PRRSV NJ-a株ORF5基因A表位的修饰与糖基化位点的突变对其DNA疫苗免疫效力的影响

Influence of Epitope A Modification and N-linked Glycosylated Site Mutation of PRRSV NJ-a Strain ORF5 Gene on the Ability to Induce Neutralizing Antibodies and T Cell Proliferation Response

摘要:

为了提高PRRSV ORF5基因的免疫效力，对ORF5基因进行了改造，将CpG序列和通用型辅助性T淋巴细胞表位插入A表位与B表位之间，并对N33与N51位糖基化位点进行了点突变，获得改造的ORF5基因。在此基础上构建了由两个CMV启动子调控的共表达改造的ORF5(MORF5)与ORF6基因的真核表达质粒pcDNA-M5A-6A。经Western-blot与IFA验证真核质粒的体外表达后，免疫6周龄Balb/c小鼠，利用微量中和试验检测免疫后的中和抗体，利用MTT法检测免疫后淋巴细胞的增生情况，并与未改造ORF5基因真核表达质粒pcDNA-5A-6A、弱毒疫苗以及灭活疫苗的免疫效果进行比较。结果表明，pcDNA-M5A-6A不但能够刺激免疫小鼠在较短的时间内产生更高水平的中和抗体，而且可以诱导产生更强烈的T淋巴细胞增殖反应。所构建的共表达PRRSV改造的ORF5基因与ORF6基因的DNA疫苗pcDNA-M5A-6A，能够较好的诱发小鼠产生较高的特异性针对PRRSV的中和抗体和细胞免疫应答，为研究能够更好地防制PRRSV的新型疫苗提供了新的思路。

Abstract:

To enhance the DNA immunogencity of PRRSV ORF5 gene, CpG sequence and the universal helper T cell antigen epitope (PADRE) sequence were inserted between the decoy epitope and the neutralizing epitope. At the same time, site-mutations were introduced at N33 and N51 to diminish the coverage effect to epitope B from the polysaccharides. Subsequently, the modified ORF5 gene (MORF5) and PRRSV ORF6 gene were cloned into the eukaryotic expression vector pcDNA3.0 under the control of two CMV promoters, respectively. With indirect immunofluorescence assay and Western-blot the expression in vitro of the two genes was confirmed, then six-week-old Balb/C mouse were immunized with the modified expression plasmid pcDNA-M5A-6A. The non-modified expression plasmid pcDNA-5A-6A, the blank eukaryotic expression plasmid pcDNA3.0, living attenuated vaccine and inactivated vaccine were used as controls. The PRRSV specific neutralizing antibodies and the T cell proliferation response were elevated with virus neutralization assay and MTT method. Results indicate that the modified plasmid pcDNA-M5A-6A can elicit not only higher titer of neutralizing antibodies in a rapid time, but also more vigorous T cell proliferation response compared with the non-modified plasmid pcDNA-5A-6A and commercial vaccines, indicating that DNA vaccine pcDNA-M5A-6A maybe a promising candidate for PRRS prevention.