输卵管特异性表达艾塞那肽的转基因鸡制备  

Production of transgenic chicken with oviduct-specific expression of exenatide

作　　者：张自富[1] 胡静[1] 赵瑜[1] 秦清明 麻冰洁 赵聘[1] 陈思睿[2] 赵云焕[1] ZHANG Zifu;HU Jing;ZHAO Yu;QIN Qingming;MA Bingjie;ZHAO Pin;CHEN Sirui;ZHAO Yunhuan(Animal Biotechnology Laboratory,College of Animal Science and Technology,Xinyang College of Agricultural and Forestry,Xinyang,Henan 464000,China;National Engineering Laboratory for animal Breeding,College of Animal Science and Technology,China Agricultural University,Beijing 100193,China)

机构地区：[1]信阳农林学院动物科技学院,河南信阳464000 [2]中国农业大学动物科技学院畜禽育种国家工程实验室,北京100193

出　　处：《中国兽医学报》2023年第9期1859-1865,共7页Chinese Journal of Veterinary Science

基　　金：河南省对外开放合作资助项目(182106000049,172102410051);河南省自然基金资助项目(182300410028);信阳农林学院高水平科研孵化器建设基金资助项目(FCL202104)。

摘　　要：艾塞那肽(exenatide)是从墨西哥巨蜥蜴毒液中分离出来的一种含有39个氨基酸的多肽序列,与胰高血糖素样肽(glucagon-like peptide 1,GLP-1)具有53%的同源性,与GLP-1同作用于G-蛋白偶联受体,是首个获准上市的肠促胰岛素类似物抗糖尿病药物。本试验构建了一个基于复制缺陷型慢病毒载体,该载体由一个克隆约2.8kb的鸡卵清蛋白基因特异性启动子调控表达的艾塞那肽基因cDNA(117bp)序列,并在鸡卵清蛋白基因特异启动子上游添加一段雌激素响应受体(estrogen response elements,ERE)(675bp)序列。利用实验室建立的生产转基因家禽技术平台,通过显微注射法把包装好的高滴度慢病毒载体注入发育至第1415期鸡胚卵黄外周静脉血管,获得生殖系嵌合的G0代鸡,扩繁后对G1代和G2代转基因鸡进行分子生物学检测,并对G2代母鸡蛋清中的艾塞那肽含量进行酶联免疫吸附试验(ELISA)检测。试验结果显示G1代有5只转基因鸡,均为单拷贝插入;G2代母鸡蛋清中的艾塞那肽平均表达量为45.19g/L。免疫组织化学检测结果显示艾塞那肽仅在输卵管上皮组织特异性表达。本试验建立了一种慢病毒载体介导的血管显微注射法制备转基因鸡的方便快捷、高效稳定的新型技术体系,为利用鸡输卵管生物反应器生产具有重要价值的药用蛋白提供了一种新方法,具有重要的商业价值和应用前景。Exenatide,the first incretin effect medicine approved for clinical use,is a 39amino acids peptide originally isolated from Heloderma suspectum lizard venom,which shares 53%homology with glucagon-like peptide 1(GLP-1).In order to generate transgenic chicken with oviduct-specific expression of exenatide,cDNA of exenatide gene(117bp)was cloned into the lentiviral vector containing ovalbumin promoter(OVP,2.8kb)with estrogen response elements(ERE,675bp).The constructed vector was microinjected into the vitelline peripheral venous vessels of chicken embryos at Hamburger-Hamilton stage 14-15(HH14-15).After G0chicks were raised to sexual maturity,G1and G2eggs were collected,and the egg whites were examined for exenatide expression by ELISA.The results showed a single copy of exenatide gene was integrated into 5of the G1 chicken;the average level of exenatide protein detected in the eggs white of G2heterozygous chicken was 45.19mg/L,and exenatide specifically expressed in oviduct epithelial tissue was detected by immunohistochemistry.In this study,a convenient,rapid,efficient and stable new technology system for the preparation of transgenic chickens by lentiviral vector mediated vascular microinjection was established,which provides a new method for the production of medical protein with important value by using chicken oviduct bioreactor,and has important commercial value and application prospect.

关 键 词：艾塞那肽 转基因鸡 慢病毒载体 卵清蛋白基因 

分 类 号：S859.7[农业科学—临床兽医学]