Construction And Eukaryotic Expression Of Nucleic Acid Vaccine From Echinococcus Granulosus In Inner Mongolia

Hydatidosis is caused by the zoonotic parasite Echinococcus granulosus.There is substantial genetic variability in different Echinococcus granulosus isolates.The fatty acid binding protein(FABP) and EG95 protein were regarded as vaccine candidates to against Echinococcus granulosus.We cloned the gene of FABP and EG95 Inner Mongolia strain and constructed nucleic acid vaccine.The construction of nucleic acid vaccine for FABP and EG95 Inner Mongolia strain may provide a effective way to control hydatidosis infection in this region.The fragment of FABP gene Inner Mongolia strain was amplified by PCR and cloned into pMD19-T vector for sequencing and analyzing.The cloned FABP gene DNA sequence was 482bp.The FABP gene DNA showed 100%homology with Echinococcus granulosus XinJang strain and 99%homology with foreign strain, which indicated that there was a certain genetic variability of FABP gene in different Echinococcus granulosus isolates.The cloned FABP gene cDNA sequence was 402bp.The deduced ORF was 399bp which encoding a protein consisted of 133 amino acids.The cDNA sequence was the same as the deduced from DNA sequence.The molecular weight of FABP predicted from the cDNA sequence was 15.095Kda.The FABP-cDNA fragment was subcloned into prokaryotic pET44a(+)vector to construct recombinant plasmid pET-FABP.The fusion protein Nus-FABP was successfully expressed in E.coli BL21 induced with IPTG and was purified by affinity chromatography.The Kunming mice were immunized with the purified protein and antiserum with high titer was produced.The FABP-cDNA and EG95-cDNA fragment were cloned into eukaryotic pEGFP-N1 vector to construct recombinant plasmids pEGFP-FABP and pEGFp-EG95.Two recombinant plasmids were transfected into sheep fetal fibroblast by lipofectamine method.Results indicated that FABP-cDNA and EG95-cDNA were expressed in the form of fusion protein with green fluorescent protein in eukaryotic cells.The FABPcDNA fragment and EG95 fragment were cloned into eukaryotic pCDNA3.1(+) vector to construct nucleic acid vaccine pCDNA3.1-FABP and pCDNA3.1-EG95.Two recombinant plasmids were transfected into sheep fetal fibroblast by lipofectamine method.The RT-PCR results indicated that FABP-cDNA and EG95-cDNA were expressed in transcription level.The nucleic acid vaccine of Echinococcus granulosus Inner Mongolia srain were successfully constructed and expressed in sheep fetal fibroblast.These results have laid foundation for the application of nucleic acid vaccine to controlling hydatidosis transmission through domestic animals.