中国人兽共患病学报2017，Vol.33Issue(2)：120-125,6.DOI:10.3969/j.issn.1002-2694.2017.02.006
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 27 kDa cysteine protease gene of Spirometra erinacei plerocercoid
摘要
Abstract
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 27kDaCP gene was amplified by PCR and cloned into pM-19T vector 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.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Key words
Spirometra erinacei/pleroceroid/cysteine protease gene/prokaryotic expression/bioinformatics分类
医药卫生引用本文复制引用
焦梦涵,刘艳丹,陈艳,李金福..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[J].中国人兽共患病学报,2017,33(2):120-125,6.基金项目
Supported by the Science and Technology Department of Guizhou Province (No.2014-3024) （No.2014-3024）
贵州省科技厅社会发展攻关项目(No.20143024)资助 （No.20143024）
