草鱼呼肠孤病毒GCRV-GD108株VP5蛋白的原核表达

Prokaryotic expression of GCRV-GD108 VP5 protein

根据草鱼呼肠孤病毒广东株GCRV-GD108编码VP5的M5基因的cDNA序列,分别设计合成带特定酶切位点的特异引物,进行PCR扩增;通过酶切与连接,构建2种重组表达载体pET30c-M5和pColdⅡ-M5,分别转化于大肠杆菌并进行诱导表达;对培养基、诱导时间、诱导剂浓度和温度等条件进行优化,确定最适表达体系。通过SDS-PAGE和Western blot对表达产物进行鉴定。结果显示,所构建的2种VP5蛋白原核表达载体,均在大肠杆菌中成功表达了分子质量大小(约80ku)与预期相符的重组蛋白,表达产物主要存在于包涵体中。诱导表达后的菌体经离心、洗涤与超声破碎,离心收集包涵体。包涵体通过变性、透析与复性,获得纯化的重组蛋白,其中pET30c-M5/BL21(DE3)工程菌的目的蛋白占全菌蛋白的22.5%,纯化后获得了高纯度的重组蛋白(>95%)。比较2种表达载体的表达结果,pET30c-M5具有诱导表达耗时少、获得重组蛋白量大的优势,可为下一步的研究提供足量的蛋白。

Grass carp reovirus（GCRV） has a genome consisting of 11 double-stranded RNA,and possesses several structural proteins.The VP5 protein is one of the structural proteins in the core capsid encoded by the M5 gene of GCRV-GD108,a reovirus strain which was isolated from grass carp cultured in Guangdong and had been identified by our lab recently.Primers with restriction enzyme digesting sites were designed based on the cDNA of M5 gene.The amplification products of M5 gene were digested and cloned into prokaryotic expression vectors.Two recombinant protein expression vectors,pET30c-M5 and pColdⅡ-M5,which had been identified by PCR,enzyme digestion and sequence analysis,were transformed into E.coli strains,respectively.Then the recombinant expression strains were induced.The effects of culture medium,induction time,temperature and IPTG levels were analyzed,and suitable conditions for the expression of the target protein were proposed.The results of SDS-PAGE and Western blot showed that the target protein VP5 with molecular weight around 80 ku was obtained,but the recombinant protein was mainly presented in inclusion bodies.The inclusion bodies were then denatured,dialyzed and renatured to get the recombinant protein.The target protein,which was expressed by pET30c-M5/BL21（DE3） accounted for 22.5% of the total bacteria proteins,and was highly purified（95.0%）.The pET30c-M5 is considered to be an engineering strain suitable for large scale expression for it cost less time of inducing and got higher production than pColdⅡ-M5.