牦牛LC3B蛋白多克隆抗体制备及在生殖器官表达检测中的应用  

Preparation of Polyclonal Antibody against Yak LC3B Protein and Its Application in Detection of Expression in Reproductive Organs

作　　者：焦正兴 潘阳阳[1,2] 王萌 王靖雷[1] 马文斌 高翔 张晖 崔燕[1,2] 余四九 王立斌[1,2] JIAO Zhengxing;PAN Yangyang;WANG Meng;WANG Jinglei;MA Wenbin;GAO Xiang;ZHANG Hui;CUI Yan;YU Sijiu;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,兰州730070 [2]甘肃省牛羊胚胎工程技术研究中心,兰州730070

出　　处：《畜牧兽医学报》2023年第6期2436-2447,共12页ACTA VETERINARIA ET ZOOTECHNICA SINICA

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

摘　　要：旨在克隆牦牛微管相关蛋白1轻链3B(microtubule associated protein 1 light chain 3B,LC3B)基因,制备牦牛LC3B多克隆抗体,为探究自噬在牦牛生殖生理过程中的作用奠定基础。本研究通过基因克隆方法获得牦牛LC 3B基因序列,利用原核表达、亲和层析法纯化牦牛LC3B重组蛋白,使用纯化的蛋白为抗原免疫10月龄日本大耳白兔,Sepharose4B柱层析法纯化制备牦牛LC3B多克隆抗体,利用酶联免疫吸附试验(ELISA)测定抗体效价,并使用蛋白质免疫印迹(WB)、免疫组织化学法(IHC)和免疫荧光技术(IF)检测牦牛生殖器官中LC3B蛋白的表达。结果显示,本研究成功克隆了牦牛LC 3B基因(登录号:OP572227),构建的重组质粒pET-32a-LC3B能够诱导产生重组蛋白,并且在包涵体和上清中均有表达;所纯化的牦牛LC3B重组蛋白大小为34 ku(含Trx和His标签),符合预期结果。通过免疫日本大耳兔后得到的兔抗牦牛LC3B蛋白血清,抗体效价分别为1∶640000和1∶320000,特异性良好,表明牦牛LC3B蛋白多克隆抗体制备成功。纯化后的抗体应用于牦牛生殖器官中LC3B的表达检测,LC3B在牦牛卵巢、输卵管和子宫中表达,并且能识别LC3B-Ⅰ和发生脂化后的LC3B-Ⅱ,主要表达于细胞质及细胞核周。本研究通过成功克隆牦牛LC 3B基因制备了牦牛LC3B多克隆抗体,使用抗体检测发现LC3B蛋白在牦牛生殖器官中均有表达,表明所制备的多克隆抗体特异性良好,能够应用于牦牛LC3B蛋白的检测和细胞自噬水平的研究。The purpose of this study was to clone the microtubule associated protein 1 light chain 3B(LC 3B)gene of yak and prepare the polyclonal antibody of yak LC3B,so as to lay a foundation for exploring the role of autophagy in the reproductive physiology of yak.The sequence of yak LC 3B gene was obtained by gene cloning.The recombinant protein of yak LC3B was purified by prokaryotic expression and affinity chromatography.The purified protein was used as antigen to immunize 10-month-old Japanese big-ear white rabbits.The polyclonal antibody against LC3B was purified and prepared by Sepharose 4B column chromatography,and the antibody titer was determined by enzyme linked immunosorbent assay(ELISA).Western blot(WB),immunohistochemistry(IHC)and immunofluorescence(IF)were used to detect the expressions of LC3B protein in yak reproductive organs.The results showed that the yak LC 3B gene(accession number:OP572227)was successfully cloned.The constructed recombinant plasmid pET-32a-LC3B induced the production of recombinant protein and expressed in inclusion bodies and supernatant.The size of purified yak LC3B recombinant protein was 34 ku(containing Trx and His tags).The rabbit anti-yak LC3B protein serum obtained by immunizing Japanese big-ear white rabbits had antibody titers of 1∶640000 and 1∶320000,respectively,with good specificity,which showed that the polyclonal antibody of yak LC3B protein was successfully prepared.The purified antibody was used to detect the expressions of LC3B in the reproductive organs of yaks.It showed that LC3B was expressed in the ovaries,oviducts and uterus of the yak,and recognized LC3B-Ⅰand LC3B-Ⅱafter lipidation,it was located mainly in the cytoplasm and perinuclear.In conclusion,LC3B polyclonal antibody was prepared successfully by cloning LC 3B gene in this study,and the expression of LC3B protein was detected in the reproductive organs.It is stated that the antibody prepared has good specificity and it can be applied to detect LC3B protein in yaks and further study on the levels

关 键 词：牦牛 LC3B 原核表达 多克隆抗体 生殖 

分 类 号：S823.85[农业科学—畜牧学]