摘要
采用RT_PCR技术扩增禽流感病毒HA1基因,将HA1结构基因克隆于pGEX_T载体上,测序结果表明插入的片段为HA1目的基因。切下目的基因HA1,重组到含有谷胱甘肽(GST)的融合蛋白原核表达载体PGEX_4T_2,获得的重组质粒经PCR、酶切以及序列分析鉴定,表明HA1基因插入的位置、大小和读码框架均正确。证明成功构建了融合表达载体pGEX_HA1。构建好的重组质粒,经1mmol/LIPTG诱导,在大肠杆菌BL21中得到了大量表达,经过4h表达量既达到高峰。融合蛋白GST_HA1的分子量为62KD,以包涵体形式存在。Western_Blot分析表明,融合蛋白能够与H5亚型AIV阳性血清发生特异性反应,表明该重组蛋白具有良好的抗原性和特异性。
The HA1 genes of AIV were cloned by reverse transcription polymerase chain reaction (RT-PCR). The HA1 gene was inserted into the pGEM-T vector and then sequenced. It showed that the insert fragment was the HA1 gene of AIV. The HA1 gene was inserted into the bacterial plasmid pGEX-4T-2 and the recombinant plasmids containing HA1 gene were identified by restriction enzyme analysis and PCR method. The recombinant fusion protein was highly expressed in E.coli BL21 four hours later induced by 1mmol/L IPTG in the form of inclusion bodies. The molecular weight of GST-HA1 is 62KD. Western-blot analysis with AIV antibodies against H5 subtype showed the recombinant protein shared good antigencity and specificity.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2004年第3期173-176,共4页
Chinese Journal of Preventive Veterinary Medicine