猪2型圆环病毒核酸疫苗免疫效应研究

Study on the Nucleotide Vaccine Immune Effect of Porcine Circovirus Type 2 Virus

PCR扩增PCV2 ORF2和BVP22基因。将ORF2基因克隆入真核表达载体pCDNA3.1(+),构建了pCORF2作为核酸疫苗免疫小白鼠。同时将具有蛋白转导功能的BVP22基因分别克隆到ORF2基因的上游和下游,使二者融合表达,命名为pCORF2BVP22和pCBVP22ORF2。分4组肌肉免疫BALB/c小白鼠,分别注射pCD-NA3.1(+)、pCORF2、pCORF2BVP22和pCBVP22ORF2,共免疫2次,间隔2周,分别于首免后2周和二免后4周采血,ELISA法检测体液抗体。同时为在体外验证ORF2和BVP22基因的真核表达,将ORF2和BVP22基因分别克隆入pEGFP-N1载体,构建了pNORF2和pNBVP22,转染Hela细胞后在荧光显微镜下观察到了ORF2和BVP22在体外的瞬时高效表达。结果显示,通过两次免疫后,各疫苗组均产生了针对ORF2的体液抗体,同时证明BVP22可增强核酸疫苗的免疫效果,其中以BVP22在ORF2上游效果更好。本研究为防制猪2型圆环病毒感染提供了较为理想的侯选疫苗。

The ORF2 of porcine circovirus type 2 and BVP22 of bovine herpesvirus were amplified by PCR. The cloned ORF2 gene was inserted into eukaryotic expression vector pCDNA3.1（＋）for constructing nucleotide vaccine vector pCORF2. Then nucleotide vaccine vectors which express BVP22-ORF2 fusion protein were constructed by inserting BVP22 gene, which has the function of protein transduction into pCORF2 at the upstream or downstream of the ORF2 gene respectively. All the above vaccines were intramuscular injected to BALB/c mice for twice at two weeks interval. 2 and 6 weeks after the first immune injection, serums were collected and the humoral antibodies were tested by ELISA. The result of ELISA showed that all the experimental groups produced anti-ORF2 antibodies, and high antibody level showed four weeks after the second immune injection. Results also showed that BVP22 could enhance the efficiency of ORF2 nucleotide vaccine. Our research provided a candidated vaccine for controlling PCV2 infection.