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  

The Fusion Expression of Avian Infectious Bronchitis Virus S1 Gene and Porcine IgG Fc Gene in HeLa Cells

作　　者：张明富[1,2] 陈汉阳[1,2] 彭博[1,2] 佟铁俦 陈焕春[1,2,3] 郭爱珍[1,2,3] 

机构地区：[1]华中农业大学农业微生物学国家重点实验室,武汉430070 [2]华中农业大学动物医学院,武汉430070 [3]华中农业大学湖北省预防兽医学重点实验室,武汉430070

出　　处：《生物技术通报》2008年第S1期276-281,共6页Biotechnology Bulletin

基　　金：973SARS防制基础研究专项(2003CB514122);湖北省科技攻关计划(项目编号:2006AA205A02)

摘　　要：根据Genbank中发表的猪IgG Fc段基因及IBV S1基因序列,设计并合成引物。以猪肝组织总RNA为模扩增出猪IgG Fc基因,以含全长IBV M41 S基因的质粒为模板扩增出IBV S1基因,分别克隆至T载体。DNA测序表明,所获得的IBV S1基因大小为1.5 kb,IgG Fc大小为1kb,序列正确。将IBV S1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)真核表达载体pcDNA3.1-tPA上,在HeLa细胞上进行瞬时融合表达。经免疫荧光和斑点杂交检测,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 taken 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 of the two genes were respectively cloned into the pMD18T vectors and sequencing were confirmed to be correct by sequencing. The results showed that we have obtained the right genes of IBV S1(1554bp)and IgG Fc(1002bp).Both IBV Sl gene and porcine lgGFc 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-IgGFcS1.HeLa cells were incubated on 6 well plates and transfected by pcDNA3.1-tPA-IgGFcS1 by lipofection.At 36h after transfection,the cells and culture medium were collected separately and detected by IFA,FA and Dot ELISA.The results showed 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 antibody to porcine IgG.

关 键 词：禽传染性支气管炎病毒 S1基因 猪IgG Fc基因 真核表达 融合表达 

分 类 号：S852.5[农业科学—基础兽医学] Q78[农业科学—兽医学]