| Serine protease inhibitor(Serine Proteinase Inhibitor, SPI) is a class of protein superfamily which can inhibit the activity of inhibits serine protease. It can change the activity of protease to affect the protein metabolism, therefore, involving in some physiological and pathological processes, for example, blood clotting, complement activation, inflammation, apoptosis, cell migration and fibrinolytic. It also plays an important role in immune system, enhancing, inhibiting or causing fierce immune response. With the continuous detecting of serine protease inhibitor in Ascarid, Brugia malayi, pig roundworm, hookworm, Onchocerca volvulus, Haemonchus contortus, Trichinella spiralis, Anisakis, schistosomes, whipworm, Clonorchis sinensis, Paragonimiasis and Echinococcus, SPI was proved to be play an important role in the interact of hosts and parasites. KaSPI(Kazal-type serine protease inhibitor) is one of the conservative serine protease inhibitor families. In structural, KaSPI has Kazal-type structural domain which makes it different from other families. In function, it is similar to others. At present, the research of KaSPI usually find in aquatic animals, such as Chinese shrimp, Procambarus shrimp, scallops, crabs, etc. KaSPI has important significance in the antimicrobial activity of them. Up to now, there was no research in Trichinella spiralis KaSPI, therefore the research of TsKaSPI not only can provide theoretical basis for the further research of serine protease inhibitors involving in the interaction of host and parasite, but also lay foundation in the prevention of trichinosis.In this study, total RNA was detected by Trichinella spiralis muscle larva. Specific primers were designed, based on Trichinella spiralis Kazal-type serine protease inhibitor(TsKaSPI) gene coding sequence in the GenBank. And then using the total RNA reverse transcription product cDNA as template to amplify TsKaSPI. Cloned the target fragment to the pEASY-T1 and then cloning into competent Tans5α. The results detected by PCR and EcoR I, Xho I digested identified. Sequencing results showed that the cloned sequence included 852 nucleotides which can encode 283 amino acids. Structure analysis revealed TsKaSPI have Kazal-FS domain, with KaSPI characteristics. In this study, expression vector pET-30a(+) was used to build the recombinant plasmid pET-TsKaSPI. The protein expression was induced by IPTG. SDS-PAGE electrophoresis analysis of fusion protein showed the predicted protein was about 38 KDa, and the result matched with the theoretical value, mainly existed in the form of inclusion bodies.Electric elution was used to purify protein, and then the purified protein was dialyzed refolded, Western Blot showed that the purified recombinant protein can be recognized by sera of mice infected with Trichinella spiralis. To detect immune protection, the purified recombinant protein was injected into the intraperitoneally of mice. Result showed worm reduction rate of Trichinella spiralis was 38.3%. Indirect ELISA showed that mice immunized with the recombinant protein to induce specific IgG antibody levels were significantly higher than control group and adjuvant group. The interaction of renatured recombinant protein and part of the protease showed it can inhibit the active of thrombin, trypsin, cathepsin G, elastase, chymotrypsin, and the granzyme B, and the higher the concentration of the recombinant protein, the better of the inhibit effect. Polyclonal antibodies in mice was produced by immunized the mice with the purified protein, immunoblotting, immunohistochemical staining experiments, western-blot analysis showed the polyclonal antiserum antibody can recognize and bind to the recombinant protein, indicating that the recombinant protein has good immunogenicity and reactogenicity; the result of immunohistochemical staining test showed TsKaSPI expressed in muscle larvae period of Trichinella spiralis and distributed in the parasite surface and internal. |
PDF Full Text Request