Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (8): 142-149.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1404

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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 Online:2022-08-26 Published:2022-09-14
  • Contact: ZHANG Ling-yun E-mail:Krystal529142879@163.com;lyzhang@bjfu.edu.cn

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