OBJECTIVES: To study the structure specificity of Echinococcus granulosus 95 (Eg95) gene and theopen reading frame (ORF) of the full-length cDNA sequence in Xinjiang, northwestern China andconstruct Eg95 Xinjiang stra...OBJECTIVES: To study the structure specificity of Echinococcus granulosus 95 (Eg95) gene and theopen reading frame (ORF) of the full-length cDNA sequence in Xinjiang, northwestern China andconstruct 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 andfull-length Eg95 cDNA were amplified by PCR from the genomic DNA and protoscolex cDNA library ofE. granulosus in Xinjiang, respectively. The Eg95 gene was cloned into pUCm-T plasmid and the Eg95cDNA into eukaryotic expression plasmid pcDNA3 for the construction of full-length ORF DNA vaccinepcDNA3-Eg95/XJ. Both Eg95 gene and Eg95 cDNA were sequenced and analyzed by DNAman andNCBI/Blast program.RESULTS: DNA sequence analysis of Eg95 Xinjiang strain (Eg95/XJ) cDNA fragment indicated thatthe coding region of the full-length of Eg95/XJ was 471bp and that encoding a peptide with 156aa and thegenomic DNA size was 1191bp. Homological comparison showed that the ORF of Eg95/XJ cDNA wasidentical to the cDNA sequence of Eg95 reported in the reading frame, but the genomic DNA was anew sequence, named Eg95/XJ and the multiple nucleotide differences, which were represented inEg95/XJ gene in comparison with those of the New Zealand strain, occurred predominantly in thenon-coding regions of the gene. The pcDNA3-Eg95/XJ positive clone was the exact recombinant plasmidand could be used ms a DNA vaccine.CONCLUSION: pcDNA3-Eg95/XJ Xinjiang strain DNA vaccine is successfully constructed.展开更多
基金This work was supported by a grant from NIH Project Foundation (No. I-RO-I-TWO-I565-01) and Xinjiang Natural Science Foundation of China (No. 990103003).
文摘OBJECTIVES: To study the structure specificity of Echinococcus granulosus 95 (Eg95) gene and theopen reading frame (ORF) of the full-length cDNA sequence in Xinjiang, northwestern China andconstruct 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 andfull-length Eg95 cDNA were amplified by PCR from the genomic DNA and protoscolex cDNA library ofE. granulosus in Xinjiang, respectively. The Eg95 gene was cloned into pUCm-T plasmid and the Eg95cDNA into eukaryotic expression plasmid pcDNA3 for the construction of full-length ORF DNA vaccinepcDNA3-Eg95/XJ. Both Eg95 gene and Eg95 cDNA were sequenced and analyzed by DNAman andNCBI/Blast program.RESULTS: DNA sequence analysis of Eg95 Xinjiang strain (Eg95/XJ) cDNA fragment indicated thatthe coding region of the full-length of Eg95/XJ was 471bp and that encoding a peptide with 156aa and thegenomic DNA size was 1191bp. Homological comparison showed that the ORF of Eg95/XJ cDNA wasidentical to the cDNA sequence of Eg95 reported in the reading frame, but the genomic DNA was anew sequence, named Eg95/XJ and the multiple nucleotide differences, which were represented inEg95/XJ gene in comparison with those of the New Zealand strain, occurred predominantly in thenon-coding regions of the gene. The pcDNA3-Eg95/XJ positive clone was the exact recombinant plasmidand could be used ms a DNA vaccine.CONCLUSION: pcDNA3-Eg95/XJ Xinjiang strain DNA vaccine is successfully constructed.
