Cloning, Recombinant Expression And Antibody Preparation Of Eg95 Gene From Echinococcus Granulosus

Cystic echinococcosis(CE) is a parasitic disease and harmful to the health of human and livestock.CE is caused by the metacestode larval of Echinococcus granulosus(Eg) when it parasitizes in the liver and lung of host.This disease acutely affects the development of human health and graziery in China and even all over the world.Now,the important way to prevent CE is the application of molecular vaccine.Eg95 gene is regarded as the effective vaccine candidate gene.Because the antigen expressed on the surface of oncosphere can coordinate with antibody and complement to mediate the dissolve of hydatid,Eg95 vaccine can generate well prevention.However,as the difference of distinct field and host, researching on more safe and more effective Eg vaccine has important significance.In this paper,the complete Eg95 gene from Qinghai strain was amplificated, cloned and sequenced using PCR method.The genomic sequence of the complete Eg95 gene is 1262bp,which includes three extrons and two introns.The length of three extrons is 70bp,306bp and 95bp,respectively.The length of the ORF of Eg95 gene is 471bp which encode 156aa protein.By comparison with the Eg95 gene from Qinghai strain,Australia strain and American strain,the Eg95 gene show 41 nucleotides difference,36 in intron and 5 in extron.There are 2 mutations at the amino acid sequences among the four Eg95 proteins.The eg95 gene was inserted into the pGEX-5X-l expressive vector.After identification with restriction endonuclease analysis,pGEX-5X-l/eg95 was transformed into E.coli BL21,and induced to express under the 37℃and 0.8mM IPTG.The SDS-PAGE results showed that there is a specific strap about 40KD, which implied that the recombinant protein maybe was expressed.Furthermore,the\ recombinant protein was identified using MS and ELISA method.The recombinant protein was injected into rabbit to prepare the antibody to eg95.The ELISA results showed the effective valence of the antibody is about 1:1600.The above results provided the base to prepare the eg95 antibody in large scale in the future.