绵羊DRB1基因外显子2酵母表面展示库的构建及鉴定  

Construction and Identification of Yeast Surface Display Library of Sheep DRB1 Gene Exon 2

作　　者：王元元 严国 罗成 齐江姣 谭君 周光普 周永顺 徐杰 高剑峰[1] WANG Yuanyuan;YAN Guo;LUO Cheng;QI Jiangjiao;TAN Jun;ZHOU Guangpu;ZHOU Yongshun;XU Jie;GAO Jianfeng(College of Life Science,Shihezi University,Shihezi 832003,China)

机构地区：[1]石河子大学生命科学学院,石河子832003

出　　处：《中国畜牧兽医》2018年第4期850-857,共8页China Animal Husbandry & Veterinary Medicine

基　　金：国家重点基础研究发展计划(973计划)(2010CB530200)

摘　　要：为了揭示布鲁氏菌病的致病机制并研制其新型分子疫苗,试验利用酿酒酵母展示系统展示绵羊白细胞表面抗原DRB1基因,构建DRB1基因外显子2的酵母表面展示库。参照GenBank中绵羊MHCⅡDRB1基因序列设计引物,以绵羊脾脏组织cDNA为模板,反转录扩增得到预期长度的产物,双酶切连接到克隆载体pEASY-T1,命名为pEASY-T1-DRB1。双酶切连接到表面展示载体pYD1上,成功构建了表面展示重组质粒pYD1-DRB1。以pEASY-T1-DRB1为模板,将DRB1基因外显子2两端进行点突变,产生新的酶切位点,然后对外显子2设计特异性引物。对500个绵羊样本进行DNA池化,扩增DRB1基因外显子2序列,双酶切后连接到经相同酶双酶切的表面展示重组突变载体pYD1-DRB1-TB上,构建酿酒酵母表面展示库。转化大肠杆菌DH5α感受态细胞,计算库容量。将其转化酿酒酵母EBY100感受态细胞,经半乳糖诱导,通过免疫荧光法在荧光显微镜下检测。结果显示,绵羊DRB1基因成功整合到酵母基因组中,酵母展示库的库容量在105个以上,DRB1基因库成功展示在酵母细胞表面。由此说明,该库可用于下一步布鲁氏菌抗原肽的筛选。In order to reveal the pathogenesis of brucellosis and develop a new molecular vaccine of brucellosis,the ovine lymphocyte antigen DRB 1 gene was designed to be displayed on yeast cell surface using yeast surface display system,explored a platform for screening antigen peptides and developing a vaccine against sheep brucellosis.A pair of primers were designed according to the published sequences of sheep MHCⅡDRB 1 gene in GenBank.DRB 1 gene was cloned using sheep cDNA extracted from spleen tissue as template,the PCR product was inserted into the cloning vector pEASY-T1 by double enzyme digestion,then named pEASY-T1-DRB1.Ligated into the yeast surface display plasmid vector pYD1 by double enzyme digestion,the recombinant plasmid pYD1-DRB1 was successfully constructed on the surface of yeast.Point mutation was made at the end of DRB 1 gene exon 2 in order to make restriction enzyme cutting site using pEASY-T1-DRB1 as a template and the specific primers of exon 2 was designed according to mutated sheep DRB 1 gene sequence.DNA pooling of 500 sheep samples was made and amplified the sequence of DRB 1 gene exon 2 which was digested by double enzyme,then connected to surface display restructuring mutation carriers pYD1-DRB1-TB by the same double enzyme digestion,the yeast surface display libraries were constructed.E.coli DH5αcompetent cells were transformed and library capacity was calculated.It was transformed into yeast EBY100 cell,after galactose induced,it was detected that DRB 1 gene library on the yeast cell surface under the fluorescence microscope by immunofluorescence assay.The results showed that DRB 1 gene was successfully integrated into yeast genome,the library capacity of librariy was more than 10 5,and DRB 1 gene library was successfully displayed on the surface of yeast cells.The results suggested that this library could be used for screening of Brucella antigen peptide.

关 键 词：绵羊 DRB1基因 外显子2 点突变 酵母展示库 免疫荧光 

分 类 号：R392.11[医药卫生—免疫学]