【摘要】 应用RT-PCR方法扩增出猪繁殖与呼吸综合征病毒国内分离株S1毒株的GP5基因序列,然后通过KpnI和XhoI酶切位点把该基因克隆入经过同样双酶切的穿梭载体pShuttle-CMV中。重组穿梭载体经过酶切和PCR鉴定后进行测序,证明所克隆入的基因以正确的阅读框插入。获得的重组穿梭载体经线性化后与腺病毒骨架载体共转化大肠杆菌BJ5183菌株。纯化的重组腺病毒质粒经酶切线性化后转染293细胞获得重组腺病毒。重组腺病毒经纯化后进行RT-PCR和间接免疫荧光鉴定,证明了用PRRSVGP5蛋白基因所构建的重组腺病毒成功的表达了GP5蛋白。猪体免疫试验后收集血清进行中和实验证明所构建的重组腺病毒在猪体内能够诱导产生中和抗体,因此我们所构建的重组腺病毒可以作为PRRSV基因工程疫苗研究候选病毒株。更多还原

【Abstract】 The GP5 gene of Porcine reproductive and respiratory syndrome virus(PRRSV)S1 strain was amplified by RT-PCR,which was digested with KpnI and XhoI and cloned into pShuttle-CMV plasmid.Sequence analysis showed that GP5 gene was cloned in correctly.Then the recombinant plasmid pShuttle-CMV-GP5 was linearized,and co-transformed into E.coli BJ5183 strain together with adenovirus backbone vector pAdEasy-1.Recombinant adenovirus plasmid pAd-GP5 was linearized with PacI,and transferred into HEK293-A cells to obtain recombinant adenovirus.The obtained recombinant adenovirus rAd-GP5 was identified with RT-PCR and IFA.The result showed that the recombinant adenovirus expressed GP5 protein of PRRSV successfully.Virus neutralization assay was carried out after callection of serum,the result demonstrated that the recombinant adenovirus can elicit effective neutralization antibody against of GP5 PRRSV,therefore,the recombinant adenovirus may be a candidate for gene engineering vaccines.更多还原