鸡源血管紧张素转换酶2(ACE2)的原核表达及其多克隆抗体的制备与应用  

Establishment of Chicken Angiotensin Converting Enzyme 2(ACE2)Prokaryotic Expression System and Preparation of It's Polyclonal Antibody

作　　者：纪晓霞 吴雨龙 陈希文 周潇 杜欣雨 张源淑[1] JI Xiao-Xia;Wu Yu-Long;CHEN Xi-Wen;ZHOU Xiao;DU Xin-Yu;ZHANG Yuan-Shu(Key Laboratory of Animal Physiology and Biochemistry,Ministry of Agriculture,Nanjing Agricultural University,Nanjing 210095,China;School of Food Science,Nanjing Xiaozhuang University,Nanjing 211171,China)

机构地区：[1]南京农业大学农业部动物生理生化重点开放实验室,南京210095 [2]南京晓庄学院食品科学学院,南京211171

出　　处：《农业生物技术学报》2024年第12期2786-2795,共10页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31972640);江苏省高校自然科学研究重大项目(20KJA416003)。

摘　　要：血管紧张素转换酶2(angiotensin-converting enzyme 2,ACE2)可作为多种病理生理过程的保护剂和调节剂,以及人冠状病毒的功能性受体,但在鸡(Gallus gallus domesticus)中的研究尚未见报道。本研究以白羽肉鸡为研究对象,首先通过PCR扩增ACE2基因编码区,与原核表达载体pET32a连接,构建鸡ACE2原核表达体系。然后以异丙基-β-D-硫代吡喃半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)诱导ACE2蛋白表达并优化诱导条件,确定其表达方式。氯化钾(KCl)染色切胶纯化获得的鸡ACE2重组蛋白,并通过SDS-PAGE和Western blot鉴定表达蛋白的特异性和分子量,管式凝胶等电聚焦(isoelectric focusing polyacrylamide gel electrophoresis,IEF-PAGE)确定其等电点(pI)。以该纯化的ACE2重组蛋白作为抗原免疫大鼠(Rattus norvegicus)制备鸡ACE2多克隆抗体,测定其效价,并对其特异性等进行验证,利用该抗体明确鸡体内ACE2的组织分布和细胞学定位。结果显示,成功构建了鸡pET32a-ACE2原核表达体系,目的蛋白最佳表达条件为1 mmol/L IPTG诱导10 h,以包涵体形式存在;获得了具有良好抗原性和特异性的鸡ACE2重组蛋白,分子量约为105 kD,pI为5.01;成功获得了具有较好特异性的鼠抗鸡源ACE2多克隆抗体,抗体效价为1∶12800;明确在白羽肉鸡心脏、肝脏、肺脏、肾脏、腺胃、十二指肠、回肠、盲肠、空肠、直肠、睾丸组织中均有ACE2的分布表达,本研究为ACE2在鸡中的进一步研究提供了实验依据和资料。Angiotensin converting enzyme 2(ACE2)can serve as a protective and regulatory agent for various pathophysiological processes,as well as a functional receptor for human coronavirus.However,there have been no reports on research related to chickens(Gallus gallus domesticus).In this study,broiler chickens were used as research subjects.Firstly,the coding region of the ACE2 gene was amplified by PCR and connected to the prokaryotic expression vector pET32a to construct a chicken ACE2 prokaryotic expression system.The expression of the ACE2 protein was induced by isopropylβ-D-1-thiogalactopyranoside(IPTG),and the induction conditions were optimized to determine the expression pattern.The purified recombinant chicken ACE2 protein obtained through KCl staining gel purification was identified for specificity and molecular weight using SDS-PAGE and Western blot techniques.The isoelectric point of the ACE2 protein was determined using isoelectric focusing polyacrylamide gel electrophoresis.The purified recombinant ACE2 protein was used as an immunogen to produce chicken ACE2 polyclonal antibodies in rats(Rattus norvegicus).The efficacy of the antibodies was measured,and their specificity was validated to determine the tissue distribution and cellular localization of ACE2 in chickens.The study successfully constructed a prokaryotic expression system for ACE2 in chickens using the pET32a vector,prepared chicken ACE2 polyclonal antibodies with good efficacy and specificity,and confirmed the widespread distribution of ACE2 in chickens for the first time.This study provides an experimental basis and data for further research on ACE2 in chickens.

关 键 词：白羽肉鸡 血管紧张素转化酶2(ACE2) 原核表达 重组蛋白 多克隆抗体 

分 类 号：S852.23[农业科学—基础兽医学]