Cloning,Expression And Enzyme Activity Study Of Serine Protease Inhibitor Of Cysticercus Pisiformis

Cysticercosis pisiformis is a kind of tapeworm caused by the larva of Taenia pisiformisCysticercus pisiformis which is parasitic on the liver,omentum,mesentery and abdominal cavity of rabbits.Cysticercosis pisiformis is one of the common parasitic diseases with high infection rate in rabbits,and has great influence on the healthy development and economic benefit of rabbit breeding.Serine protease inhibitor(serpin)is a kind of protease inhibitor with molecular weight of 40 ~ 50 kDa,which not only participates in regulating the endogenous physiological function of parasites,but also plays an important role in host-parasite interaction and immune regulation or immune evasion of the host.Therefore,serpins are considered to be an important factor involved in immune regulation and other biological process,which is expected to be a candidate target for the diagnosis,biopharmaceuticals and vaccines of the parasitic diseases.On the basis of systematic analysis of Cpserpin(Cysticercus pisiformis serpin)gene and its expression characteristics,the feasibility of indirect ELISA based on recombinant Cpserpin to diagnose Cysticercosis pisiformis was explored,and the inhibitory effect of Cpserpin on serine protease was evaluated by using eukaryotic expression Cpserpin.These studies provide a theoretical basis for revealing the biological function of Cpserpin and clarifying its role in the infection of Cysticercus pisiformis.1.Using RT-PCR and rapid amplification of cDNA ends(RACE)amplified the full-length cDNA of Cpserpin gene,and followed by the bioinformatic analysis.The results showed that the full-length cDNA of Cpserpin gene is 1 471 bp and its complete open reading frame is 1 149 bp,and had a serpin motif(DEEGAE),serpin signature(FKVDHPFIFFI)and a specific reactive center loop motif(RCL).2.The recombinant plasmid pET30a(+)-Cpserpin was successfully constructed,induced in E.coli by IPTG and obtained recombinant protein Cpserpin in the form of inclusion body.Western-blotting results suggested that Cpserpin could be recongnized by positive serum of Cysticercosis pisiformis from rabbit.By optimizing the reaction conditions,the sensitivity and specificity of indirect enzyme-linked immunosorbent assays(iELISA)based on Cpserpin as coating antigen for the diagnosis of specific antibodies of Cysticercosis pisiformis from rabbit were respectively 96.7% and 93.5%,the coefficient of variation of inter-assays and intra-assays were less than 10%.These results indicate that the Cpserpin is a promising immunodiagnostic antigen for developing an iELISA to detect Cysticercosis pisiformis.3.The results of qPCR showed that the transcription level of Cpserpin in mature proglottid was 53 times higher than that in larval stage.The recombinant protein Cpserpin purified by Ni SepharoseTM 6 Fast Flow was used to immunize New Zealand white rabbits.High titer specific IgG antibody was obtained after immuned three times.The results of immunohistochemistry showed that the expression of Cpserpin was very low in Cysticercus pisiformis,but highly expressed in mature proglottid and gravid proglottid,and was widely distributed on the body wall and eggs of adult worms,suggesting that the parasites may be involved in immune attack of the host,intake of nutrition and reproductive development.4.A recombinant eukaryotic expression plasmid pPIC9K-Cpserpin was successfully constructed and expressed in KM71.SDS-PAGE and western-blotting results showed that the recombinant protein Cpserpin could not only react with labeled antibody and Cysticercosis pisiformis positive serum.The inhibitory effects of Cpserpin on ?-chymotrypsin,trypsin and elastase activity were determined by chromogenic substrate method.The results of inhibition of protease activity showed that Cpserpin had inhibitory effect on three kinds of proteases.The inhibitory effect on elastase was the best,the inhibition rate was up to 78.8%,which indicated that Cpserpin could plays a key role in the process of resisting the damage or destruction of digestive enzymes and absorbing nutrients through the body wall of the parasites.