牛皮蝇HA基因克隆及分泌型真核表达载体的构建  

Cloning of the hypodermin A gene and construction of its secreted eukaryotic expression vector in Hypoderma bovis

作　　者：陈仁金[1] 胡安康[1] 王珍珍[2] 袁红花[2] 张腾业 吴连连[2] 朱裕华[1] 朱孝荣[1] 

机构地区：[1]徐州医学院实验动物中心,江苏徐州221004 [2]徐州医学院神经生物学研究中心,江苏徐州221004

出　　处：《黑龙江畜牧兽医》2014年第2期28-30,34,204,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(31172171);江苏省青年基金项目(BK2012138);江苏省自然科学基金项目(BK2011209);徐州医学院院长基金项目(2012KJZ20)

摘　　要：为了构建牛皮蝇的皮蝇素A(hypodermin A,HA)基因的分泌型真核表达载体pEF1α-HAAcGFP,并在Cos7细胞中表达与鉴定,试验从牛皮蝇中提取RNA,反转录成cDNA,扩增出HA基因,并在HA基因序列的上游加1段信号肽序列;通过双酶切将带有信号肽的HA基因序列克隆到真核表达载体pEF1α-IRES-AcGFP中,得到分泌型真核表达载体pEF1α-HA-AcGFP;再利用脂质体法转染Cos7细胞,并以RT-PCR和Western-blot技术在RNA和蛋白水平上检测HA基因是否在Cos7细胞中表达。结果表明:经双酶切及测序鉴定,分泌型真核表达载体pEF1α-HA-AcGFP构建成功;转染Cos7细胞后,在RNA和蛋白水平上能检测到HA基因的表达。说明成功构建了HA基因的分泌型真核表达载体PEF1α-HA-AcGFP。To construct the secreted eukaryotic expression vector （pEFI α - HA -AcGFP） of hypodermin A （HA） gene of Hypoderma boris, and identify the vector＇s expression in Cos7 ceils. RNA was extracted from Hypoderma boy/s, and then was subjected to reverse transcription eDNA. The eDNA sequence of the HA gene was obtained by RTPCR, and a signal peptide sequence was added at upstream sequence of HA,which was cloned into the eukaryotic expression vector （pEFla -IRES -AeGFP） to obtain a secreted eukaryotie expression vector pEF1α -HA - AcGFP by double digestions. The Cos7 cells were transfected using liposome method, and then RT - PCR and western blot techniques were used to detect whether or not the HA gene was expressed in Cos7 cells at the RNA and protein levels, respectively. The results showed that the secreted eukaryotic expression vector pEF1 a - HA - AcGFP was successfully constructed by double digestions and DNA sequencing. The expression of HA gene could be detected at the RNA and protein levels after transfeeting the Cos7 cells. The results indicate that the secreted eukaryotic expression vector pEF1 α - HA - AeGFP of the HA gene was successfully constructed.

关 键 词：牛皮蝇 宿主 真核表达载体 HA基因 载体构建 基因表达 

分 类 号：Q785[生物学—分子生物学]