Research On Immuno-colloidal Gold Test Strip For Detection Of High-risk Human Papillomavirus 16 Type

Objective: To explore the immuno-colloidal gold test strip against high-risk human papillomavirus16 type for the screening of cervical cancer in the early stage.Methods: According to the methods of Gene Cloning, the recombinant plasmid which contains HPV16 E6 gene was constructed and expressed. HPV16E6 proteins were purified by nickel affinity chromatography column; Rabbits were immuned by HPV16E6 protein to prepare anti-HPV16E6 protein polyclonal antibody; After the immunization of female Balb / C mice with HPV16E6 protein , hybridoma cell lines which secrete monoclonal antibody (McAb)against the desire protein were established by the hybridoma technique ,and high-titer monoclonal antibody ascites can be obtained from Balb / C mice which were intraperitoneal injected with monoclonal hybridoma cells; The IgG monoclonal antibody of mice was used to prepare high-titer rabbit anti-mouse IgG; Afterwards, the rapid diagnostic method of GICA for high-risk human nipple papilloma virus 16 type has been established by the monoclonal antibodies, polyclonal antibodies and rabbit anti-mouse IgG..Results: The engineering strain pET-28a (+)-HPV16E6-BL21 Star-DE3 PlysS was successfully constructed, and the DNA sequence was gained from the reported DNA sequencing result .The specificity of the induced protein is proved by the Western Blot; the rabbit anti- HPV16E6 protein polyclonal antibody titer was up to 1:256000; 93.2% cell fusion rate and 11 positive holes were obtained after cell fusion, by twice limiting dilution cloning, 6 hybridoma cell lines secreting stable specific anti-HPV16E6 protein antibody were screened, named respectively 1F9, 2F10, 3C5, 3C6, 4D4, 5B10,. Immunocytochemistry demonstrates that the monoclonal antibodies only reacted with CaSki but not Hela cells and immunohistochemistry proved that they only reacted with squamous cervical carcinoma (including HPV16 type)but not cervical adenocarcinoma (including HPV18 type ) . Indirect ELISA showed that the titer of rabbit anti-mouse IgG polyclonal antibody was up to 1:256000; 30nm colloidal gold particles were prepared and actual labled amount of monoclonal antibody was decided as 6ug / ml. An immuno-colloidal gold strip was developed for the detection of HPV16 type, and the monoclonal antibody was labeled with colloidal gold as the detection reagent, and the rabbit anti- HPV16E6 protein polyclonal antibodies was blotted on the test line while the rabbit anti-mouse IgG antibodies was used on the control line of the nitrocellulose membrane.Conclusion: In this paper, the engineering strain pET-28a (+)-HPV16E6-BL21 Star-DE3 PlysS is successfully constructed. The specificity of the induced protein is proved by the Western Blot, and HPV16E6 proteins were purified by nickel affinity chromatography column; High-titer polyclonal antibodies against HPV16E6 protein were successfully prepared; six monoclonal hybridoma cell lines have been successfully established. An immuno-colloidal gold strip was developed for the detection of HPV16 type, the monoclonal antibody was labeled with colloidal gold as the detection reagent, and the rabbit anti- HPV16E6 polyclonal antibodies was blotted on the test line while the rabbit anti-mouse IgG antibodies was used on the control line of the nitrocellulose membrane.