鸡LncBMP4编码小肽EPC5的生物信息学分析和多克隆抗体制备  

Bioinformatics Analysis and Polyclonal Antibody Preparation of Small Peptide EPC5 Encoded by LncBMP4 in Chicken(Gallus gallus)

作　　者：夏钱 龚威 金晶[1] 张晨[1] 高晓敏 陈晨 周舒简 胡菜 张玉 李碧春[1] 左其生[1] XIA Qian;GONG Wei;JIN Jing;ZHANG Chen;GAO Xiao-Min;CHEN Chen;ZHOU Shu-Jian;HU Cai;ZHANG Yu;LI Bi-Chun;ZUO Qi-Sheng(College of Animal Science and Technology,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]扬州大学动物科学与技术学院,扬州225009

出　　处：《农业生物技术学报》2023年第3期650-658,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(32002158);中国博士后科学基金(2020M681746);扬州大学科学创新计划(X20210663,X20210648)。

摘　　要：鸡(Gallus gallus)原始生殖细胞(primordial germ cells,PGCs)在转基因动物制备和种质资源保护领域具有广泛应用前景,为了解析鸡PGCs形成的分子机制,本研究前期鉴定了1个调节鸡PGCs形成的关键长链非编码RNA-骨形成蛋白(long noncoding RNA-bone morphogenetic protein 4,LncBMP4),可编码小肽EPC5(expression in primordial germ cells chromosome 5)。本研究通过在线预测软件初步分析EPC5小肽的化学结构并进行初步的亚细胞定位。通过化学合成法构建PET-EPC5-His-B2M原核融合表达载体并转化大肠杆菌(Escherichia coli)BL2感受态细胞,经异丙基-β-D-硫代半乳糖苷(isopropyl-beta-Dthiogalactopyranoside,IPTG)诱导后大量表达和纯化EPC5抗原。利用纯化的抗原进行动物免疫并收集抗血清(G1480,G1481),通过ELISA检测抗血清效价。纯化抗体并通过Western blot进行鉴定。结果表明,EPC5主要定位于细胞内并具有RNA聚合酶亚基。成功构建PET-EPC5-His-B2M载体,且诱导表达后重组蛋白大小约为25 kD。ELISA检测发现G1480抗血清效价为1∶5.12×105(OD_(抗血清)/OD_(免前血)≥2.1),G1481抗血清效价为1∶1.024×106(OD抗血清/OD免前血≥2.1)。对G1480抗血清进行纯化,产生的EPC5多克隆抗体浓度为10 mg/mL,ELISA检测显示效价为9.8 ng/mL时(OD_(450)>0.2)。Western blot检测结果显示制备的多克隆抗体可以特异性结合体内外表达的EPC5蛋白。本研究成功地制备EPC5多克隆抗体,为进一步研究EPC5在PGCs形成过程中的功能和机制提供了基础。Chicken(Gallus gallus)primordial germ cells(PGCs)have a wide application prospect in the fields of transgenic animal preparation and germplasm resources protection.In order to analyze the molecular mechanism of chicken PGCs formation,a key long noncoding RNA-bone morphogenetic protein 4(LncBMP4)that regulates chicken PGCs formation was identified earlier,and it can encode a small peptide,named EPC5.In this study,the chemical structure and subcellular localization of EPC5(expression in primordial germ cells chromosome 5)peptides were analyzed by online prediction software.The prokaryotic fusion expression vector of PET-EPC5-His-B2M was constructed by chemical synthesis and transformed into the BL2 competent state of Escherichia coli.After induction by isopropyl-beta-D-thiogalactopyranoside(IPTG),EPC5 antigen was expressed and purified.The purified antigen was used for animal immunization and antiserum(G1480,G1481)was collected.The titer of antiserum was detected by ELISA.The antibody was purified and identified by Western blot.The results indicated that EPC5 mainly localized in cells and had RNA polymerase subunits.The PET-EPC5-His-B2M vector was successfully constructed,and the size of the recombinant protein was about 25 kD after induction.ELISA showed that the titer of G1480 antiserum was 1∶5.12×105(ODantiserum/ODpre-immune blood≥2.1),and that of G1481 antiserum was 1∶1.024×106(ODantiserum/ODpre-immune blood≥2.1).After purification of G1480 antiserum,the concentration of EPC5 polyclonal antibody was 10 mg/mL,and the titer was 9.8 ng/mL(OD value>0.2)by ELISA.The results of Western blot showed that the prepared polyclonal antibody could specifically bind to EPC5 protein expressed in vitro and in vivo.In conclusion,the polyclonal antibody against EPC5 was successfully prepared in this study,which laid a foundation for further study on the function and mechanism of EPC5 in the formation of PGCs.

关 键 词：鸡 多克隆抗体 LncRNA 原始生殖细胞(PGCs) 

分 类 号：S831.2[农业科学—畜牧学]