中国兽医科学2013，Vol.43Issue(6)：594-598,5.
结核分枝杆菌rv3668c基因的原核表达及多抗制备
Prokaryotic expression of rv3668c gene from Mycobacterium tuberculosis and preparation of the polyclonal antibody
摘要
Abstract
To construct a recombinant prokaryotic expression vector carrying the rv3668c gene from Mycobacterium tuberculosis and to purify the recombinant protein Rv3668c expressed in Escherichia coli BL21 (DE3),the rv3668c gene was amplified by PCR using M.tuberculosis H37Rv genomic DNA and cloned into pET-30a (+) vector.The recombinant plasmid p30a-68 was transformed into E.coli BL21 (DE3) for expression under induction with IPTG(1 mmol/L).The expressed product was analyzed by SDSPAGE,purified by Ni-NTA His Bind Resin affinity chromatography and then identified by Western-blot.The reaction between the recombinant protein and positive serum from cattle diagnosed as tuberculosis was investigated by Western-blot.The polyclonal antibody against the recombinant protein was prepared by immunizing New Zealand white rabbits with the purified product.In result,restriction analysis proved that the recombinant plasmid p30a-68 was constructed correctly and sequencing results showed that the similarity of the cloned rv3668c gene to that reported in GenBank was 100％.The recombinant Rv3668c protein,with a molecular mass of about 26 ku,was mainly expressed in a soluble form and displayed good immunogenicity.In conclusion,the rv3668c gene can be expressed in E.coli in a soluble form.Polyclonal antibody against the recombinant protein,which can be used for functional study of Rv3668c,has high specificity.关键词
结核分枝杆菌/rv3668c基因/原核表达/免疫原性Key words
Mycobacterium tuberculosis / rv3668c gene/ prokaryotic expression/ immunogenicity分类
农业科技引用本文复制引用
鄢秋龙,陈利苹,刘银冰,曹俊,倪宏波,刘思国..结核分枝杆菌rv3668c基因的原核表达及多抗制备[J].中国兽医科学,2013,43(6):594-598,5.基金项目
国家重点基础研究发展计划(973)项目(2012CB518801) （973）
