猪瘟病毒cF114株E2基因在家蚕杆状病毒表达系统中的融合表达

为进一步利用家蚕杆状病毒表达系统研制猪瘟病毒(CSFV)新型的亚单位疫苗,将猪瘟病毒cF114株囊膜糖蛋白E2基因克隆至带有GST标签的分泌表达杆状病毒转移载体pAcSecG2T的EcoRⅠ和BamHⅡ位点之间,获得了带有杆状病毒囊膜蛋白gp67信号肽-GST-E2元件的重组杆状病毒转移载体pAcSecG2T-E2,与线性化家蚕杆状病毒Bm-BacPAK6 DNA共转染家蚕BmN细胞,筛选重组病毒(Bm-BacPAK6-E2),PCR证实所分离的重组病毒含有目的片段E2基因。SDS-PAGE图谱显示,感染重组病毒后,在细胞培养液上清和家蚕幼虫、蛹的血淋巴中均可检测到一条分子量为63 kD左右的特异性条带;感染Bm-BacPAK6-E2家蚕蛹的血淋巴可检测到GST活性,接种病毒148 h后重组蛋白的表达水平达到17.11μg/mL。

Fusion Expression of E2 Gene of Classical Swine Fever Virus cF114 in Bombyx mori Baculovirus Expression Vector System

Between EcoR I and BamH I sites,glycoprotein E_(2) gene of classical swine fever virus(CSFV) cF114 strain was cloned into pAcSecG2T vector,which contains a gp67 signal peptide and a GST label,for construction of the recombinant baculovirus transfer vector pAcSecG2T-E_(2).Then BmN cells were co-transinfected with pAcSecG2T-E_(2) and linear Bm-BacPAK6 DNA to generate the recombinant virus Bm-BacPAK6-E_(2).PCR confirmed that the recombinant virus screened contained the E_(2) gene.SDS-PAGE showed a special band at about(63 kD) either in the supernantant of post-infection cell-culture or in haemolymph of larvae and pupae of silkworm.GST activity was detected from haemolymph of silkworm pupae,and the expression level of the fusion protein reached(17.11 μg/mL) at 148 hours post-infection,which would be potentially benefit to develop subunit vaccine against CSFV.