Immunology Of TsAdSPI Gene From Trichinella Spiralis Adult And Biological Activity Research Of Ts AdSPI

Trichinellosis is an important zoonosis,a serious threat to public health and safety. Infected mainly through the ingestion of raw or unprocessed completely containing the infective larvae of meat and infection.Trichinosis has not been controlled fundamentally, infection rates in recent years has a tendency to rebound.Serine protease inhibitors(SPI) are structurally conserved protein family, widely exists in many organisms, is one of the main regulating protein. Play an important role in many physiological and cytological features, such as coagulation, inflammation and cell apoptosis. In a variety of parasitic infection process, SPI play an important role in the parasite and host interaction.The experiment extracted total RNA of 5 day old adult Trichinella spiralis, designing primers according to the land of serine protease inhibitor gene of trichinella spiralis in Gen Bank(registration number: EU263307.1). To obtain the c DNA through RT-PCR method, PCR method to gain serine protease inhibitor(Ts Ad SPI) gene of adult Trichinella spiralis. Cloned the target fragment to the p MD18-T vector through T4 ligase. The target fragment was connected to the expression vector p ET-28 a after identified correctly. After double enzyme digestion, PCR and sequencing appraisal, we used IPTG to induce expression on target protein. SDS-PAGE results showed that the target protein molecular weight is 44 k Da, in the form of inclusion body. The induction time, temperature, concentration of IPTG optimization results show that the induction time was 5h, temperature 37 C, the concentration of IPTG was Ts Ad SPI 1.0mmol/L the expression of recombinant protein is maximum.The Ts Ad SPI recombinant protein was purified, after emulsification with Freund’s adjuvant rabbits were immunized three times to make polyclonal antibodies. After separation of immune sera, immunogenicity of TsAd SPI recombinant proteins were analyzed by Western-blot method, the result showed polyclonal antibodies can effectively identify TsAd SPI recombinant protein and strip single. By indirect ELISA method for the preparation of polyclonal antibody titers were detected, the result showed polyclonal antibody titer reach up to 1: 51200. These results show that Ts Ad SPI recombinant protein has good immunogenicity, can induce the body to produce an efficient and highly specific immune response. We isolate the serum of mice infected with Trichinella spiralis for 5 days, Western-blot analysis showed that the serum can be identified by Ts Ad SPI recombinant protein, we prove that Ts Ad SPI recombinant protein has a good reactogenicity.Immunohistochemical localization of Ts Ad SPI recombinant protein of Trichinella spiralis in 5 day old adult and muscle larva found, the distribution of Ts Ad SPI protein was found in the two periods of the worm body and body surface. The assay of inhibitory activity by chromogenic substrate showed the recombinant Ts Ad SPI protein can effectively inhibit the activity of trypsin, chymotrypsin and elastase, the inhibition rates were 88%, 72%, 42%.The results show that, the Ts Ad SPI protein can induce good immune response, so it has good immunogenicity. Serum of mice infected with Trichinella spiralis can effectively identify the recombinant protein show that the recombinant Ts Ad SPI protein has good reactionogenicity. Through inhibitory activity of Ts Ad SPI recombinant proteins to the digestive enzyme clarified the trichinella spiralis against host digestive system and possible mechanisms of invasion of the host. The above results show that Ts Ad SPI recombinant proteins can be used as a candidate antigens in early diagnosis of trichinella spiralis infection and lay a solid foundation for the development of new vaccines and drugs. At the same time, also lay a theoretical foundation for elucidating the mechanism of Trichinella spiralis invasion of host.