牦牛ATG5多克隆抗体的制备及应用  

Preparation and application of polyclonal antibody against ATG5 protein of yaks

作　　者：高翔[1] 潘阳阳[1,2] 王萌 焦正兴[1] 王靖雷 马文斌 王军乾 余四九 崔燕 王立斌[1,2] GAO Xiang;PAN Yangyang;WANG Meng;JIAO Zhengxing;WANG Jinglei;MA Wenbin;WANG Junqian;YU Sijiu;CUI Yan;WANG Libin(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;Technology and Research Center of Gansu Province for Embryonic Engineering of Bovine and Sheep,Lanzhou 730070,China)

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]甘肃省牛羊胚胎工程技术研究中心,甘肃兰州730070

出　　处：《中国兽医学报》2023年第10期2093-2100,共8页Chinese Journal of Veterinary Science

基　　金：甘肃省自然科学基金资助项目(22JR5RA864);国家自然科学基金资助项目(32160850);甘肃省重点人才资助项目(2022-0623-RCC-0307)。

摘　　要：为了进一步研究自噬在牦牛生殖活动中发挥的作用,本研究通过合成牦牛自噬相关5(autophagy related 5,ATG5)基因,并将合成的基因插入pET-32a质粒,转化BL21(DE3)感受态细胞,异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-Thiogalactoside,IPTG)诱导表达ATG5重组蛋白并纯化,免疫日本大耳白兔,制备牦牛ATG5多克隆抗体。采用间接ELISA法检测抗体的效价,蛋白质印迹(Western blot,WB)技术检测多克隆抗体的特异性。通过Western blot、免疫组织化学法(immunohistochemistry,IHC)和免疫荧光技术(immunofluorescence,IF)应用所制备的抗体检测ATG5蛋白在牦牛生殖器官中的表达。结果成功构建了pET-32a-ATG5原核表达质粒,转化大肠杆菌IPTG诱导获得了大小约为53 kDa(含His和Trx标签)的重组蛋白;制备的牦牛ATG5多克隆抗体效价为1∶1280000,特异性良好。应用显示,ATG5蛋白在牦牛睾丸和输卵管中均有表达。本研究成功制备了牦牛ATG5多克隆抗体,特异性良好,可用于检测牦牛ATG5蛋白的表达,为进一步研究自噬在牦牛生殖生理活动中发挥的作用积累了材料。In order to further study the role of autophagy in yak reproductive activities,yak ATG5(autophagy related 5,ATG5)gene was synthesized and cloned into pET-32a plasmid,yielding pET-32a-ATG5,then it was transformed into BL21(DE3)competent cells,IPTG was used to induce the expression of ATG5 recombinant protein,after being purified,it was used to immune Japanese big ear rabbits to prepare yak ATG5 polyclonal antibody.The titer of antibody was detected by indirect ELISA,and the specificity of polyclonal antibody was detected by Western blot.The expressions of ATG5 protein were detected in reproductive organs of the yak by Western blot,immunohistochemistry and immunofluorescence.The prokaryotic expression plasmid pET-32a-ATG5 was successfully constructed,and the recombinant protein with the size of 53 kDa(including His and Trx tags)was induced by IPTG in E.coli.The titer of the prepared polyclonal antibody of yak ATG5 was 1:1280000,showing good specificity.ATG5 protein was expressed in testis and oviducts of yaks;it mainly occurs in the sperm cells and Sertoli cells of yak testis and the cytoplasm of oviduct epithelial cells.The results showed that the polyclonal antibody of yak ATG5 was successfully prepared and had good specificity,which could be used to detect the expressions of yak ATG5 protein,providing information for further research on the roles of autophagy in the reproductive physiological activities of yaks.

关 键 词：牦牛 ATG5 原核表达 多克隆抗体 自噬 

分 类 号：S852.4[农业科学—基础兽医学]