Immunoscreening Of Cysticercus Cellulosae CDNA Library And Cloning, Expression, Identification Of The Genes

Objective:Cysticercus Cellulosae cDNA Library was immunoscreened to discover genes encoding the novel molecules of diagnosis and candidate vaccine for cysticercosis.Methods:The anti-sera were collected from mixture sera of cysticercosis patients and eliminated anti-E-coli antibodies by E-coli lysate,used as antibody to screen cDNA Library constructed.The clones were selected from rescreen two times positive clones and followed sequencing.The obtained genes were compared against GeneBank datebase by BLASTn and BLASTx.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 pMD-18T vector.Then the target gene was subcloned into the eukaryocyte expression vector(pET-28a).The recombinant vector was identified by restriction analysis and transformed into E.coli BL21.The expression was induced by IPTG and the result was identified using SDS-PAGE and Western-blot.Results:The single phageblots for positive clones on the NC membrane the was initially determined after DNA library screening,which was amplified DNA fragment of different sizes in 200～1800bp;The sequence data of positive clones were analysed in GeneBank datebase.Sequence of Ts1 clone is Cysticercus Cellulosae one of the unknowed gene.Sequence of insert of Ts1 clone has 681 bp-size complete open reading frame(ORF).In PubMed the blast procedure was determined as a new gene (named Ts1),which was recorded EU009656;The size of PMD-18T-Ts1, pET-28a(+)-Ts1 digested with BamHⅠand HindⅢwas identical to the length of the PCR generated products.DNA sequencing indicated that the Ts1 gene has an open reading frame of 681bp,which encodes 227 amino acids with an approximate molecular weight of 25.86 kDa.After inducing by IPTG,the result of SDS-PAGE showed that the fusion protein was approximately 28 kDa and got more with time prolonging.Western-blot identified that the band was specially recognized by sera from cysticercosis patients infected.Conclusion:Cysticercus Cellulosae Ts1 gene was successfully screened,cloned and expressed,the protein hopefully became diagnosis antigen.