Immunoscreening Of Schistosomulum Japonicum CDNA Library And Cloning, Expression, Identification Of The Genes

Objective: Schistosomulum japonicum cDNA Library was immunoscreened to discover genes encoding the novel molecules of diagnosis and candidate vaccine for schistosomiasis Memods:The mixture patient sera of schistosomiasis were collected and eliminated anti-E-coli antibodies by E-coli lysate,used as antibody to screen cDNA Library constructed.The 3 clones were selected from rescreening three times positive clones and followed sequencing.The obtained genes were compared against GeneBank datebase by BLASTN and BLASTP.Specific primers were designed based upon the sequences of insert which have complete open reading frame. The DNA was amplified using the plasmid as template.The product from PCR was cloned into pGEM-T vector.Then the target gene was subcloned into the expression vector(pBK-CMV).The recombinant vector was identified by restriction analysis and transformed into E.coliBL21.The expression was induced by IPTG and the result was identified using SDS-PAGE and Western blotting. Results: About 7.2 X 103 recombinant phages were screened,forty-eight positive clones were obtained in which eighteen were rescreened until the twelve continuing positive clones were comfirmed.The sequence data of three positive clones were analyzed in GeneBank datebase.Sequence of H4 clone is 5)14-3-3 gene and that of H9 clone is Sj mitochondrion gene. Sequence of insert of HI clone has 1186bp and a 591bp-size complete open reading frame(ORF). The HI DNA fragment was amplified by PCR and cloned into pGEM-T vector and then subcloned into pBK-CMV vector.The same bands were obtained by double restriction of recombinant plasmids and PCR using recombinant plasmids as template.After inducing by IPTG, the result of SDS-PAGE showed that the fusion protein was approximately 26.5KDa andgot more with time prolonging. Western blotting indentified that the band was specially recognized by sera from schistosomiasis patients infected. Conclusion: Some molecules coding possibly protective immunity for schistosomiasis were obtained by immunoscreening Schistosomulum japonicum cDNA Library. The following development of protective vaccine and diagnostic antigen was base on sequencing, cloning, inducing expressing and identifying by methods of molecule biology...