摘要
针对猪脑心肌炎病毒(EMCV)非结构蛋白2C基因设计合成了1对特异性引物,通过RT-PCR扩增得到了2C基因片段。将2C基因定向克隆入原核表达载体pGEX-6P-1中,构建了重组原核表达质粒pGEX-6P-2C,并将其转化大肠杆菌BL21感受态细胞,经用0.1mmol/LIPTG诱导,成功表达了62ku的目的重组蛋白。Western-blot分析表明,表达的2C重组蛋白可以分别被猪源或兔源抗EMCV阳性血清识别;以浓度为0.5μg/mL的重组蛋白包被聚苯乙烯微量反应板,能够特异性地检测到猪抗EMCV抗体。证明表达的EMCV重组非结构蛋白2C具有良好的反应原性,可以替代EMCV全病毒进行安全、特异、敏感的基于重组诊断抗原的猪脑心肌炎血清学检测技术研究。
A pair of specific primer for nonstructural protein 2C gene of porcine encephalomyocarditis virus（EMCV） was designed and synthesized. 2C nucleotide sequence was amplified by RT-PCR and cloned into prokaryotic expression vector pGEX-6P 1 to construct recombinant expression plasmid pGEX-6P-2C. The plasmid pGEX-6P-2C was transformed into competent cells of Escherichia coli BL21. The predicted recombinant nonstructural protein 2C of 62 ku was expressed in E. coli BL21 cells after being induced by 0.1 mmol/L IPTG. Western-blotting showed that the recombinant protein 2C could be recognized by positive swine or rabbit anti-serum to EMCV. Special antibody in swine serum was detected with 0.5 μg/mL protein 2C as an antigen for coating polystyrene microreaction plate. The results demonstrated that EMCV recombinant nonstructural protein 2C was of credible reactogenicity and could replace the whole EMCV as diagnostic antigen,which may lay a foundation for the establishment of a safe, specific and sensitive serological technique for detection of porcine encephalomyocarditis based on the recombinant diagnostic antigen.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2008年第9期772-776,共5页
Chinese Veterinary Science
基金
河北农业大学校长基金项目(200511)
关键词
猪
脑心肌炎病毒
非结构蛋白2C
原核表达
鉴定
反应原性
porcine
encephalomyocarditis virus
nonstructural protein 2C
prokaryotic expression identification
reactogenicit y
