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当前位置: 首页 > 文章 > 利用酵母双杂交系统筛选与猪2型链球菌透明质酸酶(HYL)相互作用的蛋白质 中国兽医学报 2009,29 (9) 1149-1154
Position: Home > Articles > Screening for new binding proteins which interact with HYL of Streptocuccus suis serotype 2 using yeast two hybrid Chinese Journal of Veterinary Science 2009,29 (9) 1149-1154

利用酵母双杂交系统筛选与猪2型链球菌透明质酸酶(HYL)相互作用的蛋白质

作  者：
吴涛;常海涛;谭臣;付婷;司有辉;贝为成;陈焕春
单  位：
华中农业大学农业微生物国家重点实验室
关键词：
猪链球菌2型;透明质酸酶;酵母双杂交;γ内收蛋白;装配架离子转运蛋白
摘  要：
以猪链球菌2型(SS2)透明质酸酶(HYL)作为诱饵蛋白,利用CytoTrap酵母双杂交系统,从人的肺组织cD-NA文库中筛选与HYL相互作用的蛋白,探索HYL在SS2致病中的作用。首先,通过PCR扩增SS2Hyl全长基因,定向克隆到pSos载体多克隆位点中,构建诱饵载体(pSos-Hyl)。同时,将Hyl克隆到pET-28c载体中,构建重组表达载体(pET28c-Hyl)。将pSos-Hyl和人肺cDNA文库(pMyr-cDNA)共转化cdc25H酵母细胞,筛选与HYL相互作用的阳性克隆。经过筛选和重复验证,并对所得到的载体插入片段进行测序,经BLAST分析,从人肺组织cDNA文库中得到2个与HYL相互作用的阳性克隆,2个基因分别与γ内收蛋白(Homo sapiens adducin 3(gam-ma)(NT_030059.12),ADD3)和装配架离子转运蛋白(Homo sapiens chromosome 6 genomic contig,reference as-sembly ion transporter protein(NT_034880.3),AITP)基因的同源性为99%和100%。将HYL原核表达,ADD3和AITP真核细胞表达后,用免疫共沉淀试验分别验证了HYL与ADD3和AITP之间的相互作用,为进一步研究ADD3和AITP可能与SS2致病的关系奠定了前期基础。
译  名：
Screening for new binding proteins which interact with HYL of Streptocuccus suis serotype 2 using yeast two hybrid
作  者：
WU Tao1,2,CHANG Hai-tao1,TAN Chen1,FU Ting1,SI You-hui1,BEI Wei-cheng1,CHEN Huan-chun1(1.State Key Laboratory of Agricultural Microbiology,Huazhong Agricultural University,Wuhan 430070,China;2.Department of Feed Science,Wuhan Polytechenic University,Wuhan 430023,China)
关键词：
Streptococcus suis serotype 2;hyaluronidase;yeast two hybrid system;homo sapiens adducin 3;assembly ion transporter protein
摘  要：
In this study,hyaluronidase(HYL) was used as a bait protein to screen its interacted protein from the human lung cDNA library using the yeast two hybrid system to explore its function in SS2 infection.The full length Hyl gene was amplified from chromosomal DNA of SS2 strain SC21 by PCR.Hly gene was cloned directed into pSos vector to yield the bait plasmid pSos-Hyl.The recombinant expression plasmid pET-28c-Hyl was constructed by the same way at the same time.The library was screened by co-transformation of the pSos-Hyl bait constructed into a temperature-sensitive cdc25H yeast strain.By screening and testing repeatedly,two positive target plasmid was got from the human lung cDNA library.One of them was identical to Homo sapiens adducin 3 gene(ADD3).The other showed high homology to assembly ion transporter protein gene of homo sapiens(AITP).The protein-protein interaction between HYL and targets received was confirmed by ProFound Co-immunoprecipation assay.The relation between the function of ADD3 or AITP and the pathopoiesis was analyzed.

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