| Mycoplasma suis(M.suis),one kind of pathogenic microorganisms which leads Porcine Eperythrozoonosis(PE),has caused tremendous economic losses to pig industries since it was discovered.Inorganic pyrophosphatases(PPase)are important hydrolytic enzymes that catalyze the hydrolysis of inorganic pyrophosphate PPi to inorganic phosphate Pi,are an essential proteinase exist in M.suis.PPase showed better immunogenicity,which implies its feasibility in the establishments of antibody preparation and pathogen detection as an antigen of M.suis.In this experiment,M.suis ppa protein was prepared through prokaryotic expression technology in vitro,which was purified and identified.The M.suis ppa gene was directly amplified by polymerase chain reaction,and cloned into pEasy T vector.The recombinant vector was transformed into Escherichia coli DH5a for cloning.Identify and subsequently sequence the products of PCR cloning.The sequences-corrected ppa was inserted into expression plasmid pET-48b(+),then the recombined vector was transformed into E.coli BL21(DE3).Fusion protein expression ppa was induced by IPTG and purified using Ni2+ immobilized metal ion affinity chromatography(IMAC),and it was identified by SDS-PAGE and Western blot methods.Experiment results show that:(1)Cloning plasmid pEasy-ppa was successfully constructed,and the results of gene sequence corresponded with that of NCBI databases;ppa is a 495 bp gene which encodes 165 amino acids.(2)Expression plasmid pET-48b-ppa was successfully constructed,and transformed into E.coli BL21(DE3)for expression inducing.The optimal conditions of inducing are 1.0 mmol/L IPTG,30? and the best inducement time is 18 h.(3)Fusion protein ppa was identified via SDS-PAGE and Western blot,and purified under 40 mmol/L and 500 mmol/L imidazole through IMAC,its size was 25 kDa.Conclusion:soluble protein ppa with high purity was successfully obtained that enabled the establishment of M.suis' ELISA effective and rapid determination method. |
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