Establishment of Murine Tumor Cell Line Expressing HPV types 6b and 11 E6/E7 Genes / 中华皮肤科杂志

Objective To construct four expression plasmids, pcDNA3.1-GFP/HPV6bE6, pcDNA3.1-GFP/HPV6bE7, pcDNA3.1-GFP/HPV11E6, pcDNA3.1-GFP/HPV11E7 and their transfected murine cell lines. Methods The Four recombinant expression plasmids comprising HPV6bE6,HPV6bE7,HPV11E6 and HPV11E7 linked with GFP, respectively, were constructed and transfected to B16 cells by lipofectamine kit. Positive clones were selected by G418 and observed by fluorescent microscopy and identified by RT-PCR. Results The four constructed recombinant plasmids were authenticated by restriction enzyme digestion and DNA sequencing. Under the fluorescent microscope, the green fluorescence could be observed in cytoplasm and nucleus of four transfected B16 cell lines. The RNA extracted from positively transfected clones resistant to G418 were analyzed by RT-PCR, which demonstrated the presence of four expected fragments. Conclusions The transfected murine cell lines B16 can express HPV6bE6,HPV6bE7,HPV11E6 and HPV11E7 gene. These transfected cell lines can be further transplanted to mice in order to investigate the biological properties and immunological mechanisms of these genes in vivo.

Construction,Expression and Identification of Structural Gene for Porin I,the Major Outer Membrane Protein of Neisseria gonorrhoeae / 中华皮肤科杂志

Objective To construct,express,purify and identify the gene encodi ng major outer membrane protein of Neisseria gonorrhoeae (Porin I, or PI). Metho ds The gene encoding for PI of N.gonorrhoeae was amplified by PCR and cloned int o expression plasmid pGEX-4T-2 to form pGEX-4T-2/PI recombinants. A high lev el expression of GST-PI fusion protein was obtained in GST gene fusion system (GST:glutathione S transferase). The analysis indicated that the expressed pr otein was present predominantly in the insoluble form. Therefore, the induced pr otein was purified by SDS-PAGE, and bands corresponding to polypeptides of GST-PI fusion protein were excised and subjected to electroelution. A dot immunoch romatographic assay was employed to demonstrate whether the purified protein was gonococcal PI specific. Results The pGEX-4T-2/PI expression recombinants were constructed,expressed,purified and identified successfully. SDS-PAGE analysis and dot immunochromatographic assay suggested that the recombinant GST-PI fusio n protein was a 60 000 molecular weight protein andidentical in size to native PI and reacted with anti-PI monoclonal antibody. Conclusion Our results may lead to a potentiality for further study of diagnosti c kits and vaccine for Neisseria gonorrhoeae.