乙型肝炎病毒X变异体穿梭载体的成功构建及在酵母中的表达

Construction of hepatitis B virus X gene mutants shuttle vector and its expression in yeast cells

目的构建表达乙肝病毒(HBV)X变异体基因的穿梭载体。方法用PCR法扩增HBVX基因及变异体基因序列,用EcoRⅠ和PstⅠ双酶切pAS2-1和PCR产物,连接酶切片段pAS2-1及X变异体片段以构建pAS2-1-(X变异体)穿梭质粒,并通过醋酸锂转化法转化入酵母AH109中。结果已构建的pAS2-1-(X变异体)穿梭质粒经序列测定含有所需的HBV-X变异体基因片段,转入酵母细胞后经PCR证实酵母内有表达。结论成功构建了pAS2-1-X变异体穿梭质粒,为进一步在真核细胞内更全面了解HBV-X蛋白作用奠定了基础。

Objective To establish shuttle vectors expressing hepatitis B virus（HBV） X gene mutants. Methods HBV-X gene and mutants were amplified with polymerase chain reaction （PCR）. Vector pAS2-1 and PCR products were digested with EcoR Ⅰ and Pst Ⅰ. pAS2-1 and the fragments of X mutants were connected to establish the shuttle plasmid of pAS2-1-（X mutants）. Then pAS2-1-（X mutants） was transformed into the yeast AH109 using lithium acetate-mediated method. Results Shuttle plasmid pAS2-1-（X mutants） included the fragments of HBV X mutants, which were proved by auto-sequencing assay. PCR analysis showed that shuttle plasmid pAS2-1-（X mutants） expressed in yeast cells. Conclusion Shuttle plasmid pAS2-1-（X mutants） is constructed successfully, which may lay the foundation for comprehensively understanding the role of HBV X protein in eukaryotie cells.