兔出血症病毒VP60基因真核表达质粒的构建及初步评价

Development of VP60 gene vaccine against RHDV and immunity test in mice

利用pMD18-T-VP60质粒(包含RHDV-TP株VP60基因)PCR扩增并酶切回收VP60基因片段,插入到真核表达载体pcDNA3.1(+)中,构建真核表达质粒pcDNA-VP60,对重组质粒进行酶切和PCR鉴定;将重组质粒转染RK13细胞,进行间接免疫荧光和Western-blot检测;将构建好的重组质粒作为核酸疫苗,腿部肌肉注射免疫小鼠,采用间接ELISA法检测抗体水平;采用淋巴细胞增殖试验(MTT法)和流式细胞术(FACS)检测细胞免疫情况;采用PCR方法检测重组质粒在小鼠体内的生物安全性。结果显示,成功构建pcDNA-VP60真核表达质粒,并在RK13细胞中获得表达。重组质粒作为核酸疫苗能够诱导小鼠体内产生特异性抗体而且抗体水平随免疫次数增加而逐渐增高,在免疫后49d时达到峰值;脾淋巴细胞刺激指数和CD4+、CD8+淋巴细胞亚群数量百分率明显增高,且与对照组存在明显差异。经PCR鉴定VP60基因未整合到小鼠主要脏器染色体上。结果表明,构建的pcDNA-VP60真核表达质粒能够诱导小鼠产生较强的体液免疫和细胞免疫应答,重组质粒对小鼠是安全的。

The VP60 gene of RHDV was inserted into the eukaryotic expression vector pcDNA3.1 （＋） to construct nucleotide vaccine plasmid pcDNA-VP60. The nucleotide vaccine plasmid identified by PCR and digestion was trans- fected into RK13 cells for the analysis of immunofluorescence and Western blot. The constructed pcDNA VP60 was developed into nucleotide vaccine to intramuscularly inject into the mice. The serum were collected to test the anti- body level by ELISA. And cell mediated immunity was detected by MTT method and fluorescence activated cell sort- er （FACS）. To confirm the safety of pcDNA-VP60 gene vaccines,the genomes of vaccinated mice were extracted for PCR amplification. The result indicated that the nucleotide vaccine plasmid pcDNA-VP60 was constructed success- fully and the VP60 gene was expressed in RK13 cells,and pcDNA VP60 gene vaccines successfully induced humoral and cellular immune response in mice. The safety test of pcDNA-VP60 gene vaccine showed that VP60 genes could not be integrated into the mice chromosome and it was safe. The pcDNA VP60 gene vaccine successfully constructed and induced immune response in mice and was safe in mice.