Preparation of monoclonal antibody against recombinant porcine trypsin and identification of its biological characteristics
10.13200/j.cnki.cjb.004397
- VernacularTitle:重组猪胰蛋白酶单克隆抗体的制备及其生物学特性鉴定
- Author:
ZHANG Tingting
- Publication Type:Journal Article
- Keywords:
Porcine trypsin(PT);
Prokaryotic expression;
Gene recombination;
Monoclonal antibody;
Biological characteirstics
- From:
Chinese Journal of Biologicals
2025;38(1):22-26+33
- CountryChina
- Language:Chinese
-
Abstract:
Objective To prepare monoclonal antibodies against recombinant porcine trypsin(RPT) and identify the biological characteristics, so as to lay a foundation for the development of RPT detection products. Methods The PT gene was amplified by PCR and cloned into the prokaryotic expression vector pET-28a(+). The prokaryotic expression plasmid pET-28a(+)-RPT was constructed and identified by colony PCR and sequencing, which was then transformed into competent E.coli BL21(DE3), induced by IPTG, and purified by Ni-NTA affinity chromatography to obtain RPT. Mouse myeloma cells SP2/0 were fused with spleen cells of BALB/c mice immunized with RPT by hybridoma antibody preparation technique. Hybridoma cells stably secreting specific RPT monoclonal antibodies were screened and injected i.p. into the mice to prepare ascites. The positive monoclonal antibodies were screened by ELISA, and the titer, subtype, immunogenicity and specificity were identified.Results The prokaryotic expression plasmid pET-28a(+)-RPT was constructed correctly as identified by colony PCR and sequencing. RPT had a relative molecular mass of about 24 000 and mainly existed in the supernatant in soluble form, which had a purity of over 90% after purification. Two positive monoclonal cell lines, RPT-4A5C5 and RPT-4D6D11, were obtained with the antibody titers of greater than 1∶1 280 000. Both of the antibodies were IgG1 subclasses, and the light chains were Kappa chains, which reacted specifically with RPT and natural PT, but exhibited no obvious cross-reaction with recombinant carboxypeptidase B(RCPB), recombinant enterokinase(REK), sperm protein 10(SP10) and bovine serum albumin(BSA).Conclusion RPT was expressed by prokaryotic system, and monoclonal antibodies against RPT with high titer and high specificity were prepared by hybridoma technique.
