Prokaryotic Expression Of H15 ES Antigen Gene Of Haemonchus Contortus And Establishment Of Indirect ELISA Of Recombinant Protein

Haemonchosis was a parasitic disease caused by Haemonchus contortus. It could infected many ruminants including cattle,sheep,deer,poephogus grunniens,camel and so on. It was distributed all over the world. It was reported every area in China. Haemonchosis could cost great expenses in animal industry. The disease mainly used chemicals to cure, but medicine remains and environment pollution were seriously. It had drug resistance, and not show off apparente clinical symptom after infection. To control the disease effectively, it is important in the aspect of diagnosis and treatment of Haemonchosis.The primers were designed and synthesized according to sequence of Haemonchus contortus published in GenBank(U64792). H15 ES antigen gene of Haemonchus contortus was amplified by RT-PCR.The fragments of cDNA were cloned into pMD18-T plasmid and sequenced. The sequence analysis indicated that H15 ES antigen gene was made up of 453 nucleotides, including the ORF, and encoding a protein of 148 amino acid residues. The sequence analysis showed that the sequence homology was 99.78% with U64792 of GenBank, and the homology of their deduced amino acid sequence is 100%.The physico-chemical property, secondary structure, hydrophilicity, flexibility, surface probability and antigenicity of H15 ES antigen protein from Haemonchus contortus were analyzed by bioinformatics software and network platform with methods of bioinformatics, its lymphocyte epitopes with specificity of antigen were predicted and evaluated, and some potential antigenic determinants were worked out. Psort analysis indicated that the possibility of the ES antigen localized in the extracellular was 66.7%. ProtFun forecasts suggested that possibility to judge this protein as an enzyme was 0.361, and with signal transduction, immune response, stress response function of higher possibility.H15 ES antigen gene was amplified from pMD18-T-H15 ES by RT-PCR. And then it was recombinated into prokaryotic expression vector pET32a(+). The recombinant plasmid pET32a- H15 ES was transformed into E.coli BL21 and induced to express by IPTG. The result of SDS-PAGE analysis showed that H15 ES antigen gene was successfully expressed with the form of inclusion body and molecular weight of recombination protein was 35.2ku. The immunity reactivity of the recombinant protein was confirmed by Western-blot. Purified by Ni-NTA Agarose, H15 ES antigen protein was used as an detection antigen to establish the indirect ELISA method. The coating concentration of recombination protein and sera dilute strength of H15 ES antigen protein were 2μg/ml and 1:200, and cut-off value is OD450nm=0.203.This will lay the foundation to obtain diagnostic and protective antigen of Haemonchosis and study nucleic acid vaccine of Haemonchus contortus.