Construction and analysis on recombination expression vector of tegu ment membrane-associated antigen from clonorchiasis sinensis

  Objective   To identify the novel genes of clonorchiasis sinensis by screening the cDNA library and clone the screened gene-20.8kDa tegument membrane-associated antigen(CSTA 20.8)to the prokaryotic expression vectors and eukarytoyic expression vectors.

  Methods   The cDNA library of clonorchiasis sinensis were screened.The homologue of the novel sequences with a high identity was compared on amino acid and nucleotide level with blast programme on NCBI BLAST site.The motifs of the protein coded by the novel gene were searched with MotifScan in a proteins sequence on ExPA Sysite and NCBI Conserved Domain Search.Bisides, a pair of specific oligonucleotide primers via Ecoll, Xholl restriction sites respectly were designed and synthysed according to the coding region of CSTA208 gene found by screening library.The coding reg ion of CSTA20.8 gene was amplified by PCR and then cloned into the prokaryotic expression vectors PGEX-4T-1 and eukaryotyic expression vectors PCDNA3 via Ecoll and Xholl restriction sites, and transformed into E.coli BL21 respectively.The positive recombinant PGEX-4T-1-CSTA 20.8-and PCDNA3-CSTA20.8 were screened and identified by endonuclease digest ion, PCR and sequence.

  Results   A novel cDNA sequence coding CSTA20.8 was found from the cDNA library of clonorchiasis sinensis.Translation of nucleotides sequence revealed putative open reading frame of 184 amino acids with a molecular mass of 20.767 5 Kd and PI of 4.33.The predicted primary structure of CSTA20.8 shared high sequence homology with other species and contained conserved domain of Efh Motif.The recombinant plasmids PGEX-4T-1-CSTA20.8 and PCDNA 3-CSTA20.8 were constructed.

  Conclusion   Anovel gene coding CSTA20.8 of clonorchiasis sinensis was found and cloned and its prokaryotic expression vectors and eukar yotyic expression vectors were constructed successfully.