Cloning, expression of gene SjOST48 from Schistosoma japonicum and evaluation of the immunoprotective efficacy of rSjOST48 in mice.
- Author:
Yantao LIU
;
Yang HONG
;
Min ZHANG
;
Qian HAN
;
Xiaodan CAO
;
Sha LI
;
Ke LU
;
Hao LI
;
Zhiqiang FU
;
Jiaojiao LIN
- Publication Type:Journal Article
- MeSH:
Animals;
Antibodies, Helminth;
blood;
Cloning, Molecular;
DNA, Complementary;
Escherichia coli;
Female;
Genes, Helminth;
Helminth Proteins;
genetics;
immunology;
Immunoglobulin G;
blood;
Male;
Mice;
Mice, Inbred BALB C;
Real-Time Polymerase Chain Reaction;
Recombinant Proteins;
immunology;
Schistosoma japonicum;
genetics;
Schistosomiasis japonica;
prevention & control;
Vaccination
- From:
Chinese Journal of Biotechnology
2015;31(4):501-511
- CountryChina
- Language:Chinese
-
Abstract:
To identify SJCHGC01743 gene of Schistosoma japonicum and evaluate the potential of the recombinant protein as a new vaccine candidate for schistosomiasis, polymerase chain reaction (PCR) technique was used to amplify the cDNA of the gene and real-time RT-PCR was used to analyze the transcription profiles of SJCHGC01743 at different development stages. Recombinant plasmid was successfully constructed and transformed into competent Escherichia coli BL21 (DE3). Then the recombinant protein was expressed, purified and emulsified with ISA206 adjuvant to immunize BALB/c mice for three times. The immunogenicity was confirmed by Western blotting and tissue localization was detected by indirect immunofluorescent assay. The specific antibody level was detected by ELISA. The immunoprotection of rSjOST48 was evaluated by the reduction in worm and egg counts in mice. A cDNA with 1 248 nucleotides was isolated from 28-day-old schistosomes cDNAs by PCR. Sequence analysis revealed that SJCHGC01743 was a 48-kDa subunit of the oligosaccharyltransferase complex (OST48) and named as SjOST48. Real-time PCR analysis indicated that this gene was expressed in all investigated stages and had the highest expression level in 28 d worms, the level of gene transcription in female worms was significantly higher than that of male worms. Then recombinant plasmid pET28a(+)-SjOST48 was successfully constructed and expressed in E. coli BL21 (DE3). Western blotting analysis showed that rSjOST48 had good immunogenicity. Indirect immunofluorescent analysis revealed that SjOST48 was mainly distributed on the tegument of the worms. The result of ELISA indicated that the rSjOST48 vaccinated group could induce a significant increase in the level of specific IgG, IgG1 and IgG2a. An immunoprotection experiment showed that the vaccination of rSjOST48 in mice induced 32.62% (P < 0.05) reduction in the numbers of worms and 57.61% (P < 0.01) in eggs in liver, compared with that of the control group. This study provides the foundation for proceeding further research on the biological function of SjOST48 and screening new vaccine candidates for schistosomiasis.
