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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 of dengue virus type 1 (DENV-1) E protein and express it in prokaryotic cells and prepare its polyclonal antibody. Methods The DENV-1E protein sequence was amplified by polymerase chain reaction (PCR) and inserted into prokaryotic expression vector pET-32a (+) to construct recombinant plasmid pET-32-D1-E. Recombinant plasmids were transformed into E.coli Rosetta (DE3) competent cells. The target protein was induced by isopropyl-D-thiogalactoside (IPTG) and purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS-PAGE), and the purified protein was used to immunize rabbits to prepare polyclonal antiserum. The antibody titer was detected by indirect enzyme-linked immunosorbent assay (ELISA) Specificity. Results The recombinant plasmid pET-32-D1-E was successfully constructed and the expression of DENV-1E protein was highly expressed. The titer of specific antisera raised by purified recombinant protein was 1:25 600, which was confirmed by Western blot Its specificity is good. Conclusion The prokaryotic expression vector of DENV-1E protein was successfully constructed and a highly efficient polyclonal antibody for rapid detection of DENV was prepared.