摘要
为了获得表达猪细小病毒(PPV)VP2蛋白和细胞因子猪白细胞介素-18(IL-18)的重组猪伪狂犬病毒(PRV)。将PPV VP2基因和猪IL-18基因分别插入到PRV转移质粒PG中,得到重组质粒PG18-VP2。利用脂质体转染法将重组转移质粒PG18-VP2与猪PRV弱毒株DNA共转染猪睾丸(ST)细胞,以EGFP荧光标记,通过5轮蚀斑筛选纯化,成功获得表达PPV VP2蛋白和猪IL-18且带EGFP标记的重组伪狂犬病毒IL18-VP2-rPRV。用重组病毒感染ST细胞,12h后在荧光显微镜下可见明亮的绿色荧光;通过RT-PCR证实感染细胞中含有PPV VP2和猪IL-18mRNA;Western-blot试验结果显示,重组病毒能表达具有生物活性的PPV VP2蛋白和猪IL-18。重组病毒经连续20次传代后感染细胞仍能发出绿色荧光,PCR检测表明VP2及IL-18基因在重组病毒中能稳定遗传。为进一步研究表达PPV VP2蛋白和猪IL-18的重组猪伪狂犬病毒的免疫效力奠定了基础。
To obtain one recombinant pseudorabies virus （PRV） which co-expresses VP2 protein of porcine parvovirus （PPV） and porcine interleukin-18 （IL-18）, the porcine IL-18 gene and PPV VP2 gene were inserted into the PRV transferring plasmid PG respectively,obtaining the recombi- nant plasmid PG18-VP2. Then the plasmid PG18-VP2 and porcine PRV attenuated strain DNA were co-transfected into swine testicle （ST） cell by using lipofectamine. By selection of EGFP green fluorescence plaques on the ST cells overlaid with agar for five times, purified recombinant PRV （IL18-VP2-rPRV） was successfully obtained, which co-expressed porcine PPV VP2 protein and porcine IL-18. Bright green fluorescence infected ST cells can be observed using fluorescent microscope after ST cells were infected with the recombinant virus IL18-VP2-rPRV for 12 h. PPV VP2 and porcine IL-18 mRNA in the infected cells were confirmed by RT-PCR;Western-blot test results showed the recombinant virus could express active VP2 protein of PPV and porcine IL-18. After the recombinant virus was consecutively passed for 20 times, the ST cell infected by the re- combinant virus could still emit green fluorescence,and PCR detection results showed VP2 protein and IL-18 gene in the recombinant virus could steadily be inherited. This research laysa foundation of the further studying immune efficacy of the recombinant porcine pseudorabies vi- rus,
出处
《中国兽医学报》
CAS
CSCD
北大核心
2015年第9期1422-1428,共7页
Chinese Journal of Veterinary Science
基金
河南省重大科技专项资助项目(111100110300)
河南省教育厅科学技术研究重点资助项目(14B180001)
