共表达猪呼吸与繁殖障碍综合征病毒NJ-a株ORF4、ORF5与ORF6基因重组改良型痘苗病毒安卡拉株的构建  

Construction and the immunogenicity of the recombinant Modified Vaccinia Virus Ankala co-expressing ORF4、ORF5 and ORF6 genes of Porcine Reproductive and Respiratory Syndrome Virus NJ-a strain

作　　者：郑其升[1] 毕志香 李鹏[1] 陈德胜[1] 陈溥言[1] 

机构地区：[1]南京农业大学农业部动物疫病诊断与免疫重点实验室,南京210095 [2]山东省畜牧兽医职业技术学院,潍坊262101

出　　处：《微生物学报》2007年第2期345-349,共5页Acta Microbiologica Sinica

摘　　要：为探讨共表达猪繁殖与呼吸综合征病毒(Porcinerep roductive and respiratory syndrome Virus,PRRSV)保护性抗原基因的重组改良型痘苗病毒安卡拉株(Modified Vaccinia Virus Ankala,MVA)的免疫效力,将PRRSVNJ-a株ORF4、ORF5和ORF6基因插入转移载体pⅡLR中,获得了三基因共表达的转移载体pⅡLR-ORF5/ORF6/ORF4,通过同源重组的方法获得重组病毒rMVA-GP5/M/GP4。以lacZ为报告基因进行噬斑筛选和重组病毒纯化后,PCR方法证明ORF4、ORF5和ORF6成功的插入MVA基因组中;经Western blot检测与间接免疫荧光试验证实,重组病毒感染细胞能正确表达PRRSVGP4、GP5与M蛋白。用rMVA-GP5/M/GP4免疫6周龄Babl/C小鼠,首免后3周可检测到特异性PRRSV中和抗体,8周后中和抗体效价可达25,并能继续维持4周;淋巴细胞增殖试验结果表明,重组病毒免疫小鼠产生强烈的特异性细胞增殖反应。上述研究结果表明rMVA-GP5/M/GP4具有良好的免疫原性,可作为预防PRRS的候选疫苗进一步研究。To develop investigate the recombinant MVA（rMVA） vaccines against PRRSV infection, the ORF4, ORF5 and ORF6 of PRRSV NJ-a strain were subeloned into the MVA transfer vector pⅡLR and the resultant recombinant vector was called pⅡLR-ORF4/ORFS/ORF6. The rMVA was generated by transfecting MVA-infected BHK-21 cells with the recombinant vector and screened by plaque purification after X-gal staining. After six rounds of purification, insertion of PRRSV GP4, GP5 and M genes into the MVA genome was confirmed by PCR analysis and expression of the three proteins was identified by Western-blot and IFA. Each of the tested mice was inoculated with 5 × 10^5 TCID50/mouse of the rMVA-GP4/GPS/M and boosted 3 weeks later. Neutralization assay showed that PRRSV-specific neutralizing antibodies were detectable at 3 weeks and reached the highest titer （2^5 ） by 8 weeks after the primary vaccination, which maintained stable until the end of the experiment. The significant lymphocyte proliferation responses were also observed in mice immunized with rMVA-GP4/GPS/M. These results indicate the rMVA co-expressing PRRSV ORF4, ORF5 and ORF6 genes may be an attractive candidate vaccine for preventing PRRSV infection.

关 键 词：猪繁殖与呼吸综合征病毒 修饰的痘苗病毒安卡拉株 GP4 GP5与M蛋白 共表达 

分 类 号：S852.5[农业科学—基础兽医学]