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Prokaryotic Expression and Characterization of Serine Proteinase Inhibitors from Taenia solium
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摘要
Aim Taenia solium is an important parasitic zoonotic parasite which causes a great threat to human and swine. However, few studies focus on the fundamental research of pork tapeworm and no highly efficient drugs or vaccines can be used to control the disease. Serine proteinase inhibitor(Serpin) plays a key role in inhibiting blood coagulation, resentencing host protease damage, and escaping from host immune attacks. Therefore, Serpin may represent a potential target to develop new intervention tools against the parasite. Methods Five of serpins of T. solium were found from Gene DB and then specific primers were designed. The c DNA sequences of T. solium serpins were cloned by RT-PCR from total m RNA. The gene sequences were analyzed using bioinformatic tools. The constructed recombinant expression plasmids p Cold-Ts-Serpin were transformed into E. Coli BL21(DE3) and the expression condition was optimized in order to increase its solution. Western blot assay was performed to test the immune reactivity of the protein. Results The sequence analysis showed that each of the five sequences contained a typical serine protease inhibitors motif and conserved active sites of serine protease inhibitors. No signal peptide and transmembrane domain were detected. There ORFs were 1149 bp, 1131 bp, 1065 bp, 1350 bp and 1206 bp in length, respectively. Three recombinant expression plasmid p Cold-Ts-Serpins were successfully constructed and induced by IPTG. SDS-PAGE analysis showed that the recombinant proteins were expressed in inclusion body. Western blot results suggested that Tsp Cold-Serpins could be identified by rabbit lenticular cysticercus serum and anti-His serum antibody, indicating that the purified recombinant proteins have good immunoreactivities. The inhibitory activity analysis showed that the Ts-Serpins did display inhibitory activity against porcine pancreatic elastase and human neutrophil elastase. Conclusion This study demonstrated that Ts-Serpin might be involved in the adult stage of the T. solium and neutralize the digestive proteases. Ts-Serpins may also play a key role in invasion, in inflammation and host immume evasion. This will lay the foundation for the further study about the function in evade immune effect of T. solium, and provide a basis for the development of new drugs or vaccines.
Aim Taenia solium is an important parasitic zoonotic parasite which causes a great threat to human and swine. However, few studies focus on the fundamental research of pork tapeworm and no highly efficient drugs or vaccines can be used to control the disease. Serine proteinase inhibitor(Serpin) plays a key role in inhibiting blood coagulation, resentencing host protease damage, and escaping from host immune attacks. Therefore, Serpin may represent a potential target to develop new intervention tools against the parasite. Methods Five of serpins of T. solium were found from Gene DB and then specific primers were designed. The c DNA sequences of T. solium serpins were cloned by RT-PCR from total m RNA. The gene sequences were analyzed using bioinformatic tools. The constructed recombinant expression plasmids p Cold-Ts-Serpin were transformed into E. Coli BL21(DE3) and the expression condition was optimized in order to increase its solution. Western blot assay was performed to test the immune reactivity of the protein. Results The sequence analysis showed that each of the five sequences contained a typical serine protease inhibitors motif and conserved active sites of serine protease inhibitors. No signal peptide and transmembrane domain were detected. There ORFs were 1149 bp, 1131 bp, 1065 bp, 1350 bp and 1206 bp in length, respectively. Three recombinant expression plasmid p Cold-Ts-Serpins were successfully constructed and induced by IPTG. SDS-PAGE analysis showed that the recombinant proteins were expressed in inclusion body. Western blot results suggested that Tsp Cold-Serpins could be identified by rabbit lenticular cysticercus serum and anti-His serum antibody, indicating that the purified recombinant proteins have good immunoreactivities. The inhibitory activity analysis showed that the Ts-Serpins did display inhibitory activity against porcine pancreatic elastase and human neutrophil elastase. Conclusion This study demonstrated that Ts-Serpin might be involved in the adult stage of the T. solium and neutralize the digestive proteases. Ts-Serpins may also play a key role in invasion, in inflammation and host immume evasion. This will lay the foundation for the further study about the function in evade immune effect of T. solium, and provide a basis for the development of new drugs or vaccines.
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