西尼罗河病毒E蛋白结构域Ⅲ的原核表达及间接ELISA方法的建立

Prokaryotic expression of West Nile virus DⅢgene and establishment of indirect ELISA

目的建立西尼罗河病毒(West Nile Virus,WNV)抗体检测方法。方法人工合成了WNV NY99株E蛋白结构域Ⅲ(DⅢ)基因序列,构建了重组表达载体PET-28a-DⅢ。结果重组质粒转化BL21宿主菌后诱导表达,SDS-PAGE分析表明,表达产物相对分子量17kD,以包涵体形式存在。对表达产物作变性和复性及纯化处理,Western-blot鉴定结果表明,纯化产物可被WNV血清识别。将纯化重组蛋白包被酶标板,优化间接ELISA反应条件,抗原最佳包被浓度为3.75μg/mL、血清最佳稀释度为1∶1 600、酶标二抗最佳稀释度1∶6 000、阴阳血清临界值为0.148。特异性、敏感性及重复性试验结果表明,本方法特异性强、敏感性高、重复性良好。结论本研究为WNV感染的血清学诊断提供了技术储备。

E protein structural domain Ⅲ （DⅢ） gene sequences of WNV NY99 strain was synthesized in order to estab- lish the West Nile virus （WNV） antibody detection methods. The gene was cloned into PET-28a to construct recombinant ex- pression vector named PET-28a-DⅢ. The vector was transformated into E. coli BL21 and induced by IPTG. SDS-PAGE anal- ysis showed that the recombinant protein which relative molecular weight was 17 kD existed in the form of inclusion body. Af- ter denaturation, renaturation, and purification of expression products,lthe protein could be combined with specific antibody ac- cording to western-blot result. The purified recombinant protein was Used to coat 96 well plate to establish the indirect-ELISA assay for detecting the antibody against WNV. The optimized concentration to the antigen was 3.75 ug/mL, the detected ser- um dilution was 1 ： 1 600, the concentration of HRP-Goat anti-rabbit IgG was 1 ： 6 000, and cut-off value was 0. 148. The re- sults of specificity, sensitivity and repeat test to evaluate the indirect-ELISA detection system were satisfactory. This study supplied the technique preparation for diagnosis of WNV infection.