生物技术通报 ›› 2022, Vol. 38 ›› Issue (8): 142-149.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1404

• 研究报告 • 上一篇    下一篇

青杄PwHAP5基因原核表达及多克隆抗体制备

覃雪晶(), 王雨涵, 曹一博, 张凌云()   

  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
  • 收稿日期:2021-11-10 出版日期:2022-08-26 发布日期:2022-09-14
  • 作者简介:覃雪晶,女,硕士研究生,研究方向:经济林栽培理论与利用;E-mail: Krystal529142879@163.com
  • 基金资助:
    北京林业大学中央高校基本科研业务费专项资金项目(2021ZY01)

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

QIN Xue-jing(), WANG Yu-han, CAO Yi-bo, ZHANG Ling-yun()   

  1. Key Laboratory of Forestry Silviculture and Conservation of Ministry of Education,The College of Forestry,Beijing Forestry University,Beijing 100083
  • Received:2021-11-10 Published:2022-08-26 Online:2022-09-14

摘要:

制备青杄(Picea wilsoniiPwHAP5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∶729 000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。

关键词: PwHAP5基因, 原核表达, 蛋白纯化, 多克隆抗体

Abstract:

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∶729 000 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.

Key words: PwHAP5 gene, prokaryotic expression, protein purification, polyclonal antibody