摘要
采用RT～PCR技术扩增禽流感病毒M1基因，将M1结构基因克隆于pMDl8一T载体。测序结果表明，插入的片段为M1目的基因。切下目的基因M1，重组到含有谷胱苷肽（GST）的融合蛋白原核表达载体pET一32a（＋），获得的重组质粒经PCR、酶切以及序列分析鉴定表明，M1基因插入的位置、大小和读码框架均正确，证明成功构建融合表达载体pET32aM1。构建好的重组质粒经1mmol／LIPTG诱导，在大肠杆菌BI，21中得到大量表达，经过6h表达量达到高峰。融合蛋白M1的分子质量为29．8ku，以包涵体形式存在。Western—Blot及ELISA分析表明，融合蛋白能够与M1单克隆抗体发生特异性反应，说明该重组蛋白具有良好的抗原性和特异性。
The M1 gene of AIV was cloned by reverse transcription polymerase chain reaction （RT PCR）. The amplified fragment was cloned into the pMD18-T vector and then confirmed that it was the M1 gene of AIV by sequencing. The M1 gene was sub-cloned into the bacterial expression plasmid Pet32a（q-） and the recombinant plasmids containing M1 gene were confirmed by restriction enzyme digestion and PCR method. After six hours inducing with lmmol/L IPTG, the recombinant fusion protein was highly expressed in t3. coli BL21 in the form of inclusion bodies, The molecular weight of M1 is 29. 8 ku, Western-blot analysis showed the recombinant protein shared good antigencity and specificity.
出处
《西北农业学报》
CAS
CSCD
北大核心
2012年第1期26-29,共4页
Acta Agriculturae Boreali-occidentalis Sinica
关键词
禽流感病毒
M1基因
原核表达
Avian influenza M1 gene Prokaryotic expression
