摘要
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,以含全长IBV M41S基因的质粒为模板扩增出IBVS1基因,分别克隆至T载体。DNA测序表明,所获得的IBV S1基因大小为1.5kb,IgG Fc大小为1kb,序列正确。将IBVS1与IgG Fc基因串连,插入含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pcD-NA3.1-tPA上,在HeLa细胞上进行瞬时融合表达。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 Fc活性。
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 liver was used as the template to amplify IgG Fe 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 Sl 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 FeSl 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.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2008年第10期1145-1149,共5页
Chinese Journal of Veterinary Science
基金
973SARS防制基础研究专项基金资助项目(2003CB514122)
湖北省科学技攻关计划资助项目(2006AA2052)
