人类乳头瘤病毒16型E7C亚基因在耻垢分枝杆菌中的表达

Expression of the Human papillomavirous type16 E7C subgene in Mycobaterium smegmatis mc^2 155

目的在耻垢分枝杆菌Mycobaterium smegmatis mc2155中表达人类乳头瘤病毒16型E7C亚基因,为其重组BCG疫苗的研究奠定基础。方法采用电穿孔转化法将重组质粒pBCG-E7C导入耻垢分枝杆菌(M.smegmatismc2155)中,通过卡那霉素抗性筛选经PCR鉴定的重组M.smegmatis mc2155,培养于middlebrook7H9 broth(M7H9)培养基中,并添加10%M7H9 enrichment ADC和0.05%Tween 80,37℃培养48 h^72 h,42℃诱导表达5 h,表达产物进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)及Western blot分析。结果成功构建pBCG3000-E7C质粒,SDS-PAGE显示表达产物的分子质量单位约为6.5 ku,与预期理论值相符,Western blot分析表达产物能被宫颈癌患者血清识别。结论人类乳头瘤病毒16型E7C基因在M.smegmatis mc2155中成功表达。

Objective To express the C-terminal E7 subgene of human papillomavirous typel6 in Mycobaterium smegmatis mc^2 155 in the hope of laying a solid foundation for the further recombinant BCG vaccine research. Methods The recombinant plasmid pBCG- E7C was introduced by electroporation into M. smegmatis mc^2 155 which was first selected by kanamycin resistance and identified by PCR. The recombinant M. smegmatis mc^2 155 is later cultivated in liquid middlebrook 7H9 medium with 10% middlebrook 7H9 enrichment ADC and 0.05% Tween 80 （M-ADC-TW broth） at 37 ℃ for 48-72 hours. Then the cultures were induced at 42 ℃ for 5 h with its expressing product being analyzed by sodium dodecyl benzene sulfonate polyacrylamide gel electrophoresis （SDS-PAGE） and Western blot. Results Plasmid of pBCG- E7C was successfully constructed. SDS-PAGE showed that the quantum unit of the molecule in the expressed products was about 6.5 ku, which fits the theoretical assumption. Western blot result also indicated that the expressed products could be recognized by the sera of cervical cancer patient. Conclusion E7C gene of human papillomavirous typel6 can be expressed in M. smegmatis mc^2 155.