摘要
以乙型脑炎病毒 SA1 4 - 1 4 - 2株基因组 RNA为模板 ,采用RT- PCR一步法扩增 pr M/ E基因的全长 c DNA(约 2 kb) ,将其克隆至 p MD1 8- T载体 ,获得了克隆质粒 p Tpr M/ E,并对其进行了测序。序列分析结果表明 ,与已报道的 SA1 4 强毒株和 SA1 4 - 1 4 - 2疫苗株的核苷酸比较 ,pr M基因的序列同源性为 1 0 0 %,E发生了 4个点突变 ,其中 1个为回复突变 ,并导致了 3个氨基酸的改变。以伪狂犬病病毒 Ea株 TK- / g G- / L ac Z+突变株为载体 ,构建了 1株乙脑病毒 pr M/ E基因的重组伪狂犬病病毒 TK- / g G- / pr M/ E+。乙脑病毒 pr M/ E基因的克隆及重组伪狂犬病病毒的成功构建 ,为开展乙脑病毒分子生物学及猪猪伪狂犬病一乙脑二价基因工程疫苗的研究打下了坚实基础。
Based on the nucleotide sequence of Japanese encephalitis virus(JEV) SA_(14)-14-2 strain,a pair of primers was designed.The prM/E gene of JEV was cloned by RT-PCR.The sequence analysis showed that the prM genes of SA_(14) and SA_(14)-14-2 strains had 100% homology,while there are 4 mutants in the E gene,and it cause 3 amino acids change.The prM/E gene was digested with BamHⅠ and EcoRⅠ,and the fragment was inserted into the same sites of the universal vector pPgG-uni.A recombinant virus transfer vector pPgG-prM/E was constructed,the pPgG-prM/E digested with KpnⅠ was co-transfected PK-15 cells with PRV Ea TK^(-)/gG^(-)/LacZ^(+) mutant strain genomic DNA digested with EocRⅠ using liposome method.The recombinant virus TK^(-)/gG^(-)/prM/E^(+) was gained by the plaque purification and PCR amplification.This recombinant virus strain can be used for the research of genetic engineering vaccine against PRV and JEV.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2005年第2期162-165,共4页
Chinese Journal of Veterinary Science
基金
教育部重大项目(0113)
