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高危型人乳头瘤病毒16型免疫胶体金诊断试纸条的研制

发布时间:2018-12-16 01:05
【摘要】:目的:研制高危型人乳头瘤病毒16型免疫胶体金诊断试纸条,以用于宫颈癌的早期筛查。 方法:利用基因克隆技术构建和表达HPV16E6基因,用镍柱亲和层析纯化HPV16E6蛋白;用HPV16E6蛋白免疫家兔,制备抗HPV16E6蛋白多克隆抗体;用HPV16E6蛋白免疫雌性Balb/C小鼠,用杂交瘤技术建立抗HPV16E6蛋白的单抗杂交瘤细胞株,用体外诱生法制备大量的单抗腹水;用鼠源单克隆抗体作为抗原免疫家兔制备高效价的兔抗鼠IgG即二抗;用获得的单克隆抗体、多克隆抗体和二抗建立了高危型人乳头瘤病毒HPV16型的胶体金免疫层析快速诊断方法。 结果:利用基因克隆技术成功构建含HPV16E6基因原核表达质粒的工程菌pET-28a(+)-HPV16E6-BL21 Star- DE3 PlysS,DNA测序正确,Western Blotting鉴定诱导表达出的蛋白为HPV16E6蛋白;兔抗HPV16E6蛋白多抗的效价高达1:256000;细胞融合率达93.2%,筛选出阳性克隆11孔,两次有限稀释法克隆化培养,筛选出了6株能稳定分泌特异性抗HPV16E6蛋白单克抗体的杂交瘤细胞株,分别命名为1F9、2F10、3C5、3C6、4D4、5B10。免疫细胞化学检测时这些单抗仅与CaSki细胞(含HPV16型)发生特异性反应,而不与其Hela细胞(含HPV18型)发生反应,免疫组织化学检测时这些单抗只与宫颈癌鳞癌组织(含HPV16型)发生反应,而不与宫颈癌腺癌(含HPV18型)发生反应;间接ELISA方法检测兔抗鼠单抗IgG的多克隆抗体血清的效价高达1:256000;制备了30nm的胶体金颗粒,单克隆抗体(4D4株)的实际标记量为6ug/ml。将金标抗体固定于结合垫(玻璃纤维膜)上作为显色源,将兔抗HPV16E6蛋白多抗和兔抗鼠IgG的抗抗体喷画在硝酸纤维素膜上分别作为捕获线和质检线,通过正交试验确定三种抗体的最佳喷涂量后制成了HPV16型免疫胶体金诊断试纸条。 结论:本研究成功地构建了含HPV16E6基因的原核表达质粒工程菌pET-28a(+)-HPV16E6-BL21 Star- DE3 PlysS,Western Blotting鉴定诱导表达的蛋白为HPV16E6蛋白,用镍柱亲和层析纯化了HPV16E6蛋白;制备了高效价的抗HPV16E6蛋白的多克隆抗体;获得6株能稳定分泌抗HPV16E6蛋白单克隆抗体的杂交瘤细胞株;制备了高效价的兔抗鼠IgG抗抗体。将这三种抗体应用在HPV16的诊断上,研制完成高危型人乳头瘤病毒16型的免疫胶体金诊断试纸条。
[Abstract]:Objective: to develop a high-risk human papillomavirus 16 immuno-colloidal gold diagnostic test strip for early screening of cervical cancer. Methods: HPV16E6 gene was constructed and expressed by gene cloning technique, HPV16E6 protein was purified by nickel column affinity chromatography, HPV16E6 protein was immunized with HPV16E6 protein to prepare polyclonal antibody against HPV16E6 protein. Female Balb/C mice were immunized with HPV16E6 protein. Hybridoma cell lines against HPV16E6 protein were established by hybridoma technique. A large number of monoclonal antibody ascites were prepared by in vitro induction method. High titer rabbit anti-mouse IgG (second antibody) was prepared by immunizing rabbits with murine monoclonal antibody (McAb) as antigen, and high risk human papillomavirus (HPVV) HPV16 type was quickly diagnosed by colloidal gold immunochromatography with the obtained monoclonal antibody, polyclonal antibody and second antibody. Results: the engineering strain pET-28a ()-HPV16E6-BL21 Star- DE3 PlysS,DNA containing the prokaryotic expression plasmid of HPV16E6 gene was successfully constructed by gene cloning technique, and the expressed protein was identified as HPV16E6 protein by correct, Western Blotting sequencing. The titer of rabbit polyclonal antibody against HPV16E6 protein was 1: 256000; The cell fusion rate was 93.20.The positive clones were screened out for 11 wells and cloned twice by limited dilution method. Six hybridoma cell lines which could stably secrete the specific antibody against HPV16E6 protein were selected and named as 1F9F102F103C5C6O3C6D4D5B10. In immunocytochemistry, these McAbs only reacted specifically with CaSki cells (including HPV16 type), but not with Hela cells (including HPV18 type). These monoclonal antibodies reacted only with cervical squamous cell carcinoma (including HPV16 type) and not with cervical carcinoma adenocarcinoma (including HPV18 type) by immunohistochemistry. The titer of polyclonal antibody of rabbit anti-mouse monoclonal antibody (IgG) detected by indirect ELISA method was as high as 1: 256000.The colloidal gold granules of 30nm were prepared, and the actual labeling amount of monoclonal antibody (4D4 strain) was 6ugpml. The gold-labeled antibody was fixed on the binding pad (glass fiber membrane) as the color source, and the rabbit anti-HPV16E6 protein polyantibody and the rabbit anti-mouse IgG antibody were sprayed on the nitrocellulose membrane as the capture line and the quality inspection line, respectively. The HPV16 immuno-colloidal gold diagnostic test strip was made after determining the best spraying amount of three kinds of antibodies by orthogonal test. Conclusion: the prokaryotic expression plasmid pET-28a ()-HPV16E6-BL21 Star- DE3 PlysS,Western Blotting containing HPV16E6 gene was successfully constructed to identify the expressed protein as HPV16E6 protein, and the HPV16E6 protein was purified by nickel column affinity chromatography. High titer polyclonal antibodies against HPV16E6 protein were prepared, six hybridoma cell lines secreting monoclonal antibodies against HPV16E6 protein were obtained, and high titer rabbit anti-mouse IgG antibodies were prepared. The three antibodies were applied to the diagnosis of HPV16, and the high risk human papillomavirus type 16 immuno-colloidal gold diagnostic test strip was developed.
【学位授予单位】:重庆理工大学
【学位级别】:硕士
【学位授予年份】:2009
【分类号】:R392.1

【引证文献】

相关硕士学位论文 前1条

1 王亮;IL-9重组表达及其单克隆抗体的制备[D];重庆理工大学;2012年



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