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。
Objective To clone and express the EgM123 gene of Echinococcus granulosus（Eg）in E.coli. MethodsPrimers specific for EgM123 were designed in accordance with the nucleotide sequence published in GenBank,and the plasmid pET41b-EgM123 with a GST tag was used as a template to amplify the gene with PCR.The amplified fragment was inserted into a pET28 aplasmid vector to construct the recombinant plasmid pET28-EgM123 with 6His as a tag.The plasmid was transformed into E.coil BL21.The purified plasmid was sent to a company for sequencing.The transformed E.coli was cultured and expression of the recombinant protein was induced with IPTG.The expressed protein was analyzed using SDS-PAGE and Western blotting. Results The EgM123 gene is a 594-bp fragment encoding 198 amino acids.Sequencing indicated that the amplified gene fragment has 99.83% similarity to the published nucleotide sequence and 100% similarity to the published protein sequence.SDS-PAGE analysis indicated that E.coli expressed a 29-ku protein.Western blot analysis indicated that the expressed EgM123 protein was recognized by anti-EgM123-GST-specific rabbit polyclonal antibody. Conclusion The EgM123 gene was cloned and expressed in E.coli.The expressed protein specifically binds to anti-EgM123 antibodies,providing a basis for further study of vaccines against echinococcosis.
作者
王慧
齐文静
何黎
郭宝平
张文宝
李军
WANG Hui QI Wen-jing HE Li GUO Bao-ping ZHANG Wen-bao LI Jun(Base to Foster a State Key Lab for Major Disease Research in Xinjiang and Xinjiang Key Laboratory of Echinococcosis, First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Chin)
出处
《中国病原生物学杂志》
CSCD
北大核心
2017年第1期38-41,共4页
Journal of Pathogen Biology
基金
国家自然科学基金项目(No.U1303203)
