狂犬病病毒核蛋白的原核表达及纯化

Prokaryotic Expression and Purification of Nucleoprotein in Rabies Virus CVS Strain

采用RT-PCR方法扩增出狂犬病病毒(RV)CVS株核蛋白(N)基因,并将目的基因亚克隆入原表达载体pET-28a(+)中,经PCR、双酶切以及序列分析,结果表明:已成功构建了重组质粒。将重组质粒转化至大肠埃希氏菌Rossetta(DE3),在37℃,1 mmol/LIPTG条件下进行诱导表达。诱导产物经SDS-PAGE分析,在分子量约为54 KD附近出现较粗的目的带,与预期的目的蛋白分子量相符合。经表达条件优化,用1 mmol/LIPTG在37℃诱导表达5h,表达量达到最高,Western-Blotting鉴定目的蛋白有较强的免疫原性。为狂犬病毒的N蛋白的新型疫苗的制备奠定了基础。

N gene of RV CVS strain was amplified by reverse transcriptase-polymerase chain reaction （RT-PCR）. The product was purified and cloned into the prokaryotic expression vector pET-28 a （＋）, double digestion and sequence analysis were carried after. Results indicated that the recombinant plasmids were successfully obtained. They were transformed into E. coli Rossetta （DE3） competent cells for expression. IPTG was added to final concentration of lmmol/L at 37℃ to induce the expression of N gene. SDS-PAGE was performed to analyze the N production. Results showed that N gene was highly expressed in E. coli, the molecular weight of the protein is 54 KD, which was same as expected. Western- Blotting analysis showed that the expression protein was recognized specifically by rabbit anti-RV polyclonal antibody. This study could provide reference for further study on anti-N protein of rabies virus monoelonal antibodies.