鸡源CD40L基因克隆、蛋白表达及生物活性检测

Gene cloning,protein expression and examination of biological activity of chicken CD40L

为获得鸡源CD40L(chCD40L)蛋白,以鸡脾细胞制备cDNA并以之为模板扩增chCD40L基因,构建pFastBac-chCD40L供体重组质粒,转化感受态细胞DH10Bac,通过筛选及鉴定获得Bacmid-chCD40L重组质粒,转入真核表达系统sf9昆虫细胞进行蛋白表达与纯化,获得His-chCD40L蛋白。此外,构建pQM01-chCD40L质粒,转染HEK293T细胞进行蛋白表达与纯化,获得Strep-chCD40L蛋白。亲和层析纯化的chCD40L蛋白浓度为0.01 mg/mL。为检测chCD40L蛋白的生物活性,分离和培养3周龄SPF雏鸡的法氏囊组织原代细胞,将chCD40L加入细胞培养液刺激细胞增殖,通过Western blotting试验、间接免疫荧光试验、流式细胞术检测,发现该蛋白能够与法氏囊B淋巴细胞表面的CD40结合,说明chCD40L具有生物活性。成功获得chCD40L蛋白,为原代B淋巴细胞体外培养及IBDV野毒分离与诊断奠定了基础。

To obtain chicken CD40L protein,the cDNA was prepared from chicken splenic cells and used as a template to clone and amplify CD40L by PCR.The target gene was cloned into pFastBac vector to construct a pFastBac-chCD40L donor plasmid.Recombinant plasmid was transformed into DH10 Bac and recombinant Bacmid-chCD40L was obtained.The Bacmid-chCD40L plasmid was transfected into sf9 insect cells to obtain His-chCD40L protein.In addition,the target gene was cloned into pQM01 vector to construct a pQM01-chCD40L plasmid,recombinant plasmid was transfected into HEK 293 T cells to obtain Strep-chCD40L protein.The chCD40L protein was purified by affinity chromatography,and the concentration of purified chCD40L protein was determined to be 0.01 mg/mL.Primary cells were isolated from the bursal tissue of 3-week old SPF chickens,and the chCD40L protein was added to the culture medium to stimulate cells.The chCD40L could bind to CD40 on B cells as examined by Western blotting,indirect immunofluorescence assay and flow cytometry,suggesting that chCD40L protein is biologically active.We successfully obtained chicken CD40L protein of biological activity,which laid the foundation in the in vitro culture of primary B lymphocytes for the isolation and diagnosis of virulent IBDV.