鸡传染性支气管炎病毒S1基因与猪IgG Fc基因在HeLa细胞中的融合表达

根据GenBank中发表的猪的IgG Fc段基因及鸡传染性支气管炎病毒(IBV)S1基因序列,设计并合成引物.以猪肝组织总RNA为模板扩增出猪IgG Fc基因,以舍全长IBV M41 S基因的质粒为模板扩增出IBV S1基因,分别克隆至T裁体.DNA测序表明,所获得的IBV S1基因大小为1.5 kb,lgG Fc大小为1 kb,序列正确.将IBVS1与IgG Fc基因串连,插入舍有人组织型纤维蛋白溶酶原激活物分泌信号肽序列(tPA)的真核表达载体pcDNA3.1-tPA上,在HeLa细胞上进行瞬时融合表迭.经免疫光和斑点杂交检测,表达产物同时具有IBV S1蛋白和IgG Fe活性.

The fusion expression of avian infectious bronchitis virus S 1 gene and porcine IgG Fc gene in HeLa cells

According to the published gene sequences of IBV M41 S1 gene and porcine IgG heavey chain Fc fragment DNA in GenBank,we synthesized two pairs of specific primers.Total RNA of swine 1iver was used as the template to amplify IgG Fc gene by RT-PCR.IBV M41 S1 gene was amplified by PCR from the plasmid encoding full length of S gene.Both the two genes were respectively cloned into the pMD18T vectors and confirmed to be correct by sequencing.The results showed that we have obtained the right genes of IBV S1(1 554 bp)and IgG Fc(1 002 bp).Both IBV S1 gene and porcine IgG Fc gene were subcloned into the same pcDNA3.1-tPA vectors with the tissue plasminogen activator(tpA)leader sequence and the resulting plasmid was designated as pcDNA3.1-tPA-IgG FcS1.HeLa cells were incubated on 6 well plates and transfected by pcDNA3.1-tPA-IgG FcS1 by lipofection.At 36 h after transfection the cells and culture medium were collected separately and detected by IFA,FA and Dot ELISA.The results indicated that the S1-IgG Fc fusion protein was expressed within the HeLa cells.The fusion protein can bind to both the rabbit antiserum to IBV and goat antiboby to porcine IgG.