摘要
采用RT-PCR技术扩增禽呼肠孤病毒(ARV)S1133毒株和广西分离株R 1的2σ基因。将2σ基因克隆至PGEX-4T-1载体上,测序结果表明,插入的片段为2σ目的基因。切下目的基因2σ重组到含有谷胱甘肽(G ST)的融合蛋白质原核表达载体PGEX-4T-1,获得重组质粒。经PCR、酶切以及序列分析鉴定,表达2σ基因插入的位置、大小和读码框架均正确,表明成功构建了融合表达载体PGEX-S1133-2σ和PGEX-R 1-2σ。构建好的重组质粒,在大肠杆菌JM 109α中经1 mm o l/L IPTG诱导得到了表达。融合蛋白G ST-2σ和G ST R 1-2σ的相对分子质量为65 100,以包涵体形式存在。W estern-b lot分析表明,融合蛋白能够与ARV阳性血清发生特异性反应,表明该重组蛋白具有良好的反应原性。
The σ2 gene of ARV Sl133 ＆ R1 isolates in Guangxi province were amplified by reverse transcription chain reaction（RT-PCR）. The σ2 gene was inserted into the bacterial plasmid PGEX-4T-1 vector and then sequenced. It showed that the insert fragment was the σ2 gene of ARV. The σ2 gene was inserted into the bacterial plasmid PGEX-4T-1 and the recombinant plasmid containing σ2 gene were indentified by restriction enzyme analysis and PCR method. It indicated that fusion expression vector PGEX-S1133-σ2 ＆. PGEX-RI-σ2 was constructed. The recombinant fusion protein was highly expressed in E. coli JM 109 induced by 1 mmol/L IPTG in the form of inclusion bodies. The molecular weight of GST-σ2 ＆ GST R1-σ2 is 65 100. Westernblot analysis with ARV antibodies against the fusion protein showed the recombinant protein has a good reactivity.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2006年第2期133-135,共3页
Chinese Journal of Veterinary Science
基金
广西留学回国人员基金项目(桂科回0144014
0342006)
广西科技攻关项目(0235001-4
桂科自0542034)
