关键词: 日本血吸虫, 磷酸甘油酸变位酶, 烯醇酶, 多表位疫苗, 免疫保护

Abstract:

Objective To construct and express the recombinant plasmid pET32a-SjPGAM-SjEnol and evaluate its immuno-protective efficacy against the infection of Schistosoma japonicum in mice. Methods The peptides of SjPGAM and SjEnol containing the multivalent epitopes with higher binding capacity of human MHCⅡand mouse H2-dⅡ but low homology with the host were analyzed and screened through bioinfomatics. The corresponding nucleotide sequence of selected epitopes was spliced and the recombinant plasmid pET32a-SjPGAM-SjEnol was constructed and expressed in Escherichia coli BL21 cells. The antigenicity of the recombinant protein was detected by Western blotting and the protective effect induced with the recombinant was evaluated in mice. 55 BALB/c mice were randomly divided into 5 groups each with 11. Mice from groups A, B and C were injected with a mixture of recombinant protein （27 μg） pET32a-SjPGAM-SjEnol （A）, pET28a-SjPGAM （B） and pET28a-SjEnol （C） respectively together with 206 adjuvant, mice from groups D and E received adjuvant or PBS only, all injected for three times at two-week intervals. Mice were then challenged with 40±2 cercariae of S. japonicum at two weeks after the last vaccination, and sacrificed for perfusion by 6 weeks post infection. Adult worms were collected, the number of eggs in a gram of liver tissue was counted, and the rates of worm reduction and egg reduction were calculated. Serum samples were collected before vaccination, every one week after each inoculation and before sacrifice, and specific IgG was detected by ELISA. Results The sequences encoding the 96-147 aa of SjPGAM and 233-312 aa of SjEonl were chosen for constructing the recombinant plasmid, a cDNA fragment with the length of 447 bp was amplified by PCR. The recombinant plasmid was expressed in E. coli with a molecular weight of M_r 33 000. Western blotting revealed that the fusion protein was recognized by the rabbit serum specific to SjSWAP, and showed an adequate antigenicity. Vaccination experiment showed that when compared with those of the blank control, the worm reduction rate in group A was 39.7%, significantly higher than that of groups B （18.5%） and C （14.7%） （P<0.05）. The liver egg reduction rate in group A was 64.9%, also higher than that of groups B （47.5%, P<0.05） and C （30.5%, P<0.01）. ELISA showed that the serum specific IgG in group A （2.372±0.268） was much higher than that of groups D （0.490±0.138） （P<0.01） and E （0.220±0.088） （P<0.01）. Conclusion The recombinant plasmid pET32a-SjPGAM-SjEnol has been constructed, and recombinant protein pET32a-SjPGAM-SjEnol induces higher immuno-protection against S. japonicum than that of SjPGAM and SjEonl.

Key words: Schistosoma japonicum, Phosphoglyceromutase, Enolase, Multivalent epitopes vaccine, Immunoprotection