东北白鹅CD4基因的克隆及其胞外区的表达与抗血清的制备  

Molecular Cloning and Expression of Extracellular Region of Northeast White Goose CD4 Gene and Preparation of Its Antiserum

作　　者：张雪莲[1] 魏双施[1] 邵建伟[1] 母晓宇[1] 高明春[1] 张文龙[1] 马波[1] 王君伟[1] 

机构地区：[1]东北农业大学动物医学学院,哈尔滨150030

出　　处：《畜牧兽医学报》2014年第4期639-646,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：黑龙江省"十二五"科技攻关项目(GA09B302);高校博士学科点专项基金(20102325110004);黑龙江省青年基金(QC04C32)

摘　　要：本研究根据GenBank公布的绿头鸭CD4(AF378701)基因序列设计引物,RT-PCR获得东北白鹅CD4基因。根据测序结果设计特异性引物,克隆得到东北白鹅CD4胞外区基因,并在pET-32a(+)/Rosetta(DE3)pLysS系统中进行原核表达。经IPTG诱导重组蛋白获得表达,Ni-NTA柱亲和层析获得纯化的重组蛋白,以纯化的重组蛋白为免疫原制备兔抗鹅CD4胞外区抗血清。I-ELISA、Western blot和流式细胞仪分析表明抗血清可特异识别重组蛋白以及分离获得的东北白鹅外周血T淋巴细胞,间接免疫荧光试验证实纯化的抗血清可特异性识别瞬时真核表达的鹅CD4胞外区蛋白。以上结果说明制备的抗血清可作为鹅CD4+T淋巴细胞的检测试剂。The aim of this study was to prepare antiserum against the goose CD4protein.Specific primers for goose CD4gene were designed according to public Mallard duck reference sequence(AF378701)in GenBank.Then we obtained Northeast White Goose CD4gene by RT-PCR successfully.According to goose CD4 ORF sequence,specific primers were further designed and goose CD4extracellular region gene was cloned.We produced recombinant goose CD4extracellular region protein in prokaryotic expression system(pET-32a(+)/Rosetta(DE3)pLysS).The recombinant protein was induced by IPTG and was purified by Ni-NTA column affinity chromatography.As immunogen,the purified recombinant protein prepared rabbit anti-goose CD4extracellular region serum.I-ELISA,Western blot and Flow cytometric analysis showed that antiserum could identify recombinant goose CD4extracellular region protein and peripheral blood T lymphocytes respectively.Indirect immunofluorescence test confirmed the purified antiserum could identify goose CD4extracellular protein by eukaryotic expression system specifically.The above results show that antiserum can be used as detection reagent to detect goose CD4+T lymphocytes.

关 键 词：东北白鹅 CD4 基因表达 T淋巴细胞 

分 类 号：S835[农业科学—畜牧学]