Secreening, Cloning, Expression And Identification Of A Novel CDNA Encoding An Protein Of Cysticercus Cellulosae

Objective:In order to find new diagnostic antigen and candidate molecules vaccine of Cysticercus cellulosae,the cDNA library was immuno-screened.Methods:The cDNA library was immuno-screened by using cysticercosis patients' antiserum.After the second rescreening,nine clonies were isolated and sequenced respectively.Compare the result of sequencing in GenBank,four gene fragments with complete ORF were selected.According which,primers were designed and the insertion fragments were amplified using PCR.The ORF were then cloned to PMD-18T and subcloned into PET28a.The recombinants were induced to express the target protein, and were analyzed by SDS-PAGE and Western blotting.Results:Six thousand and one hundred recombinant phages were first screened.Then 9 clones and 4 clones were selected by the first rescreening and the second rescreening, respectively.Four positive clones were obtained and sequenced,which were unknown sequence of Cysticercus cellulosae.Among which,a fragment numbered 5H with a complete 528bp ORF was amplified by PCR,the positive recombinant plasmids pET28a-5H were identified by restriction endonuclease digestion and PCR,and the size of gene fragment was in accordance with the expected one.Then,the objective fragment was high expressed in vector PET28a.The size of expressed protein was about 24kDa with the same size as putative peptide together with the His-tag in the vector. The recombinant could be recognized by serum from cysticercosis patients' antiserum.Conclusion:Some candidate molecules vaccine were obtained from the cDNA library by immunoscreening.One clone numbered 5H was sequenced,cloned and could be induced to express successfully,which will be benefit to carry out new diagnostic antigen and candidate molecules vaccine of Cysticercus cellulosae by molecular biology methods.