猪口蹄疫O型合成肽疫苗抗原多肽的串联表达及活性鉴定  

Tandem Expression and Activity Analysis of Antigen Peptide of Swine Foot and Mouth Disease(Type O) Synthetic Peptide Vaccine

作　　者：卢清侠[1] 郭军庆[1] 郝慧芳[1] 杨苏珍[1] 邢广旭[1] 杨继飞[1] 王世红[1] 张改平[1] 

机构地区：[1]河南省农业科学院农业部动物免疫学重点实验室/河南省动物免疫学重点实验室,河南郑州450002

出　　处：《河南农业科学》2011年第10期126-130,共5页Journal of Henan Agricultural Sciences

基　　金：国家"863"计划(2007AA100606);中国博士后科学基金面上资助项目(20110491000)

摘　　要：为获得含合成肽疫苗抗原多肽的重组表达蛋白,根据猪口蹄疫O型合成肽疫苗中抗原多肽的氨基酸序列,使用大肠杆菌偏好性密码子人工合成该抗原多肽的双拷贝基因序列,并克隆至表达载体pET32a中,构建原核表达质粒pET32a-P2。将重组质粒转化BL21(DE3),经IPTG诱导表达,收集诱导的菌液进行SDS-PAGE电泳和Western-blot分析。结果显示,在分子量27 kD处有1条明显的蛋白表达条带,且能被O型FMDV阳性血清识别。对表达产物进行可溶性分析,结果显示,表达蛋白几乎全部以可溶形式存在。表达蛋白经Ni-NTA树脂亲和纯化,SDS-PAGE显示纯化的目的蛋白在27 kD处有单一目的条带,具有较高的纯度。ELISA检测结果显示,纯化的重组蛋白具有较高的活性。结果表明,该抗原多肽的双拷贝串联重复序列在大肠杆菌中得到了可溶性高效表达,为开发诊断试剂和基因工程疫苗奠定了基础。In order to get the recombination protein which contained antigen peptide of synthetic peptide vaccine,two-copy gene was synthesized using E.coli optimal codons according to the amino acid sequence of antigen peptide.The recombinant expression vector pET32a-P2 was constructed by cloning two-copy gene of antigen peptide into the prokaryotic expression plasmid pET32a.The BL21（DE3） was transformed with pET32a-P2,and the protein corresponding to P2 gene was expressed after induction with IPTG.SDS-PAGE and Western-blot with the product of bacterial culture induction with IPTG showed that the molecular mass of the protein was approximately 27 kD,which was identified by positive sera of FMDV serotype O.The soluble analysis showed that the recombination protein was almost solubly expressed.The protein was purified by Ni-NTA resins and assayed by ELISA,and the results showed that it demonstrated high purity and activity.So the recombination protein which contained antigen peptide of synthetic peptide vaccine was expressed solubly and efficiently in E.coli,and the purified protein could be developed asdiagnosis antigen or vaccine.

关 键 词：猪口蹄疫 合成肽疫苗 抗原多肽 串联表达 蛋白纯化 活性鉴定 

分 类 号：S852.65[农业科学—基础兽医学]