含CpG序列PRRSV ORF5基因真核质粒构建及其免疫原性的研究

Study on Construction and Immunogenicity of Eukaryotic Expression Vector Containing PRRSV ORF5 and CpG Motifs

【目的】提高猪繁殖与呼吸综合征病毒(PRRSV)基因免疫的效果,构建含CpG基序和PRRSVORF5的真核重组表达质粒CpG-pVAX1-ORF5。【方法】经酶切鉴定和序列测定,将重组质粒转染COS-7细胞,经间接免疫荧光试验证实CpG-pVAX1-ORF5可表达PRRSVGP5蛋白。免疫SPF小鼠,检测鼠的特异性抗体滴度、脾T淋巴细胞亚群数量(CD4+和CD8+)以及淋巴细胞增殖反应(MTT法)水平。【结果】本试验构建的含CpG基序真核重组表达质粒CpG-pVAX1-ORF5能诱导小鼠产生针对PRRSV的体液免疫与细胞免疫。【结论】CpG-ODN可提高PRRSV基因疫苗的免疫效果。

[Objective] To improve the effect of the gene immunization against porcine reproductive and respiratory syndrome virus （PRRSV）, the eukaryotic expression vector CpG-pVAX1- ORF5 containing PRRSV ORF5 gene coding region and CpG motifs were constructed. [ Method ] After identified by enzyme analysis and nucleotide sequencing test, the recombinant expression vector plasmid CpG-pVAX1-ORF5 was transfected into COS-7 cells. The transient expression of PRRSV ORF5 nucleocapsid protein was detected by indirect immunofluorescence assay （IFA）. SPF mice were inoculated with the recombinant expression plasmid. The PRRSV antibody in serum, the number of CD4^＋, CD8^＋ and the lymphocyte proliferation test （MTT） in peripheral blood of mice vaccinated were detected. [ Result ] The recombinant plasmid CpG-pVAX 1- ORF5 could induce higher response of cellular immune response and specific humoral immune responses in mice. [Conclusion] CpG-ODN could significantly enhance the immune effect of PRRSV gene vaccine.