| Trichinellosis caused by the Trichinella spiralis is an important food-borne parasitic zoonosis.T.spiralis can infect more than 150 animal species, Such as humans, pigs, dogs, cats, rats, tigers and wolves. Trichinellosis in humans can cause fever, muscle aches, diarrhea, edema, rash,metabolic disorders, and bleeding; It also can lead to the nervous system, cardiovascular system,respiratory system complications; severe cases can lead to death. Animal infection can lead to acute catarrhal enteritis, fever, movement disorders, breathing difficulties and other symptoms.Throughout its life cycle, the parasite is exposed to the digestive enzymes in the stomach, intestine,and body fluids. Evidence shows that the secretory protease inhibitors of many parasitic intestinal nematodes can neutralize the damage of these digestive proteases within the immediate environment of the nematodes, facilitate feeding, and regulate the immune response of the host.The serine protease inhibitors（SPIs） constitute the most important of these protease inhibitor families.SPIs, which can inhibit one or more serine proteases, play important roles in the development,survival, and reproduction of animals, plants, parasites, insects, and viruses. Many SPIs have been isolated from various nematode genera including Schistosoma, Trichuris, Ancylostoma, Taenia,and other parasites. It has been suggested that SPIs play an important role in protecting nematodes from host digestive enzymes and interact with host cellular components involved in the regulation of inflammation. These SPIs can counteract the digestive activities of various target proteases including trypsin, chymotrypsin, pepsin, pancreatic elastase, and neutrophile lactase. According to the primary sequence, structural motifs and mechanism of binding, SPIs are divided into at least 18 families, such as kunitz, kazal, Serpin, TAP, and tumor-infiltrating lymphocytes（TIL） family.The experiment obtained full-length c DNA sequence from Gen Bank and designated Tsp03044, Tsp Ad5, Tsp03548（XM 003379333 and EU263307, XM003379851）. The basic physical and chemical properties, transmembrane region, signal peptide secondary structure of the evolutionary position, etc. are analyzed in detail using a variety of bioinformatics software. Based on the binding protein transmembrane region, signal peptide, we found Tsp03044, Tsp Ad5,Tsp03548 are likely to be secreted proteins, and each has a typical Ascaris, Serpin, Kazal type domain, belonging to Ascaris, Serpin, Kazal type serine protease inhibitor. Meanwhile, according to the typical structure of their family we predict the important site of action of the three proteins.In addition, the phylogenetic relationship results of Tsp03044, Tsp Ad5 and Tsp03548 indicated that: three proteins are genetically related to their family members, and have a high homology.Tsp03044, Tsp Ad5, Tsp03548 in one evolution of the branch with the serine protease inhibitor of other nematodes.In this study, we designed and synthesized primers of Tsp0304, and amplified Tsp03044 gene from adult T. spiralis genome. After sequencing, we cloned the Tsp03044 to the p ET-30 a vector.Then we used the condition of 1 m M IPTG, 18 ℃ and overnight to express soluble protein Tsp03044. In addition, we use the condition of 1 m M IPTG, 37 ℃ and overnight to express Tsp Ad5 and Tsp03548. After purification and desalting treatment, the concentrations of the purified proteins are greater than 1 μg/μl.We determined the fluorescence value of enzyme-substrate reaction by fluorescence in 10 m M Tris-HCl, calculated the reaction rates, then calculate the enzyme kinetic parameters Km and kcat values by double reciprocal method, and then get the Ki values of the inhibitory activities of Tsp03044, Tsp Ad5 and Tsp03548 against trypsin, α-chymotrypsin, and pepsin. Results showed that32 n M of Tsp03044 could significantly inhibit the serine proteases trypsin, α-chymotrypsin and pepsin. The Ki value for Tsp03044 activity against typsin, α-chymotrypsin, and pepsin was 10.05,12.53, and 1.21 n M, respectively. These data indicate that the inhibitory effect of Tsp03044 against pepsin is stronger than the inhibitory effect against the other two enzymes. 32 n M of Tsp Ad5 also inhibited the activity of α-chymotrypsin（Ki= 9.85 n M） and pepsin（Ki= 5.92 n M）. These data showed that Tsp Ad5 inhibits pepsin more strongly than α-chymotrypsin. Similarly, 32 n M of Tsp03548 also inhibited the activity of α-chymotrypsin（Ki = 139.31 n M） and pepsin（Ki = 3.83 n M）, but had no inhibition against trypsin. In addition, we also examined the inhibitory of Tsp03044, Tsp Ad5, Tsp03548 against thrombin, elastase, cathepsin G and granzyme B,respectively, by spectrophotometry.The inhibitory activities of Tsp03044 against trypsin and α-chymotrypsin, and that of Tsp Ad5 against α-chymotrypsin were investigated at different temperatures. The results showed that 37 ?C is the optimum temperature for each reaction, Tsp03044 and Tsp Ad5 could maintain their inhibitory activity against related proteases in a temperature range between 28 and 48 ?C.To identify the active site of three serine protease inhibitors, we prepared the recombinant protein which active sites were mutanted by site-directed mutagenesis, and detected the inhibition efficiency of each mutant protein against trypsin and α-chymotrypsin, the results show that the inhibition efficiency of mutanted protein against trypsin and α-chymotrypsin were lower than the inhibition efficiency of the original protein, indicating that the mutated amino acid were the active sites of the serine protease inhibitors.The expression levels of Tsp03044 and Tsp Ad5 in adult worm and muscle larvae were determined using SYBR Green I real-time RCR. Tsp03044, Tsp Ad5 and Tsp03548 were expressed at adult and muscle larvae phase, but they were much more highly expressed at the muscle larvae phase. These results indicate the important role of the two inhibitors in helping muscle larva resist host-protease digestion. The results of western blot further confirmed this conclusion.The successful cloning, expression and purification of Tsp03044, Tsp Ad5 and Tsp03548,which is belongs of Ascaris, Serpin and Kazal type serine protease inhibitor, respectively, laid the material foundation for the detection of their function. Detection of the inhibitor activity of Tsp03044, Tsp Ad5, Tsp03548 against trypsin, α-chymotrypsin, pepsin and other protease inhibitors,and determination of the expression levels of Tsp03044, Tsp Ad5, Tsp03548 laid foundation for further study the functions in protecting T. spiralis against host digestive enzyme digenstion. In summary, our results show that Tsp03044, Tsp Ad5, Tsp03548 arebeffective serine protease inhibitor, this results will provide evidence for the study the function of Tsp03044, Tsp Ad5 and Tsp03548. |
PDF Full Text Request