通过自诱导方法对乙型肝炎病毒聚合酶TP区进行可溶性表达及鉴定

Soluble expression of TP domain of hepatitis B virus polymerase by auto-induction and identification of expressed product

目的构建包含乙型肝炎病毒(hepatitis B virus,HBV)聚合酶TP区段的重组原核表达质粒,转化表达型大肠埃希菌,以自诱导培养方法获得可溶性表达,并对其进行鉴定。方法分析HBV(A型)聚合酶N-末端1-192 AA区域的DNA序列,通过网络工具(http://www.jcat.de/)在线优化,并在5′和3′端分别添加NdeⅠ和XhoⅠ限制性内切酶位点后,送英潍捷基(上海)贸易有限公司进行人工合成;将人工合成的TP-DNA双酶切后,插入pET32a(+)原核表达载体,构建重组原核表达质粒,转化大肠埃希菌BL21(DE3)pLysS进行自诱导表达。结果重组原核表达质粒pET32a(+)/POL-TP-Opt经双酶切及测序证明构建正确;在表达菌的破菌上清中有特异性表达的蛋白,相对分子质量约20 000,经SDS-PAGE和Western blot鉴定,为可溶性的重组TP蛋白。结论通过自诱导培养方法,直接成功获得可溶性的重组TP蛋白,改变了长期以来依靠包涵体复性对TP蛋白相关功能进行研究的状况。

Objective To construct the prokaryotic expression vector for TP domain of hepatitis B virus polymerase（HBV POL-TP）,transform to E. coli for soluble expression by auto-induction,and identify the expressed product. Methods The DNA sequence of amino acids（AA） 1 ~ 192 at N-terminus of HBV POL type A was analyzed and optimized by the network tool（http：//www.jcat.de/）. The optimized POL-TP sequence was synthesized,with restriction endonuclease sites added to 5＇-and 3＇-terminuses,digested with Nde Ⅰand Xho Ⅰ,and inserted into vector pET32a（＋）. The constructed recombinant plasmid was transformed to E. coli BL21（DE3）pLysS for expression by auto-induction. Results Recombinant plasmid pET32a（＋）/POL-TP-Opt was constructed correctly as proved by restriction analysis and sequencing. Specific protein with a relative molecular mass of about 20 000 was expressed in supernatant,which was identified as soluble recombinant TP by SDS-PAGE and Western blot. Conclusion Soluble recombinant TP was obtained successfully by auto-induction,which changed the persistent status of study on the function of TP depending on the re-naturalization of inclusion body.