Clonging And Construction Of The Prokaryotic Expressionvector Of Eg95 Gene From Echinococcus Granulosus In Inner Mongolia

Cystic Echinococcus(CE) is a serious zoonosis caused by infection with the metacestode stage of the typeworm Echinococcus granulosus in humans and many livestock such as sheep, goat, cattle and horse. CE is a global verminosis and is particularly epidemic in the developed region of animal husbandry. In China, CE mainly occurs in the western and northern areas such as Inner Mongolia, Xinjiang, Gansu, Ningxia, Qinghai, Sichuan and Xizang. Vaccinotherapy using CE vaccines, such as natural protein vaccine, recombinant protein vaccine, synthetic peptide vaccine, DNA vaccine and recombinant Salmonella typhimurium vaccine, is important way to control this disease. Eg95 genes are highly conservative among all strains of Echinococcus granulosus, and are expressed at the oncosphere stage. Thus Eg95 is an effective antigen candidate which can trigger a high immunoreaction to preventpeople from CE. The Eg95 genomic DNA was amplified from the hydatid cyst By PCR and ligated with pMD19-T plasmid to construct pMD19-T/Eg95 DNAvector for sequencing. The coding regions of Eg95-NM sequence was found to be identical to those of Eg95 genes reported in GenBank.The Eg95 cDNA was also amplified by RT-PCR from the hydatid cyst and sequenced. Sequence analysis showed that 5 bp of cDNA sequence were different from the coding regions of Eg95-NM genomic DNA and henceled to 4 amino acid variation. Antigen epitope analysis of the amino acid sequence deduced from the coding regions of genomic DNA sequence demonstrated that the varied amino acid 58 and 122 located in the epitopes. So the varied base pairs which coding the amino acid 58 and 122 were rectified by PCR based site-directed mutagenesis to assure the correct structure of Eg95 protein.Finally a corrected 493bp cDNA fragment carrying an open reading frame(ORF) of 471bp, which encodes a protein of about 16.9KDa, was cloned and inserted into the prokaryotic expression vector pET44a(+). A putative NusA fusion protein of about 83.3KDa, in which NusA tag is 66.4KDa and Eg95 is about 16.9KDa, was induced to be expressed by IPTG in E.coli BL21(DE3). SDS-PAGE and western blotting analysis with the positive sera of echinococcosis patients as the primary antibody confirmed that the target band of about 83.3KDa was NusA-Eg95 fusion protein. Our work may provide an alternative way to generate recombinant Eg95 antigen for inoculation against CE.