摘要
为了利用绵羊卵巢酵母双杂交cDNA文库筛选与FHL2蛋白相互作用的宿主蛋白,构建重组诱饵载体pGBKT7-FHL2,试验从绵羊卵巢组织中提取总RNA并反转录为cDNA,依据FHL2基因核苷酸序列设计特异性引物,利用PCR技术扩增FHL2目的片段,连接到经限制性内切酶EocRⅠ和BamHⅠ酶切后的酵母空载体pGBKT7上,并转化至大肠杆菌DH5α感受态细胞中,进行PCR鉴定及测序比对分析,将构建好的重组诱饵载体pGBKT7-FHL2转化到酵母Y2HGlod感受态细胞中,利用酵母双杂交系统检测其表达情况、自激活活性及细胞毒性。结果表明:PCR扩增出大小约为876 bp的目的条带;成功构建出重组诱饵载体pGBKT7-FHL2,测序结果与NCBI中预测的FHL2碱基序列同源性为100%;重组诱饵载体pGBKT7-FHL2可在酵母Y2HGold感受态细胞中表达FHL2蛋白,对酵母Y2HGold感受态细胞无自激活活性和毒性作用。说明试验成功构建出重组诱饵载体pGBKT7-FHL2,通过检测证明该重组诱饵载体能够用于酵母双杂交系统筛选互作蛋白。
In order to screen host protein interacting with FHL2 protein by using ovine ovary yeast two hybrid cDNA library,a recombinant bait vector pGBKT7-FHL2 was constructed,the total RNA was extracted from sheep ovarian tissue and was reversely transcribed into cDNA,specific primers were designed according to the nucleotide sequence of FHL2 gene,and the fragment of FHL2 was amplified by PCR,then connected to yeast empty vector pGBKT7 which was digested by restriction endonuclease EocRⅠand BamHⅠ,and then transformed into E.coli DH5αcells for PCR identification and sequencing analysis,then a recombinant bait vector pGBKT7-FHL2 was transformed into yeast Y2 HGlod cells,and the bait protein was detected by self-activation,cytotoxicity assays and expression.The results showed that the PCR amplification size was about 876 bp,the recombinant bait vector pGBKT7-FHL2 was constructed successfully,and the sequence homology was 100%with the predicted FHL2 base sequence in NCBI;the recombinant bait vector pGBKT7-FHL2 can express FHL2 protein in yeast Y2 HGold cells,but it has no self-activating activity and toxic effect on yeast Y2 HGold cells.The result showet that the yeast bait vector pGBKT7-FHL2 was successfully constructed,the bait vector could be used to screen the interaction protein in yeast two hybrid syatem.
作者
张丽萌
聂晓宁
李闰婷
陈龙欣
王林青
邢真真
宋月
刘爱菊
田树军
马润林
ZHANG Limeng;NIE Xiaoning;LI Runting;CHEN Longxin;WANG Linqing;XING Zhenzhen;SONG Yue;LIU Aiju;TIAN Shujun;MA Runlin(College of Animal Science and Technology,Hebei Agricultural University,Baoding 071001,China;Laboratory of Molecular Biology,Zhengzhou Normal University,Zhengzhou 450044,China;The Research Center of Cattle and Sheep Embryonic Technique of Hebei Province,Baoding 071001,China)
出处
《黑龙江畜牧兽医》
CAS
北大核心
2020年第21期25-29,175-176,共7页
Heilongjiang Animal Science And veterinary Medicine
基金
河北省羊产业技术体系项目(HBCT2018140202)
