细粒棘球绦虫成虫相关基因EgM123的克

发布时间：2018-01-19 21:16

  本文关键词： 包虫病 细粒棘球绦虫 EgM 蛋白表达 疫苗　出处：《中国病原生物学杂志》2017年01期 　论文类型：期刊论文

【摘要】：目的构建细粒棘球绦虫成虫相关基因EgM123基因原核表达载体,诱导表达并鉴定EgM123蛋白。方法提取pET41b-EgM123重组质粒,以此为模板PCR扩增EgM123基因片段,克隆至原核表达载体pET28a中,经限制性内切酶双酶切和测序鉴定。将重组菌质粒转化大肠埃希菌BL21,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导目的基因表达,采用SDS-PAGE对表达蛋白进行分析,Western blot鉴定其免疫反应性。结果 PCR扩增EgM123基因片段长度为594bp,经酶切分析和测序鉴定证明pET28a-EgM123原核表达载体构建正确。重组质粒转化BL21在37℃条件下,经IPTG(终浓度为0.4mmol/L)诱导3h,获得分子质量单位约为29ku的EgM123蛋白,与理论值相符。该蛋白能被EgM123-GST免疫兔抗血清识别。结论成功构建了pET28a-EgM123原核表达载体,表达蛋白具有免疫反应性,为研制包虫病疫苗奠定了基础。
[Abstract]:Objective to construct the prokaryotic expression vector of the EgM123 gene associated with Echinococcus granulosus and to express and identify the EgM123 protein. Methods the recombinant plasmid of pET41b-EgM123 was extracted. The EgM123 gene fragment was amplified by PCR and cloned into prokaryotic expression vector pET28a. The recombinant plasmid was transformed into Escherichia coli BL21 and induced by isopropyl- 尾 -D- thiogalactoside (IPTG). The immunoreactivity of the expressed protein was identified by SDS-PAGE and Western blot. Results the length of EgM123 gene fragment amplified by PCR was 594bp. The construction of pET28a-EgM123 prokaryotic expression vector was confirmed by restriction endonuclease analysis and sequencing. The recombinant plasmid was transformed into BL21 at 37 鈩，

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