捻转血矛线虫酵母双杂交cDNA文库的构建及鉴定  

Construction and identification of yeast two-hybrid cDNA library of Haemonchus contortus

作　　者：张惠 黄艳 周静茹 吴飞 童丹妮 陈学秋[1] 杨怡 马光旭 杜爱芳[1] ZHANG Hui;HUANG Yan;ZHOU Jingru;WU Fei;TONG Danni;CHEN Xueqiu;YANG Yi;MA Guangxu;DU Aifang(Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine,Institute of Preventive Veterinary Medicine,College of Animal Sciences,Zhejiang University,Hangzhou 310058,China)

机构地区：[1]浙江大学动物科学学院动物预防医学研究所,浙江省动物预防医学重点实验室,杭州310058

出　　处：《浙江大学学报（农业与生命科学版）》2022年第2期247-253,共7页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：国家自然科学基金(32002304);国家重点研发计划(2017YFD0501203);浙江省基础公益研究计划(LGN20C180005)。

摘　　要：为构建捻转血矛线虫cDNA文库,进一步研究捻转血矛线虫蛋白互作机制以及为筛选捻转血矛线虫互作蛋白提供依据,本研究以捻转血矛线虫L3期幼虫为材料,利用TriZol法提取捻转血矛线虫总RNA;使用试剂盒构建捻转血矛线虫的cDNA文库,并对cDNA进行均一化处理,再将纯化后的cDNA与线性化的pGADT7-Rec重组构建的文库质粒转化至Y187酵母中,构建出酵母转录激活结构域(activationdomain,AD)文库。结果表明:本研究构建了重组率为100%,插入片段平均长度为1000 bp,工作液细胞密度大于3.5×10^(7)CFU/mL的捻转血矛线虫均一化酵母AD文库;所构建的表达文库各项指标均达标,符合酵母双杂交筛选要求。本文库为捻转血矛线虫的分子机制研究以及疫苗的开发奠定了基础。This experiment aimed to construct a cDNA library of Haemonchus contortus,which could provide the basis for further research on the protein interaction mechanism and screening out the interactive proteins of H.contortus.The L3 stage larvae of H.contortus were used to extract the total RNA by the TriZol method.Besides,the kit was adopted to construct a cDNA library of H.contortus and the cDNA was normalized.Then,the purified cDNA were transformed into yeast Y187 cells together with the linear pGADT7-Rec,and a yeast two-hybrid cDNA library of H.contortus was constructed by homologous recombination.The results showed that a homogenized yeast activation domain(AD)library with the recombinant rate of 100%,average inserted fragment length of 1000 bp,and working fluid cell density of more than 3.5×10^(7)CFU/mL was constructed.The constructed expression library met the requirements of yeast two-hybrid screening.This library lays a foundation for the molecular mechanism study and vaccine development of H.contortus.

关 键 词：捻转血矛线虫 CDNA文库 酵母双杂交系统 蛋白质间相互作用 

分 类 号：S855.9[农业科学—临床兽医学]