, and purify 6His-IgA protein and neutralization epitope fusion protein, and detect the immunogenicity of fusion protein.
(2) we constructed the eukaryotic expression vector of Neisseria gonorrhoeae IgA protease neutralization epitope, and expressed the corresponding products in the animal body through the inoculation of genital tract mucosal channel, and induced mucosal immunity, so as to provide experimental evidence for developing highly effective anti gonorrhea nucleic acid vaccine.
[method]:
(1) IgA neutralizing epitope prokaryotic expression vector construction, expression, purification and immunogenicity identification of Neisseria gonorrhoeae strain MS11 IgA protease encoding gene was amplified by PCR from 1601 to 2722 sequences, construct pQE30-IgA neutralizing epitope prokaryotic expression vector; after sequencing in Escherichia coli M15, inclusion body group of protein by nickel agar gel FF purification by 8M urea denaturation, analysis and identification of SDS-PAGE and Western-Blot of the purified protein; the latter by urea gradient dialysis refolding, SDS-PAGE after correct identification, with the adjuvants in New Zealand multi point injection induced immune rabbit polyclonal antibody against gonococcal IgA protease neutralizing epitope; package board with recombinant protein antigen after refolding, an indirect ELISA method, detection of rabbit immune serum neutralization antibody titer of a IgA protease.
(2) to construct IgA neutralizing epitope of eukaryotic expression vector of mice induced by mucosal immune IgA protease encoding gene of Neisseria gonorrhoeae strain MS11 1 601-2 722 bit sequence was amplified by PCR to construct pcDNA3.1 (-) -IgA neutralizing epitope of eukaryotic expression vector; after identification, preparation of recombinant plasmid chitosan mixed particles. Through the vagina perfusion in mice to induce mucosal immunity, the immune control group without recombinant plasmid chitosan solution and pcDNA3.1 (-) chitosan mixed particle solution; a second immunization for third weeks and fifth weeks, to detect the expression of target antigen indirect immunofluorescence method in mouse vaginal tissue, serum ELISA? Vaginal washing liquid and anti IgA protease IgA and IgG antibody level.
[results]:
(1) construct a prokaryotic expression vector of gonococcal IgA protease neutralizing epitope. Gonococcal IgA protease amplified by PCR neutralizing epitope sequence reported in GenBank. The cloned gene was expressed in Escherichia coli, the protein in the cell in the form of inclusion bodies, the results of Western-Blot the purified protein can be anti 6-His monoclonal antibody recognition, renaturation after refolding protein dissolved state. IgA protease immunohistochemistry in rabbit serum neutralizing epitope antibody titer was 1:6 400.
(2) the successful construction of gonococcal IgA protease neutralizing epitope of eukaryotic expression vector. Using chitosan as vaginal delivery system fourth days after immunization, by indirect immunofluorescence assay, in immunized mice vaginal epithelial cells can be observed in certain brightness green fluorescence, suggesting that IgA neutralizing epitope the expression in murine vaginal tissue. Immunized mice vaginal washing fluid was detected in the anti IgA neutralizing epitope of sIgA was significantly higher than control group.
[Conclusion]:
(1) the prokaryotic expression vector of Neisseria gonorrhoeae pQE30-IgA protease neutralization epitope was successfully constructed. After immunization, the neutralizing epitope protein of purified IgA protease was obtained.
(2) we successfully constructed the eukaryotic expression vector of pcDNA3.1 (-) -IgA protease neutralization epitope, and its chitosan mixed particles could induce effective mucosal immunity in mice through immunization with vagina.
(3) the pcDNA3.1 (-) -IgA protease neutralization epitope protein can be expressed in the mouse vaginal epithelial tissue.
【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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