OBJECTIVES: To study the structure specificity of Echinococcus granulosus 95 (Eg95) gene and the open reading frame (ORF) of the full-length cDNA sequence in Xinjiang, northwestern China and construct Eg95 Xinjiang strain DNA vaccine.
METHODS: Primers of Eg95 were designed on the basis of the sequence of Eg95 antigen cDNA. Genomic DNA was extracted from E.granulosus protoscoleces (sheep) in Xinjiang. The Eg95 gene and full-length Eg95 cDNA were amplified by PCR from the genomic DNA and protoscolex cDNA library of E.granulosus in Xinjiang, respectively. The Eg95 gene was cloned into pUCm-T plasmid and the Eg95 cDNA into eukaryotic expression plasmid pcDNA3 for the construction of full-length ORF DNA vaccine pcDNA3-Eg95/XJ. Both Eg95 gene and Eg95 cDNA were sequenced and analyzed by DNAman and NCBI/Blast program.
RESULTS: DNA sequence analysis of Eg95 Xinjiang strain (Eg95/XJ) cDNA fragment indicated that the coding region of the full-length of Eg95/XJ was 471bp and that encoding a peptide with 156aa and the genomic DNA size was 1191bp. Homological comparison showed that the ORF of Eg95/XJ cDNA was identical to the cDNA sequence of Eg95 reported in the reading frame, but the genomic DNA was a new sequence, named Eg95/XJ and the multiple nucleotide differences, which were represented in Eg95/XJ gene in comparison with those of the New Zealand strain, occurred predominantly in the non-coding regions of the gene. The pcDNA3-Eg95/XJ positive clone was the exact recombinant plasmid and could be used as a DNA vaccine.
CONCLUSION: pcDNA3-Eg95/XJ Xinjiang strain DNA vaccine is successfully constructed.