鸡Stra8原核表达载体的构建及多克隆抗体血清的制备  

Construction of Stra8 Prokaryotic Expression Vector and Preparation of Multiple Antiserum in Chicken

作　　者：赵宗仪 王颖洁[1] 靳锴 左其生[1] 张亚妮[1] ZHAO Zongyi;WANG Yingjie;JIN Kai;ZUO Qisheng;ZHANG Yani(Key Laboratory for Animal Genetics Breeding Reproduction and Molecular Design of Jiangsu Province,College of Animal Science and Technology,Yangzhou University,Yangzhou,Jiangsu 225009)

机构地区：[1]扬州大学动物科学与技术学院,江苏省动物遗传繁育与分子设计重点实验室,江苏扬州225009

出　　处：《中国家禽》2021年第10期10-16,共7页China Poultry

基　　金：江苏高校优势学科建设工程资助项目。

摘　　要：试验旨在探究视黄酸介导的Stra8信号在生殖细胞发生过程中的作用和调控机制。以如皋黄鸡睾丸cDNA为模板扩增Stra8基因CDS区,并与pET-28a载体相连获得原核重组表达载体pET-28a-Stra8,然后转染大肠杆菌BL21感受态,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导外源基因表达,离心收集菌液,并用超声波破碎等方法收集表达的蛋白,用纯化的蛋白作为抗原制备兔源抗体,产生带有免疫抗体的兔血;取全血,经血凝离心等步骤后获取血清,通过SDS-PAGE检测抗体的纯度,将获得的含有多克隆抗体的血清进行ELISA检测效价。结果显示:成功构建pET-28a-Stra8重组载体,IPTG诱导后在大肠杆菌BL21中表达,表达产物经SDS-PAGE分析后显示,STRA8蛋白大小约为55 ku;ELISA检测显示在血清稀释比为1∶256000时P/N=3.3>2.1,达到阳性血清合格标准;Western blotting结果显示制备的多克隆抗体血清可结合STRA8蛋白,该结果为进一步研究Stra8在生殖细胞发生过程中的作用和调控机制提供了基础。The aim of this paper is to study the exact role and regulatory mechanism of retinoic acid-mediated Stra8 in germ cell progression.The CDS region of the Stra8 was amplified using Rugao yellow chicken testicular’s cDNA as a template,and it was linked with pET-28a vector to obtain the prokaryotic recombinant expression vector pET-28a-Stra8.pET-28a-Stra8 was transfected into E.coli BL21 competent cells,and exogenous gene was induced to express by IPTG,the bacterial fluid was collected by centrifugation,and the expressed protein was collected by ultrasonic fragmentation.The whole blood and the obtain serum were taken by blood coagulation centrifugation.The purity of antibody was detected by SDS-PAGE.The results showed that pET-28a-Stra8 recombinant vector was successfully constructed and expressed in E.coli BL21 by IPTG induction,and the size of STRA8 protein was about 55 ku according to SDS-PAGE.The ELISA test showed that the serum dilution ratio was 1∶256000 P/N=3.3>2.1,met the positive eligibility criteria.Western blotting results showed that the prepared polyantibody serum could bind STRA8 protein,which would provide a foundation for the study of the function of Stra8 in germ cell development and its regulatory mechanism.

关 键 词：Stra8 原核表达载体 IPTG诱导 多克隆抗体 

分 类 号：S831.2[农业科学—畜牧学]