Prokaryotic expression of norovirus P2 domain and preparation of its antiserum
10.13200/j.cnki.cjb.003912
- VernacularTitle:诺如病毒P2结构域的原核表达及其抗血清制备
- Author:
CHEN Jianwei
- Publication Type:Journal Article
- From:
Chinese Journal of Biologicals
2024;37(8):957-963+969
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo express and purify the P2 domain of 5 genotypes(GⅠ.1,GⅡ.2,GⅡ.3,GⅡ.4,and GⅡ.17)of norovirus(NV)in prokaryotic expression system and prepare their antisera.MethodsThe bioinformatics method was used to analyze the sequence conservation of P2 domains of different genotypes of NV. Then the P2 DNA sequence was synthesized and subcloned into pET24a vector to construct the recombinant prokaryotic expression plasmid,which was transformed into E.coli BL21(DE3)competent cells and induced by IPTG. 30 female BALB/c mice were immunized with the purified recombinant P2 protein to prepare antiserum. The titer and cross reaction of antiserum were detected by ELISA,the specificity was detected by Western blot,and the recognition and binding ability to wild-type NV were analyzed by dotblot.ResultsThe expressed recombinant P2 proteins showed purity of over 90% with the relative molecular mass of about15 000. The expression levels of P2 protein of GⅠ.1,GⅡ.2,GⅡ.3,GⅡ.4,and GⅡ.17 were 60,26. 4,19. 8,16. 3 and33. 3 mg/L,respectively. The antiserum titers against five genotypes of NV P2 antigens were all over 1∶128 000,which well recognized their corresponding P2 antigens,while showed no interaction with unrelated P2 antigens and no crossreaction with recombinant human group A rotavirus VP7 antigen and recombinant Helicobacter pylori uroenzyme antigen,and had good recognition and binding ability to wild-type NV.ConclusionFive genotypes of NV P2 antigens epidemic in China were successfully expressed and purified by prokaryotic expression system. The prepared antisera had high titer and good specificity,which laid a foundation of the establishment of rapid and on-site diagnosis technology of NV.
