鲫鱼TNFSF13b(BAFF)基因的克隆、可溶性表达及生物学功能分析  

Molecular Cloning,Expression and Functional Analysis of TNFSF13b( BAFF) Gene in Goldfish,Carassius auratus

作　　者：裴丽丽[1] 黄芳[2] 李瑰婷 庞书英[2] 任文华[2] 

机构地区：[1]南京医科大学康达学院,连云港222000 [2]南京师范大学生命科学学院,南京210046

出　　处：《中国生物工程杂志》2016年第12期21-27,共7页China Biotechnology

基　　金：国家自然科学基金资助项目(31370401)

摘　　要：目的:获得鲫鱼B细胞活化因子(BAFF)基因的全长序列,并构建鲫鱼B细胞活化因子(BAFF)基因的可溶性表达体系,研究其蛋白表达产物对鲫鱼B淋巴细胞的作用。方法:通过RTPCR技术克隆鲫鱼BAFF(简称CaBAFF)的全长cDNA序列,并利用RACE技术快速扩增cDNA末端,将CaBAFF的可溶性片段(简称CasBAFF)与表达载体sumo连接,并在大肠杆菌BL21中高效表达和纯化,Western bloting对产物Nus-His-CasBAFF蛋白进行鉴定。激光扫描共聚焦显微镜分析表明,Nus-His-CasBAFF蛋白可以与鲫鱼B淋巴细胞结合。体外,通过MTT检测纯化的NusHis-CasBAFF蛋白能否促进鲫鱼B淋巴细胞的存活。结果:成功克隆鲫鱼BAFF基因,构建了重组表达载体,获得可溶性Nus-His-CasBAFF蛋白。体外实验证明,可溶性Nus-His-CasBAFF蛋白能够促进鲫鱼B淋巴细胞的存活。结论:可溶性Nus-His-CasBAFF蛋白能够促进鲫鱼B淋巴细胞的存活/增值,为鲫鱼免疫系统的研究奠定了基础。Objective： To clone the goldfish B cell activating factor（ BAFF） gene and construct soluble BAFF expression vector,and investigate the effect of the expression product on goldfish lymphocytes B cells.Methods： The full-length cDNA of BAFF from the goldfish（ designated CaBAFF） was cloned using RT-PCR techniques. Using Rapid amplification of cDNA ends（ RACE） to obtain the full-length cDNA of CaBAFF including the 5＇ and 3＇ untranslated regions（ UTRs）,and its soluble BAFF gene（ designated CasBAFF） was linked with SUMO,efficiently expressed in Escherichia coli BL21（ DE3） and confirmed by Western blot analysis. Laser scanning confocal microscopy analysis showed that SUMO-CasBAFF could bind to the surfaces of goldfish lymphocytes B cells. In vitro,the 3-（ 4,5-dimethylthiazol-2-yl）-2,5-diphenyl tetrasodium bromide（ MTT） assay analysis if the purified Nus-His-CasBAFF protein could induce the survival/proliferation of goldfish splenic B cells. Results： The goldfish BAFF was successfully constructed and obtained a soluble Nus-HisCasBAFF protein. In vitro,the soluble Nus-His-CasBAFF protein could induce the survival/proliferation of goldfish splenic B cells. Conclusions： The purified soluble Nus-His-CasBAFF protein could induce the survival/proliferation of goldfish splenic B cells. These results about goldfish BAFF gene would provide a basis for understanding the characteristics of the immune system of the goldfish.

关 键 词：BAFF 鲫鱼(Carassius auratus) RACE 细胞活性 

分 类 号：Q789[生物学—分子生物学]