猬裂头蚴27kD半胱氨酸蛋白酶基因的克

发布时间：2018-02-11 19:16

本文关键词： 猬迭宫绦虫裂头蚴半胱氨酸蛋白酶基因原核表达生物信息学　出处：《中国人兽共患病学报》2017年02期 　论文类型：期刊论文

【摘要】：目的克隆、表达猬裂头蚴27kDa半胱氨酸蛋白酶(Cysteine protease,CP)基因,并分析其生物学特性。方法提取蛇源猬裂头蚴总RNA,逆转录合成cDNA,PCR扩增27kDa半胱氨酸蛋白酶(27kDa CP)基因,克隆入pMD-19T载体,测序正确后将27kDa-CP基因亚克隆入表达载体pET-28a(+),构建pET-28a(+)-27kDa-CP重组质粒,转入大肠埃希菌Transetta(DE3)中,经IPTG诱导,表达目的蛋白27kDa CP。用镍离子柱亲和层析法纯化目的蛋白。表达产物用SDS-PAGE及Western Blotting进行分析,并运用NCBI和ExPASy等有关的生物信息学分析工具,对27CP基因及其编码蛋白进行预测和分析。结果 CP基因序列的开放阅读框长为1 011bp,去除信号肽序列长954bp,登录到GenBank,获得登录号ANA52569。该基因编码一个含317个氨基酸的蛋白多肽,属于Peptidase_C39_like超家族,理论分子量约35 669.9Da,等电点5.92。pET-28a(+)-27kDa-CP重组质粒经IPTG诱导后,蛋白在大肠埃希菌中成功表达,经纯化后的蛋白利用Western blotting检测,证明与预期大小相符,且与猬裂头蚴感染阳性血清有较好的结合反应。结论成功克隆、表达了猬裂头蚴27kDa CP基因,获知CP基因及其编码的氨基酸序列和生物信息学。
[Abstract]:Objective to clone and express the 27kDa cysteine protease (CPN) gene and analyze its biological characteristics. Methods the 27kDa cysteine protease 27kDa CPG gene was amplified by reverse transcription synthesis and cloned into pMD-19T vector. After sequencing correctly, the 27kDa-CP gene was subcloned into the expression vector pET-28a (pET-28a). The recombinant plasmid pET-28a (pET-28a CP) was transformed into E. coli TransettaDa-DE3 and induced by IPTG. The expressed protein was purified by nickel ion column affinity chromatography. The expressed protein was analyzed by SDS-PAGE and Western Blotting, and the bioinformatics analysis tools such as NCBI and ExPASy were used. Results the open reading frame length of CP gene sequence was 1 011bp. the length of signal removing peptide sequence was 954bp. the gene was registered to GenBankand obtained the accession number Ana52569. the gene encodes a protein polypeptide containing 317 amino acids. It belongs to the Peptidase_C39_like superfamily, the theoretical molecular weight is about 35669.9Da. the isoelectric point is 5.92.pET-28a. after the recombinant plasmid was induced by IPTG, the protein was successfully expressed in Escherichia coli. The purified protein was detected by Western blotting and proved to be in accordance with the expected size. Conclusion the 27kDa CP gene was cloned successfully and the amino acid sequence and bioinformatics of CP gene and its encoding were obtained.
【作者单位】：贵州医科大学人体寄生虫学教研室;
【基金】：贵州省科技厅社会发展攻关项目(No.20143024)资助~~
【分类号】：R383

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