The Primary Epitope Study On Echinococcus Multilocularis Antigen 18

Objective:Screen special mimic epitopes of Echinococcus multilocularis antigen Eml8 by phage 7-mer peptide library and molecular biology.Method:The primers of truncated Eml 8 were designed by DNAman biosoftware and the truncated fragment were amplified by PCR from pMD18-T/Eml8,and were cloned into prokaryotic expression plasmid pET41a to construct the pET41a-Eml8-4,.The recombinant plasimid were analyzed by sequenceing.The rEml8.4-GST fusion protein were expressed by induction with IPTG and purified using His-tag column,and then were detected by Western Blot and ELISA.Use the rEml8-GST fusion protein immunizing New-Zealand White Rabbits. Then the His-Bind Resin was used to purified anti rEml8-GST in order to delete anti GST,and obtain the specific anti Eml8.Use the anti-Eml8 to screen the phage 7-mer peptide library for 5 rounds.Clones were picked out randomly and 46 specific clones were sequenced and their amino acid sequences were compared with Eml8.Results:The pET41a-Eml8.4 positive clones were the exact recombinant plasmid and the expressed rEml8.4-GST recombinant protein can be detected as a band of 41KDa by SDS-PAGE and Western blot result confirmed that the recombinant protein could not specifically react with the serum samples from patients with alveolar echinococcosis(AE).The crossing reaction with GST was deleted by purified using the His Bind Resin. After screening the phage 7-mer peptide library with anti-Eml8,The amino acid sequences of clones showed no homology with Eml8.Conclusion:The epitope of antigen Em 18 may be not on 81aa-160aa.The amino acid sequences of clones may be not the mimic epitope of antigen Eml 8...