The Influence Of T.spiralis SPI On The Progress Of The Infective Larva Invading Into Intestinal Epithelial Cell Of The Host

Trichinosis is a serious disease caused by Trichinella spiralis which is one of the three most important food-borne parasitic zoonosis. People get this disease by digest the incomplete cooked meat containing the infectious larvae.The progress of the infective larvae invade into intestinal epithelial cell is the first step of the infection, Evidence shows that many parasitic intestinal nematodes secretory E/S proteins to neutralize and regulate the immune response of the host to facilitate the inf ection, and SPI super family were the most important one among these E/S proteins which had some conserved structural motifs.It has been suggested SPIs of nematodes could be involved in resistance to the digestive enzymes of hosts and interact with host ce llular components involved in the regulation of inflammation.At first,we isolate the total RNA of the muscle larvae 45 dpi and the cDNA was prepared by RT-PCR. Then the c DNA was tend to PCR using the primers designed according to the GENEBANK. The PCR production attached to 18 T vector were sequenced and the most perfect one was reserved for the following assay.The target fragment was cut from 18 T vector and connected to p ET-28 a expressing vector then turn to E.coli BL-21 and identified by PCR as well as dual-enzyme digestion.The IPTG was used to induce the expression on target protein.the results showed that the target protein expressed as inclusion body has the molecular weight of 42 k Da.and the most perfect condition of induction was inducted along with 1m M IPTG at 37℃ for 5h.After purified by chelating resin column,the recombinant protein was turn to Western-blot by anti-his antibody and the result showed that it was expressed perfectly.After emulsification with Freund’s adjuvant,the recombinant prote in was immunized to BALB/c mouse for four times to obtain polyclonal antibodies.then turn to Western-blot by antiserum antibody.the result showed that the Ts11-1 recombinant protein can be identified by polyclonal antibodies effectively and the stripe was single.the polyclonal antibody titers were detected by ELISA, the result said that the polyclonal antibody titer reach up to 1:1×105.all the results show that the TS11-1 recombinant protein has good immunogenicity and good reactogenicity.The enzyme activity inhibition assay was performed by chromogenic peptide substrate method using related chromogenic substrate,the result show that the TS11-1 recombinant protein could inhibit the activity of trypsin, chymotrypsin and pepsin effectively and the inhibition rates were 71.1% 73% and 72.5% respectively.We have also constructed the infectious larvae-intestinal epithelial cell invasion model and optimized the condition of invasion.the larvae co-cultured with the TS11-1 and the TS03044 recombinant protein along with their antiserum in different density were turned to invasion model to explore the their influence against the invasion.the result of TS11-1 experiment group show that the IR grown along with the increase of the density and the invasion rate was turn to supreme as 82.5% by 200μg/ml TS11-1 recombinant protein. The IR turn down along with the increase of the antiserum and the minimum was 73.5% by 10% antiserum. his result indicate that the TS11-1 recombinant protein has some accelerate influence on the p rogress of the T.spiralis infectious larvae invading into the intestinal epithelial cell of host.in addition,we have study the influence of the Tsp03044 recombinant protein which had been expressed by the other members of our experiment group using the same method,the result show that the invasion rate of the TS03044 recombinant protein group had no obvious relate with the increase of the recombinant protein as well as the antiserum. which indicated that this protein has little influence on the progress of invasion.