狂犬病病毒核衣壳蛋白的原核表达与鉴定

Prokaryotic expression and identification of rabies virus nucleocapsid protein

为实现狂犬病病毒核衣壳蛋白在原核系统中高效表达,参考GenBank中发布的RV N基因序列(登录号为1489853),在不改变RV N蛋白氨基酸序列的情况下,对该基因的密码子进行优化,化学合成N基因,并将其克隆至原核表达载体pET28a(+)中,构建重组质粒pET28a(+)/RV N,将该质粒转化至大肠杆菌BL21(DE3)感受态细胞中进行诱导表达,利用His-tag镍柱进行重组RV N蛋白的纯化,对纯化后的RV N蛋白进行SDS-PAGE鉴定及Western blot分析。结果表明:重组质粒双酶切后在1359 bp处有目的条带,经测序后与其所对应的已知基因序列完全相符,当重组菌在IPTG浓度0.1mmol/L、30℃诱导6 h,RV N蛋白表达量最高;经过镍柱纯化获得了RV N重组蛋白;BCA法测得重组RV N蛋白浓度达1.783 mg/mL;在51KD处可见明显的蛋白印迹,与预期目的条带相符;使用该方法成功在大肠杆菌系统高效表达了RV N蛋白,为后续狂犬病的检测与新型疫苗的研制奠定了基础。

In order to realize efficient expression of nucleoprotein(N)from rabies virus(RV)in the prokaryotic system,this paper refers to the RV N gene sequence published by GenBank(accession number 1489853).Without changing the amino acid sequence of the RV N protein,the codon of the gene is optimized,and the N gene is chemically synthesized and cloned into the prokaryotic expression vector pET28a(+).The recombinant plasmid pET28a(+)/RV N is constructed and transformed into E.coli BL21(DE3)competent cells for expression.The recombinant RV N protein is purified by His-tag nickel column.The purified RV N protein is then identified by SDS-PAGE and analyzed by Western blot.The results show that the recombinant plasmid is identified by double enzyme digestion,and a band appears at 1359 bp,which is completely consistent with the corresponding known gene sequence after sequence tests.When the recombinant bacteria are induced in an IPTG concentration of 0.1 mmol/L at 30℃for 6 hours,the RV N protein expression is the highest.The RV N recombinant protein is obtained through the purification of the nickel column.The concentration of the recombinant RV N protein measured by BCA method is 1.783 mg/mL.An obvious western blot appears at 51 kD,which is consistent with the expected target band.It shows that RV N recombinant protein is successfully expressed in E.coli BL21(DE3)competent cells,which lays a foundation for the subsequent rabies detection and the development of a new vaccine.