Prokaryotic expression and polyclonal antibody preparation of LRRC15 protein, an excretory-secretory antigen of Cysticercus cellulosae
10.3760/cma.j.cn231583-20221216-00407
- VernacularTitle:猪囊尾蚴排泄分泌抗原LRRC15蛋白的原核表达及多克隆抗体制备
- Author:
Qianqian WANG
1
;
Shimin WANG
;
Qianfei WANG
;
Fengling YUAN
;
Wei HE
;
Lizhu LI
;
Biying ZHOU
Author Information
1. 贵州省遵义医科大学第一临床学院，遵义 563000
- Keywords:
Taenia solium;
Cysticercus cellulosae;
Excretory-secretory antigens;
Leucine-rich repeat containing 15 protein;
Prokaryotic expression;
Polyclonal antibody
- From:
Chinese Journal of Endemiology
2023;42(9):704-709
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct a recombinant plasmid pET30a-leucine-rich repeat (LRR) containing 15 (LRRC15) of Taenia solium, prokaryotically express and purify the LRRC15 recombinant protein, and prepare a rabbit polyclonal antibody. Methods:The LRRC15 protein encoding gene of Taenia solium was obtained by whole gene synthesis; it was cloned into pET30a vector, and the recombinant plasmid pET30a-LRRC15 was constructed and identified by double-enzyme PCR; the recombinant plasmid was transformed into competent cells of Escherichia coli BL21 (DE3), and the recombinant protein LRRC15 was induced to express by isopropyl-beta-D-thiogalactopyranoside (IPTG), the expression product was analyzed and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); the LRRC15 recombinant protein was purified by Ni-IDA affinity columns, the purified recombinant protein was analyzed and identified by SDS-PAGE, and the specificity of the purified recombinant protein was identified by Western blot (WB); the New Zealand rabbits were immunized with purified LRRC15 recombinant protein to prepare polyclonal antibodies against LRRC15, and the potency of the purified polyclonal antibody was determined by indirect enzyme-linked immunosorbent assay (ELISA). Results:After PCR identification, a band with a length of 1 506 bp was amplified, which was consistent with the LRRC15 gene; after SDS-PAGE and WB identification, the LRRC15 target protein with a relative molecular mass ( Mr) of about 55.36 × 10 3 was obtained; after immunizing New Zealand rabbits with purified LRRC15 recombinant protein, a polyclonal antibody against LRRC15 was obtained, and its potency was 1∶1 587 200. Conclusion:The recombinant plasmid pET30a-LRRC15 is successfully constructed, the LRRC15 recombinant protein of Taenia solium is prepared, and a high purity and high potency rabbit anti polyclonal antibody against LRRC15 recombinant protein is obtained.
