| Cystic echinococcosis in hunmans and livestock animals is caused by infection with the cestode parasite Echinococcus.The disease is an important issue in public health globally and has a high prevalence and high mortality in China.The Vaccine immunization is the best way to control the diseases.Recombinant antigens Eg95 have been used to induce more than 90%protection against against hydatid disease in sheep.Objectives:To clone Eg95 gene into pET30a for expression and purification of recombinant antigens;To construct and identify the recombinant BCG-Eg95.Methods:(1)Eg95 gene was cloned into the vector pET30a to construct the pET30a-Eg95(N)and pET30a-Eg95(E),which were confirmed by PCR amplification and restriction endonucleasse digestion and sequencing.The recombinant plasmid were transformed into Ecoli.BL21 for expression.(2)Eg95 gene was cloned into Ecoli-Mycobacteria shuttle vector pMV261 to construct the recombinat plasmid pMV261-Eg95,which were introduced into BCG successfully by electroporation.The recombinant BCG postive clones were screened by Kan+ and identified by PCR.Results:(1)The 471bp Eg95 sequence was successfully cloned into the vector pET30a.The recombinant plasmid was introduced into E.coli.BL21 for protein expression.The SDS-PAGE result found that IPTG could not induce Eg95 express,while his-Eg95 could express in existence of 0.5mM IPTG.His-Eg95 had a higher productivity after culture 6h in 25℃.The best IPTG concentration was 0.05mM.We also found that his-Eg95 had a good solubility in PB.(2)We successfully constructed the recombinant Ecoli-Mycobacteria shuttle vector pMV261-Eg95,which were transformed into BCG.PCR result showed rBCG-Eg95 were constructcd successfully.The recombinant BCG-Eg95 expressed a protein approxiimately 1 8kDa after 42 ℃ incubacton.Conclusions:(1)The fusion protein Eg95 was produced in E.coli.BL21.which had a good solubility in PB.(2)We successfully constructed the Echinococcus granulosus recombinant BCG-Eg95. |
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