青杄PwHAP5基因原核表达及多克隆抗体制备  

Prokaryotic Expression and Preparation of Polyclonal Antibody of PwHAP5 Gene in Picea wilsonii

作　　者：覃雪晶 王雨涵[1] 曹一博[1] 张凌云[1] QIN Xue-jing;WANG Yu-han;CAO Yi-bo;ZHANG Ling-yun(Key Laboratory of Forestry Silviculture and Conservation of Ministry of Education,The College of Forestry,Beijing Forestry University,Beijing 100083)

机构地区：[1]北京林业大学林学院森林培育与保护教育部重点实验室,北京100083

出　　处：《生物技术通报》2022年第8期142-149,共8页Biotechnology Bulletin

基　　金：北京林业大学中央高校基本科研业务费专项资金项目(2021ZY01)。

摘　　要：制备青杄(Picea wilsonii)PwHAP5基因的特异性多克隆抗体,为研究PwHAP5基因功能、分子机制奠定基础。经PCR扩增获得青杄花粉PwHAP5基因片段与载体pET-48b相连构建原核表达载体pET-48b-PwHAP5。经诱导产生重组蛋白PwHAP5,通过His-NTA镍柱纯化蛋白产物。蛋白经SDS-PAGE检测后再免疫兔子制备兔抗血清。血清经ProteinA纯化后,通过SDS-PAGE和Western blot检测抗体纯度和特异性。结果表明,成功构建了原核表达载体pET-48b-PwHAP5,制备了分子质量约为45 kD的重组蛋白PwHAP5。获得的青杄PwHAP5兔抗血清经间接ELISA法检测效价达1∶729000以上;制备的PwHAP5蛋白兔多克隆抗体能特异性识别抗原,效价高,满足后续研究PwHAP5蛋白在青杄生长发育中的功能等需求。Specific polyclonal antibodies against PwHAP5 gene of Picea wilsonii were prepared to provide the experimental data for follow-up studies of PwHAP5 protein localization,expression and function.The PwHAP5 gene fragment from P.wilsonii pollen was amplified by PCR and then was inserted into vector pET-48b for constructing the recombinant plasmid pET-48b-PwHAP5.The recombinant protein of PwHAP5 was induced and was purified by His-NTA nickel column.The expressed recombinant protein was tested by SDS-PAGE and injected into New Zealand rabbits as an antigen to obtain antiserum.Antiserum was purified by ProteinA,the purity and specificity of the antibody was identified by SDS-PAGE and Western blot method.As results,the prokaryotic expression vector pET-48b-PwHAP5 was successfully constructed,and the PwHAP5 protein with molecular weight of 45 kD was prepared.The titer of PwHAP5 rabbit antiserum was more than 1∶729000 via indirect ELISA.The prepared PwHAP5 protein of rabbit polyclonal antibody has a high titer and specifically recognize antigen,which can meet the requirements of subsequent studies on the functions of PwHAP5 in the growth and development of P.wilsonii.

关 键 词：PwHAP5基因 原核表达 蛋白纯化 多克隆抗体 

分 类 号：S792.99[农业科学—林木遗传育种] Q943.2[农业科学—林学]