Effect of molecular chaperone on prokaryotic expression level and activity of tetanus toxin fragment C
10.13200/j.cnki.cjb.004433
- VernacularTitle:分子伴侣对破伤风毒素C蛋白原核表达水平及活性的影响
- Author:
WU Huiyu
- Publication Type:Journal Article
- Keywords:
Tetanus toxin fragment C(TTC);
Molecular chaperone;
Prokaryotic expression;
Adjuvant
- From:
Chinese Journal of Biologicals
2025;38(03):272-278
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the effect of molecular chaperone on the prokaryotic expression level and activity of tetanus toxin fragment C(TTC) in order to provide new ideas for the development of new tetanus vaccines.Methods The gene sequence of TTC was optimized for E.coli adaptive codons,and the recombinant plasmid pET30a-HC was constructed,which was identified by double enzyme digestion.The recombinant plasmid pET30a-HC and molecular chaperone pGro7were co-transformed into competent E.coli BL21(DE3) and induced by IPTG to express TTC.The obtained TTC was identified by 12% SDS-PAGE and Western blot,and purified by Ni FF metal chelate affinity chromatography column.Female Kunming mice were immunized with TTC,TTC+Al,TTC+CpG,PBS,and tetanus toxoid(TT) by subcutaneous injection,10 mice in each group.After immunization,blood samples were collected by tail cutting or eyeball picking,and the serum was separated.The antibody titer,antibody typing,IL-4 and IFNγ levels were detected by ELISA.The lung and liver of mice were harvested aseptically,and the safety of TTC was evaluated by histopathological method.Results The recombinant plasmid pET30a-HC was constructed correctly as identified by double enzyme digestion.After adding molecular chaperone pGro7,the soluble expression of the target protein accounted for 40% of the total supernatant expression.After 42 days of the initial immunization,the specific IgG antibody titer in serum of TTC+CpG group was significantly higher than that of PBS,TTC,and TT groups(t=4.753-8.624,each P <0.01),which was also higher than that of TTC+Al group,but there was no significant difference(t=0.966,P> 0.05).There was no significant difference in serum IgG1,IgG2a,IgG2b,IgG3,and IgGM antibody levels among the groups(t=0.163-0.203,P> 0.05),IgG1/IgG2a> 1,indicating that the immune pathway was biased towards Th2 type.Compared with TTC group,the serum IL-4 and IFNγ levels in TTC+Al group had no significant difference(t=2.666 and 2.470,respectively,each P> 0.05),while significantly increased in TTC+CpG group(t=18.080 and8.440,P <0.001 and <0.05,respectively).Compared with TT group,the serum IL-4 level in TTC+CpG group increased significantly(t=8.561,P <0.01),but there was no significant difference in IFNγ level(t=0.472,P> 0.05).No obvious pathological changes were found in lung and liver of mice in each group.Conclusion Molecular chaperone can improve the soluble expression level of TTC with good safety,and combined with CpG adjuvant,it can induce strong immune response.
