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目的构建羧基端缺失30个氨基酸的HBx蛋白的真核表达质粒,并在真核细胞内稳定表达。方法提取HBV-DNA阳性血清总DNA,PCR扩增截短变异的HBx基因(HBx),克隆到真核质粒pEGFP-N3中,酶切及测序鉴定。利用脂质体将重组质粒转染HepG2细胞,通过W estern blot方法检测细胞内截短变异的HBx蛋白的表达。结果构建的重组质粒pEGFP-N3/x经酶切及测序鉴定结果正确。质粒pEGFP-N3/x转染细胞后可检测到目的蛋白。结论成功构建在真核细胞内稳定表达的重组质粒pEGFP-N3/x。
Objective To construct a eukaryotic expression plasmid containing a 30-amino acid deletion of the carboxyl terminus of HBx protein and to express it in eukaryotic cells. Methods The total DNA of HBV-DNA positive serum was extracted. The truncated variant of HBx gene (HBx) was amplified by PCR, cloned into eukaryotic plasmid pEGFP-N3, digested and sequenced. The recombinant plasmids were transfected into HepG2 cells by liposome, and the expression of truncated intracellular variant of HBx protein was detected by Western blot. Results The constructed recombinant plasmid pEGFP-N3 / x was confirmed by enzyme digestion and sequencing. After the plasmid pEGFP-N3 / x transfected cells can detect the target protein. Conclusion The recombinant plasmid pEGFP-N3 / x was successfully constructed in eukaryotic cells.