Cloning, Expression And Characterization Of Cathepsin B From Taenia Solium

Taenia solium metacestodes,that can cause cysticercosis in pigs and humans, seriously affect the production of pigs and the health of human beings. Cathepsin B is one of the improtant members of cysteine proteases. It was proved by many previous studies that cathepsin B had a variety of functions in parasite pathogenicity and facilitated parasite evasion from host immune responses, essential nutrient uptake and tissue penetration, which contributes the enzyme a promising target for chemotherapy or immunoprophylaxis. Therefore, this research was focused on the cloning, expression and characterization of cathepsin B gene from T.solium. We have achieved the following results:1. Making use of the c DNA of T.solium as a template, we first amplified cathepsin B gene. We also used bioinformatic tools to analyze the gene sequence. The sequence analysis showed that its ORF was 1074 bp in length,encoding 357 amino acids. The theoretical molecular mass of its encoded protein was appropriately 39.27 ku. The results showed that Tscp B owned typical cysteine protease domain and conserved active sites of cathepsin B. The result of real-time PCR indicated that Tscp B was higher expressed in the adult stage, compared with the larval stage.2. The recombinant expression plasmid p ET30a-Tscp B was successfully constructed. After inducing with IPTG, we got the recombinant protein whose size was about 37 ku as insoluble inclusion bodies. Western blotting analysis showed that r Tscp B was recognized by the serum of swine infected with porcine cysticercosis. We immunized rabbits with the r Tscp B. After 4 times immunization, a high titer of specific Ig G antibodies was produced in rabbits. Immunohistochemical results showed that r Tscp B was mainly expressed in the adult worm tegument and reproductive organs, which indicated that Tscp B may be involved in the essential nutrient uptake and growth.3. The recombinant expression plasmid p PIC9K-Tscp B in P.pastoris was also successfully constructed. The recombinant protein showed the catalytic activity to cleave gelatin. Furthermore, y Tscp B showed typical properties of cysteine proteases and could efficiently hydrolyze Z-Phe-Arg-AMC, and the optimal activity was observed at p H 5.5. In addition, swine Ig G as humoral molecules and fibronectin as extracellular were chosen to investigate the ability of y Tscp B to degrade macromolecules. All of the protein substrates used was completely digested by y Tscp B and degradation of protein substrates was almost completely inhibited by adding a cysteine peptidase inhibitor, E-64. Protein substrate digestion assay revealed that Tscp B may play important roles in invasion and nutrition.4. The structure of Tscp B was predicted using homology modeling approaches. And the interaction between Tscp B and E-64 was predicted through molecular docking method.