东北白鹅CD8α基因的克隆及其胞外区基因的表达与抗血清的制备  

Molecular Cloning of Northeast White Goose CD8α Gene,Expression of Its Extracellular Region and Preparation of Corresponding Antiserum

作　　者：张雪莲[1] 魏双施[1] 刘晓玫[1] 邵建伟[1] 张树栋[1] 李珊珊[2] 高明春[1] 张文龙[1] 邢育钢 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 [2]黑龙江中医药大学附属第二医院,哈尔滨150001 [3]哈药集团生物疫苗有限公司,哈尔滨150069

出　　处：《畜牧兽医学报》2015年第4期672-680,共9页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：黑龙江省教育厅科学技术面上项目(12541042);黑龙江省青年基金(QC04C32)

摘　　要：根据GenBank公布的绿头鸭CD8α(AF378373)基因序列设计引物,通过RT-PCR获得东北白鹅CD8α基因。根据测序结果设计特异性引物,克隆得到东北白鹅CD8α胞外区基因,并在pET-30a(+)及pET-28a(+)/Rosetta(DE3)pLysS系统中进行原核表达。经IPTG诱导重组蛋白质获得表达,Ni-NTA柱亲和层析获得纯化的重组蛋白质,以纯化的重组蛋白质(pET-30a-CD8ex)为免疫原制备兔抗鹅CD8α胞外区抗血清。I-ELISA、Western blot分析表明抗血清可特异识别分离获得的东北白鹅外周血T淋巴细胞与重组蛋白质(pET-28a-CD8ex),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CD8α胞外区蛋白质。利用激光共聚焦显微镜观察到分离获得的鹅外周血淋巴细胞的细胞膜处出现明显的荧光信号。本研究结果说明制备的抗血清可作为鹅CD8+T淋巴细胞的检测试剂。The aim of this study was to prepare antiserum against the goose CD8αprotein.Specific primers for goose CD8αgene were designed according to public Mallard duck reference sequence(AF378373)in GenBank.Then Northeast White Goose CD8αgene was cloned by RT-PCR successfully.According to goose CD8α ORF sequence,specific primers were further designed and goose CD8αextracellular region was cloned.The recombinant goose CD8αextracellular region protein was expressed in prokaryotic expression system,pET-30a(+)and pET-28a(+)/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 stimulated the rabbit toproduced anti-goose CD8αextracellular region serum.I-ELISA,Western blot analysis showed that antiserum could identify recombinant goose CD8αextracellular region protein and peripheral blood T lymphocytes respectively.Indirect immunofluorescence test confirmed that the purified antiserum could identify goose CD8αextracellular protein expressed by eukaryotic expression system specifically.The laser confocal scanning microscope had observed the fluorescence signal on the cell membrane.The above results show that antiserum can be used as detection reagent to detect the goose CD8+T lymphocytes.

关 键 词：东北白鹅 CD8Α 基因表达 T淋巴细胞 

分 类 号：Q78[生物学—分子生物学]