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目的构建人端粒酶逆转录酶启动子(hTERT)调控的、携带幽门螺杆菌细胞空泡毒素A(Vacuolatingcytotoxin A,VacA)基因的选择性表达重组杆状病毒。方法应用酶切、连接方法将hTERT连接于杆状病毒穿梭质粒pFastBac~(TM)DUAL载体P10启动子序列之后,和SV40分别调控目的基因VacA的表达和终止;将重组穿梭质粒电转入DH10Bac~(TM)E.coli感受态细胞,经转座重组获得重组杆粒Bacmid-VacA。利用脂质体Cellfectin~将重组杆粒转染Sf9昆虫细胞,包装重组杆状病毒。结果应用PCR验证、双酶切分析、序列测定等方法验证重组质粒基因片段连接正确无突变,通过转座重组获得携带有VacA基因片段的重组杆粒。结论携带VacA基因的选择性复制重组杆粒成功构建。
Objective To construct a recombinant baculovirus that regulates the expression of human telomerase reverse transcriptase promoter (hTERT) and carries the Vacuolating cytotoxin A (VacA) gene of Helicobacter pylori. Methods The hTERT gene was ligated into pFastBac ~ (TM) DUAL vector p10 promoter by restriction enzyme digestion and ligation, and the expression of VacA was regulated by SV40 and terminated respectively. The recombinant shuttle plasmid was transformed into DH10Bac ~ (TM) E.coli competent cells, and recombinant bacmid Bacmid-VacA was obtained by transposon recombination. The recombinant bacmid was transfected into Sf9 insect cells using liposome Cellfectin ~,, and the recombinant baculovirus was packaged. The results of PCR validation, double enzyme digestion analysis, sequence analysis and other methods to verify the recombinant plasmid gene fragment connection correct non-mutated recombinant plasmid carrying VacA gene by transposon recombinant bacmid. Conclusion The recombinant plasmid carrying VacA gene was successfully constructed.