猬裂头蚴27kD半胱氨酸蛋白酶基因的克隆、表达及生物信息学分析

Cloning,expression and bioinformatics analysis of 27kDa cysteine protease gene of Spirometra erinacei plerocercoid

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。

To clone and express 27 kDa cysteine protease（CP）gene of Spirometra erinacei plerocercoid,and analyze the biology characteristics,a total of RNA was extracted from the plerocercoids and reversely transcribed into cDNA.The 27 kDaCP gene was amplified by PCR and cloned into pM-19 Tvector for sequencing.The accurate sequence was subcloned into the expression vector pET-28a（＋）.The recombinant plasmid was transformed into Transetta（DE3）and the expression protein induced by IPTG.The recombinant protein was purified by Ni2＋ affinity chromatography,and analyzed by SDS-PAGE and Western blotting.The 27 kDa CP gene and its expression protein were predicted and analyzed by bioinformatics analysis tools such as NCBI and ExPASy.Results showed that the ORF length of 27 kDa CP gene sequence was 1 011 bp,and the removed signal peptide sequence was 954 bp with the submission number of ANA52569 in GenBank.The whole sequence of 27 kDa CP（Mr 35 669.9,pI 5.92）was 317 amino acids conferred from cDNA,which belongs to the Peptidase_C39_like superfamily.The pET-28a（＋）-27kDa-CP was expressed under the induction of IPTG.Western blotting analysis showed that the purified protein reach expectancy,and had better response with positive serum of Spirometra erinacei plerocercoid infection.In conclusion,the 27 kDa CP gene of Spirometra erinacei plerocercoid is successfully cloned and expressed and knowing coded sequences and bioinformatic.