Analysis And Characterization Of Antigenic Epitopes Of Newborn Larvae Stage-specific T668 Protein In Trichinella Spiralis

Trichinosis is a parasitic disease caused by Trichinella spiralis, and can infect almost all mammals, including humans. It has been more than 170 years since the discovery of Trichinella spp. The lack of rapid,specific,sensitive and effective diagnosis methods makes the outbreaks of human trichinellosis emerge. occasionally., Trichinellosis of slaughtered animals is made to be the principal, imperative and mandatory inspection item in all countries of the world. The official methods for slaughtered animals trichinosis recommended by International des Epizooties (OIE) are microscopy and pooled sample digestion, however, both of them possess shortages such as time-consuming and low sensitivity. Immunology methods like ELISA are currently the most sensitive approaches, but by the limitations of antigens (non-specificity, etc), they are still beside the regulatory methods. For this reason, screening specific and sensitive antigen in order to establish a rapid and specific detection (diagnostic) method is becoming the focus of many scholars at home and abroad.Because of the complex structures of Trichinella spiralis polypide antigens and immune escape artifices formed in long-term parasitism, using the complex polypide antigens is always difficult to achieve the desired diagnosis,precaution and therapy efficacy. In recent years, in order to solve these problems,scholars transfer their eyesight to genetic engineering antigen. By constructing cDNA library, screening the effective antigens and predicting the epitope, the rapid and broad-spectrum Trichinella spiralis detection methods can be explored. T668 gene (Serine Protease) is a high reactionogenicity gene discovered by our laboratory, in order to further improve the detection of antigen-specific, we screen its epitope for the further study to develop early stage diagnosis reagent of Trichinella spiralis.We analysed the hydrophilicity, antigenicity and surface accessibility of T668 sequence (GeneBank ID: AF331160.1) by DNAstar software, then designed 4 pairs of specific primers to amplify three active points of T668 protein (His, Asp, Ser )and hydrophilic domain in C end( named T668H). The result of 1.0% agarose gel electrophoresis show the length of amplified fragments agree with the expectation. We ligated the amplified fragments to expression plasmid pGEX-4T-1. After confirmation by PCR, restriction endonuclease digestion with EcoRI/XhoI, BamHI/XhoI and gene sequencing, the results of identification were all correct. The recombined expression plasmids wer named pGEX-His, pGEX-Asp, pGEX-Ser, pGEX-T668H. The prokaryotic expression plasmids were transferred to E.coli BL21 and induced by IPTG, optimized cultural condition and analyzed existence and solubility of the GTS-fusion protein by SDS-PAGE. The SDS-PAGE show purpose bonds of pGEX-His,pGEX-Asp,pGEX-T668H in expecting positions, whose relative molecular weights were 34.6kD, 34.3kD and 40.8kD respectively, While the pGEX-Ser was failed to express the recombined protein. We suppose this Ser active point may be the toxic point of Serine protein, which lead to the unexpression. The other three kinds of recombined proteins could exist in both dissolubility and inclusion body forms. The optimizing induce condition was 25℃in temperature and 9h in time. After magnify expression, collected by centrifugating, the bacteria were crushed by ultrasonic wave, purified soluble protein in supernatant of E.coli BL21 lysate by Gluthathione-Sepharose 4B affinity purification. Three kinds of GST-fusion protein was purified. ELISA and WB analyse of the GST-fusion protein using swine-to-Trichinella spiralis antiserum confirmed the T668H region was the major epitope determin domain.We designed 16 short peptids with 5 amino acids frame shift superposed according to amino acid sequence of T668H region. Then artificial synthesissed 16 pairs of oligonucleotide chains according to amino acid sequence of 16 short peptids, annealed, formed small pieces of DNA, and directly cloned into pGEX-4T-1 expression vector Gene sequencing indicated 16 recombined plasmids which named pGEX-N5EM1-16. constructed successfully. The recombined plasmids were transfered to E.coli BL21 and induced by IPTG, express productions were identified by SDS-PAGE. After magnify expression, collected by centrifugating, then the bacterium was crushed by ultrasonic wave, purified soluble protein in supernatant of E.coli BL21 lysate by Gluthathione-Sepharose 4B affinity purification, 16 kinds of recombined proteins was purified. ELISA and WB analyse of the 16 recombined proteins using swine-to-Trichinella spiralis antiserum definited seven antigen epitopes (N5EM3, N5EM4, N5EM5, N5EM7, N5EM8, N5EM9, N5EM11), among the total, N5EM5, N5EM7, N5EM11 show high response signal, which could be used as candidates for Trichinella spiralis diagnostic reagent.