摘要:
[目的]构建猪圆环病毒2型(Porcine circovirus 2,PCV2)ORF2 DNA核酸疫苗,并评价其免疫效果和安全性。[方法]设计一对特异性引物,扩增PCV2-ORF2,利用T载体对其进行克隆,与真核表达载体PcDNA3.1在同等条件下进行双酶切,并进行连接,转化感受态细菌DH5α并克隆,经双酶切鉴定筛选阳性重组质粒,即DNA核酸疫苗,命名为PcDNA3.1-ORF2。将该核酸疫苗瞬时转染Vero细胞,通过RT-PCR鉴定其在细胞内的表达情况;并且将该核酸疫苗按100μg.只-1腿部肌肉注射免疫Balb/c小鼠,同时设PBS和PcDNA3.1空载体为对照,免疫2次,间隔2周。分别于首免后第14 d和第28 d采血,用ELISA方法检测其血清抗体。取首免后56 d小鼠的心、肝、脾、肺、肾和脑等实质器官,采用PCR方法检测该核酸疫苗的安全性。[结果]成功地构建了PcDNA3.1-ORF2核酸疫苗,在体外Vero细胞中得到了瞬时表达,并且能诱导小鼠产生较强的免疫应答。安全性试验表明该核酸疫苗未整合到小鼠染色体上。[结论]PcDNA3.1-ORF2核酸疫苗可以诱导小鼠产生较强的免疫应答,并且是安全的。
Abstract:
This study was to develop ORF2 DNA vaccine against PCV2 and evaluate its immunity effect and safety.[Method] A pair of specific primers was synthesized to amplify the PCV2-ORF2 by PCR.PCV2-ORF2 was cloned into T vector and named T-ORF2.T-ORF2 and PcDNA3.1 were double digested under same conditions,connected by using T4 DNA ligase,and cloned.The positive recombinant plasmids(DNA vaccine) were confirmed by double digestion and named PcDNA3.1-ORF2.The DNA vaccine was transfected in Vero cells with lipofectamine 2000 for RT-PCR analysis.At the same time,100 μg of the vaccine was intramuscular injected into Balb/c mice at two-week intervals.On the 14th and 28th day after the first vaccination,serums were collected for ELISA test.On the 56th day,the genomes of parenchymal organ including heart,liver,spleen,lung and kidney of the vaccinated mice were extracted for PCR amplification to determine safety of the DNA vaccine.[Result] The results indicated that the DNA vaccine was successfully made,PCV2-ORF2 gene was expressed in Vero cells in vitro,and the vaccine did induce immune response in mice.The safety test showed that ORF2 genes could not be integrated into the mice chromosome.[Conclusion] The resultant DNA vaccine successfully induced immune response in mice and was confirmed to be safe.
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