绵羊肺腺瘤病毒内蒙毒株囊膜蛋白基因在大肠杆菌中的分段表达

Cloning and Expressing of Capsid Protein Gene of Jaagsiekte Sheep Retrovirus Nei Meng Strain in E.coli

为了建立原核高效表达体系,从自然感染绵羊肺腺瘤病毒内蒙毒株的肺肿瘤组织中提取基因组DNA,应用PCR技术分别扩增编码囊膜蛋白(env)基因的表面蛋白(surface protein,SU)和跨膜蛋白(transmembrane protein,TM)区域基因,通过T-A克隆的方法分别克隆入pGEM-TEasy载体中,然后利用设计的起始密码、终止密码以及相应酶切位点的引物,把env基因的SU区和TM区基因亚克隆到表达载体pGEX-4T-1中,分别构建SU和TM的重组表达质粒,经酶切、PCR及DNA测序鉴定后,将阳性质粒转化入大肠杆菌BL21 CodonPlus中并在IPTG的诱导下表达,表达产物用SDS-PAGE分析鉴定。结果表明,该基因可以在大肠杆菌中以稳定的包涵体形式高效表达,表达的SU和TM融合蛋白表观分子质量约为68 ku和46 ku,表达量分别占全菌蛋白的25%和30%,并且从包涵体分离纯化得到的表达蛋白具有较高纯度,是一种理想的免疫原。

In order to construct surface protein（ SU ） and transmembrane protein（TM ） of envelope gene expression vector that directs the synthesis of protein in E. coli, genome DNA were extracted from lung tumour tissues of sheep with naturally affected pulmonary adenomatosis were amplified with gene - specific primers designed from the JSRV - NM sequences by polymerase chain reaction. The PCR product was cloned into T vector, pGEM - T Easy. Expression con- struction for SU and TM were engineered by inserting corresponding DNA into a pGEX - 4T - 1 vector. The recombinant plasmid was transformed into E. coli BL21 CodonPlus cells, and target protein was induced by IPTG. Samples of the expression product were subjected to SDS - PAGE analysis. The product yield was 68 ku and 46 ku in size. The expression outputs was approximate 25% and 30% of total bacterial proteins, respectively, and ocuurred in the form of inclusion bodies. The fusion protein was purified and obtained high purity of SU and TM fusion protein. The results lay foundation for the development of monoclonal antibody and diagnostic reagents and vaccines against the SU and TM fusion protein of JSRV - NM strain.