登革病毒1型E蛋白的原核细胞表达及多克隆抗体的制备

Prokaryotic expression of E protein of dengue virus type 1 and preparation of polyclonal antibody

目的构建登革病毒1型(DENV-1)E蛋白的原核细胞表达载体并进行原核细胞表达,并制备其多克隆抗体。方法利用聚合酶链反应(PCR)扩增DENV-1E蛋白序列,经酶切后将其插入原核细胞表达载体pET-32a(+),构建重组质粒pET-32-D1-E。重组质粒转化E.Coli Rosetta(DE3)感受态细胞,目的蛋白经异丙基-D-硫代半乳糖苷(IPTG)诱导表达,纯化后采用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)初步分析蛋白表达情况,并用纯化后的表达蛋白免疫家兔,制备该蛋白的多克隆抗血清,用间接酶联免疫吸附测定(ELISA)检测抗体的效价,Western blot检测抗体的特异性。结果成功构建了pET-32-D1-E原核细胞表达重组质粒,DENV-1E蛋白获得高效表达;纯化后的重组蛋白免疫家兔获得的特异性抗血清效价为1∶25 600,Western blot证实其特异性良好。结论成功构建了DENV-1E蛋白的原核细胞表达载体,并制备了可用于DENV快速检测的高效多克隆抗体。

Objective To construct prokaryotic expression vector for E protein of dengue virus type 1 （DENV-1） and induce its expression and prepare its polyclonal antibody.Methods Polymerase chain reaction （PCR） was used to amplify DENV-1Eprotein sequences which were inserted into prokaryotic expression vector pET-32a （＋） after restriction enzyme digestion.Recombinant plasmid pET-32-D1-E was constructed and were transformed into E.Coli Rosetta （DE3） competent cells.The target protein was in- duced to expression by isopropyl-beta-D-thiogalactopyranoside （IPTG）and preliminarily analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE） after purification.The purified expression protein was adopted to immunize rabbits in order to prepare its polyclonal antibody.Enzyme-linked immunosorbent assay （ELISA） was employed to detect the antibody titer and Western blot to test its specificity.Results Recombinant plasmid pET-32-D1-E plasmid expression in prokaryocyte was suc- cessfully constructed and DENV-1 Eprotein was highly expressed.The titer of the polyclonal antibody obtained by immunization was 1∶25 600 and its high specificity was confirmed by Western blot.Conclusion Prokaryotic expression vector of DENV-1 E protein has been successfully constructed and efficient polyclonal antibody for rapid DENV detection has been prepared.