Cloning, Expresson And Functional Analysis Of A Novel Stage-specific Gene Sj314C10 Of Schistosoma Japonicum

Using the method of rapid amplifiction cDNA ends (RACE) acqured Schistosoma japonicum genes expression sequence tags (ESTs) full length sequences of adult worm stage specific expression gene. In order to discover genes encoden the novel molecules of diagnosis and candidate vaccine for schistosomiasis. After analysising a serials of expressing sequence tags of adult worms stage specific expressing gene,we screened one EST from them and designed nest PCR primers based upon this one. Then first RACE was carried, purifiction objective seqence, linking clone vector,transform into host bacteria,selecting postive cloning and sequencing were conducted. The sequence was linked to the origenal EST,and then extended electronically. As a result, a novel EST was acquired. The second RACE was continued at the same way till the DNA sequence was no longer extended. Upon the biological informatics analysis, the DNA sequence was obtained and accessed in gene GenBank after two RACE amplifications. Specific primers with double enzyme sites were designed based on the sequence which had complete open reading frame (ORF). The DNA was amplified using the total RNA as template. The product from PCR treated by enzymes and purified was cloned into expression pET28a(+) vector, which was treated by the same enymes. The recombinant plasmid was identified by restriction enzymes analysis and transiformed into E. coli BL21. The expression was induced by IPTG and the fusion protein expression was observed under different conditions. The fusion protein was collected and purified, and its biological activity was studied, its function was analysed at last. The results showed that a full length gene including a complete ORF was acquried after two RACEs. Sequence analysis revealed that the gene was a novel gene of S. japonicum(GenBank accession number was AY847290),and it was named as Sj314C10 (S. japonicum 314C10 cloning). Analysing the structure and functional motifs of Sj314C10 coden protein showed the amino acid sequence of the protein had 78% homology compared with the baculovirus IAP repeated domain. After inducing by IPTG,the result of SDS-PAGE showed that the fusion protein was approximately 50ku and got more with time prolonging. The change of cultivating tempreture,concentrations of IPTG,different expression time had no remarkable effects on the insoluble inclusion body. Function anlalysis as follows: western blotting identified that the band was specific recognized by serum from rabbites immuned by the crude antigon of S. japonicum adult worm. ELISA result indicated the mice had high antibody titer after immuned by purification fusin protein. The immunological experiments in mice showed that recombinant protein antigen did induce partial protection against S. japonicum cercaria challenge.