Expression And Characterization Of Serine Proteases Of Echinococcus Granulosus And E.multilocularis

Objective: 1)To express serine protein(SP)of Echinococcosis by prokaryotic expression system and product polyclonal antibody;2)To detect the proteins from different developmental stages of protoscolex by antibodies against Echinococcus serine proteins;3)To analyze serine proteins expression at different developmental stages of Echinococcus;4)To analyze the hydrolase activity of the recombinant serine proteases and its effect on cell chemotaxis.Methods: 1)EmSP and EgSP gene fragments were amplified using PCR and sequenced.According to the sequence analysis,functional domain sequence as a part of the gene(SP1)and the full-length gene(SP2)were cloned and expressed.Recombinant plasmid vector pET30a-SP1 and pET30a-SP2 were constructed with NdeI/HindIII restriction enzymes.E.coli BL21(DE3)was transformed and PCR positive clones were selected.Sequencing analysis confirmed that the open reading frames were corrected.Recombinant proteins were expressed in BL21 induced by IPTG;2)The recombinant proteins were purified by Ni-IDA affinity column;the molecular weights were determined by SDS-PAGE and Western blotting;3)BALB/c mice were immunized with recombinant SP to prepare polyclonal antibodies,and the titers of the antibodies were detected by ELISA;The immunogenicity of recombinant antigen was detected by Western blotting;4)The hydrolytic activity of proteins was detected by hydrolyzing Z-Phe-Arg-AMC and Z-Arg-AMC releasing AMC;5)The effect of SP on C5 a chemotaxis was analyzed by cell migration experiment;Results: 1)The basic characteristics of EgSP and EmSP were compared by bioinformatics software.it was found that they had the same molecular weight,signal peptide,transmembrane region,spatial structure and functional domain,38 KDa contains a trypsin-like serine protein conserved domain;2)PCR amplified both SP fragments were 1455 bp.SP1 and SP2 genes were synthesized after sequencing and identification.Restriction enzyme digestion and sequencing analysis showed that the inserted fragment was right in size and open reading frame was corrected.Recombinant plasmid pET30a-SP1 and pET30a-SP2 weresuccessfully constructed.SP1 and SP2 were expressed in E.coli induced by IPTG in LB medium;3)SDS-PAGE analysis showed that the proteins were insoluable and in form of inclusion bodies,with a relative molecular weight of about 38 kDa and 54 kDa.Western blot analysis showed the purified band of recombinant protein was recognized by both anti-His-Tag antibodies and pooled sera from AE patients;4)The relative quantitative results of qRT-PCR showed that the expression of SP was higher in cystic germinal layer and adult stage than that in protoscolex.The difference was statistically significant(P<0.001).The expression of SP was the highest in germinal layer cells and secondly was in adult stage.In E.multilocularis,the expression of Antigen5 was opposite,and the expression of SP decreased compared with protoscolex(P < 0.001).The results of Western blotting were consistent with qRT-PCR;5)The ability of SP to hydrolyse Z-Phe-Arg-AMC and Z-Arg-AMC to release free AMC were strongly effected by pH.SP1 and SP2 were most active in sodium phosphate buffer(pH7.2),followed by Tris-HCl(pH8.6),it has almost no activity in sodium acetate solution(pH5.0)and was not hydrolyze Z-Phe-Arg-AMC/Z-Arg-AMC into free AMC.The activity of HCF in sodium phosphate and Tris-HCl was the same as that of SP1 and SP2,but the ability of hydrolyzing Z-Phe-Arg-AMC in sodium acetate was significantly higher than that of Z-Arg-AMC;6)Recombinant SP reduced the attraction of chemokine C5 a for cell migration.The extracted natural protein also reduced the ability of chemotaxis,but the effect was not as higher compared to the recombinant SP.Conclusion: The cloned SP genes were successfully expressed in prokaryotic expression system.The recombinant proteins were recognized by AE and CE patient sera,indicating the proteins have good reactivity and immunogenicity.SP genes are present in different stages of two Echinococcus,and are increasing in the process of E.granulosus development from protoscolexs to cysts while reducing in E.multilocularis.SP of Echinococcus has serine activity and can reduce cell chemotaxis,suggesting that the differential expression of SP in the larvae of the two types of Echinococcus and the activity of SP in the two species of Echinococcus are the key regulators in the interface pathological phenotypes between host and parasite.