高危型人乳頭瘤病毒16型免疫膠體金診斷試紙條的研制
發(fā)布時間：2018-12-16 01:05
【摘要】：目的:研制高危型人乳頭瘤病毒16型免疫膠體金診斷試紙條,以用于宮頸癌的早期篩查。 方法:利用基因克隆技術構(gòu)建和表達HPV16E6基因,用鎳柱親和層析純化HPV16E6蛋白;用HPV16E6蛋白免疫家兔,制備抗HPV16E6蛋白多克隆抗體;用HPV16E6蛋白免疫雌性Balb/C小鼠,用雜交瘤技術建立抗HPV16E6蛋白的單抗雜交瘤細胞株,用體外誘生法制備大量的單抗腹水;用鼠源單克隆抗體作為抗原免疫家兔制備高效價的兔抗鼠IgG即二抗;用獲得的單克隆抗體、多克隆抗體和二抗建立了高危型人乳頭瘤病毒HPV16型的膠體金免疫層析快速診斷方法。 結(jié)果:利用基因克隆技術成功構(gòu)建含HPV16E6基因原核表達質(zhì)粒的工程菌pET-28a(+)-HPV16E6-BL21 Star- DE3 PlysS,DNA測序正確,Western Blotting鑒定誘導表達出的蛋白為HPV16E6蛋白;兔抗HPV16E6蛋白多抗的效價高達1:256000;細胞融合率達93.2%,篩選出陽性克隆11孔,兩次有限稀釋法克隆化培養(yǎng),篩選出了6株能穩(wěn)定分泌特異性抗HPV16E6蛋白單克抗體的雜交瘤細胞株,分別命名為1F9、2F10、3C5、3C6、4D4、5B10。免疫細胞化學檢測時這些單抗僅與CaSki細胞(含HPV16型)發(fā)生特異性反應,而不與其Hela細胞(含HPV18型)發(fā)生反應,免疫組織化學檢測時這些單抗只與宮頸癌鱗癌組織(含HPV16型)發(fā)生反應,而不與宮頸癌腺癌(含HPV18型)發(fā)生反應;間接ELISA方法檢測兔抗鼠單抗IgG的多克隆抗體血清的效價高達1:256000;制備了30nm的膠體金顆粒,單克隆抗體(4D4株)的實際標記量為6ug/ml。將金標抗體固定于結(jié)合墊(玻璃纖維膜)上作為顯色源,將兔抗HPV16E6蛋白多抗和兔抗鼠IgG的抗抗體噴畫在硝酸纖維素膜上分別作為捕獲線和質(zhì)檢線,通過正交試驗確定三種抗體的最佳噴涂量后制成了HPV16型免疫膠體金診斷試紙條。 結(jié)論:本研究成功地構(gòu)建了含HPV16E6基因的原核表達質(zhì)粒工程菌pET-28a(+)-HPV16E6-BL21 Star- DE3 PlysS,Western Blotting鑒定誘導表達的蛋白為HPV16E6蛋白,用鎳柱親和層析純化了HPV16E6蛋白;制備了高效價的抗HPV16E6蛋白的多克隆抗體;獲得6株能穩(wěn)定分泌抗HPV16E6蛋白單克隆抗體的雜交瘤細胞株;制備了高效價的兔抗鼠IgG抗抗體。將這三種抗體應用在HPV16的診斷上,研制完成高危型人乳頭瘤病毒16型的免疫膠體金診斷試紙條。
[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.
【學位授予單位】：重慶理工大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392.1
本文編號：2381595
[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.
【學位授予單位】：重慶理工大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392.1
【引證文獻】
相關碩士學位論文 前1條
1 王亮;IL-9重組表達及其單克隆抗體的制備[D];重慶理工大學;2012年
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