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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 a recombinant prokaryotic expression plasmid containing the TP segment of hepatitis B virus (HBV) polymerase and transform the expressed Escherichia coli to obtain soluble expression by self-induced culture and identify it. Methods The DNA sequence of the N-terminal 1-192 AA region of HBV (type A) polymerase was analyzed by online tools (http://www.jcat.de/) and NdeI And Xho I restriction endonuclease site, sent to Invengit (Shanghai) Trading Co., Ltd. for artificial synthesis; the synthetic TP-DNA double digestion, inserted into the prokaryotic expression vector pET32a (+), the construction of recombinant prokaryotic The expression plasmid was transformed into Escherichia coli BL21 (DE3) pLysS for self-induced expression. Results The prokaryotic expression plasmid pET32a (+) / POL-TP-Opt was confirmed by double enzyme digestion and sequencing. The recombinant protein was expressed specifically in the supernatant of the bacterium. The relative molecular mass was about 20,000, -PAGE and Western blot identification, soluble recombinant TP protein. Conclusions Soluble recombinant TP protein can be directly and successfully obtained by self-induction culture method, which has changed the situation of TP protein-related function relying on the refolding of inclusion body for a long time.