Cloning, homological analysis and construction of Eg95 Xinjiang strain DNA vaccine.
OBJECTIVE
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.


CONCLUSIONS
pcDNA3-Eg95/XJ Xinjiang strain DNA vaccine is successfully constructed.
Lin
Ren-Yong
RY
Xinjiang Hydatid Basic Medical Key Lab, First Teaching Hospital, Xinjiang Medical University, Urumqi 830054, China.
Ding
Jian-Bing
JB
Wen
Hao
H
D000818:Animals; D002681:China; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis; D004443:Echinococcosis / Q000517:prevention & control*; D004446:Echinococcus / Q000235:genetics*; D019353:Endemic Diseases; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012756:Sheep; D019444:Vaccines, DNA / Q000494:pharmacology*
D018076:DNA, Complementary; D019444:Vaccines, DNA