Preparation and identification of rabbit polyclonal antibody against human FAM21
10.3969/j.issn.1000-484X.2025.06.037
- VernacularTitle:兔抗人FAM21多克隆抗体的制备与鉴定
- Author:
Tuo TANG
1
;
Yanji LU
;
Wenlong LI
;
Tao WANG
;
Xian HONG
;
Zhihui DENG
Author Information
1. 齐齐哈尔医学院医学技术学院,齐齐哈尔 161006;齐齐哈尔医学院医药科学研究院蛋白质结构与功能研究室,齐齐哈尔 161006
- Publication Type:Journal Article
- Keywords:
FAM21;
Prokaryotic expression;
Protein expression and purification;
Polyclonal antibody
- From:
Chinese Journal of Immunology
2025;41(6):1484-1489
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To prepare rabbit polyclonal antibody against human FAM21 and analyze antibody specificity.Methods:Using the plasmid encoding human FAM21 full-length gene as a template,the nucleotide sequence of its 2 431～3 006 base was amplified by PCR and connected to the pGEX-6p-1 prokaryotic expression vector to construct pGEX-6P-1-FAM21 recombinant plasmid expressing the 811～1 002 amino acid fragment of FAM21.The recombinant plasmid was transformed into BL21(DE3)compe-tent Escherichia coli and was expressed inductively,and the protein was purified using GST fusion protein purification magnetic beads.The purified GST fusion protein was used as an antigen to immunize New Zealand rabbits,and the collected antiserum was purified by an agarose column containing GST protein.The specificity of antibody was detected by Western blot and immunofluorescence assay in stable FAM21 knockdown HeLa cells.Results:The pGEX-6p-1-FAM21 prokaryotic expression plasmid was successfully constructed and induced to express in BL21(DE3)competent Escherichia coli.The purified GST fusion protein had a molecular weight of approxi-mately 50 kD,and the purified antibody titer from immunized New Zealand rabbits was greater than 1∶128 000,with high specificity.Conclusion:The pGEX-6p-1-FAM21 prokaryotic expression plasmid is successfully constructed,and the rabbit polyclonal antibody against human FAM21 is prepared for Western blot and immunofluorescence assay.
