Application Of High-quality Genetic Recombination Proteins In Diagnosis Of Trichinellosis Of Slaughter Animals

As a zoonotic parasitic disease, trichinellosis show a worldwide distribution and has a high prevalence in China, could be infected over 150 mammals besides human, not only lead to enormously economic loss of husbandry and meat industry but also severely threat to public health. Recently, this disease has been extention as a reason of the transportation of pigs and uninspected contaminative pork. Therefore, it's very important to enhance the rapid test of trichinellosis and sanitary control of contaminative pork. However, effective method to test the trichinellosis of slaughter animals has not been occurrence so far, traditional and exhaustive microscopy and digestive methods are still used as official method. ELISA has many advantages such as sensitive, specific in testing the antibody against Trichnella. There were reports on the application of antigens based on the body of worms in testing trichinellosis of pigs. However, these antigens are very complicated, although they can be purified by affinity chromatography using monoclonal antibodies to reduce even eliminate the false positive reactions and to enhance the detection rate, the procedures are much more tedious and time-consuming and the quantity of antigens can hardly be satisfied. The fundamental method to work out a solution must be simplex genetic recombination antigens. However, considering the advance on the research, it won't work if we continue following the traditional idea that finding one native antigen without cross reaction to test infection of trichinella at developmental stages.Therefore, the idea was changed in this work. Based on the characterization of reactionogenicity, three antigen genes with high reactionogenicity were selected to identify the transcription level and expression trait, and the value of the recombinant protein in immunodiagnosis, and the overcome-blind-area ELISA method in diagnosis of trichinellosis of pigs was established. Furthermore, the characterizations were useful for further study on development, invasion, evasion and encapsulation mechanism of the worms. First of all, the transcription levels of the four antigen genes of developmental stages were systematically identified. After isolation of total RNA of developmental stages worms and reverse transcription using primer Oligo dT18, the real-time PCR were performed by using SYBR Green dye and specific primers designed according to the sequences of antigen genes and reference gene. The results showed that the transcription of the four genes was stage-specific. (1)Ts-clp gene was transcripted in every developmental stage in this assay; however, the transcription level was obviously different. Ts-clp gene was largely restricted to inML6, inML24, ML and Ad (Ad2, Ad3 and Ad5). The lowest value was in NBL and was significantly lower than any other stages (P <0.05). (2)Ts-serpin gene was transcripted in every developmental stage in this assay; however, the transcription level was obviously different. There were two higher transcription periods that one was inML24/Ad2/Ad3 and another was ML. There was one lower transcription period that was Ad5 and NBL. The minimum was Ad5 which was not significantly different with NBL (P > 0.05) and was significantly different with any stage of higher transcription periods (P < 0.05). No transcription signal of Ts-sp-T668 gene was detected in ML. The low transcription period appeared in inML24 and Ad (Ad2, Ad3 and Ad5), there was no significant difference within the four stages. The level of inML6 was significantly higher than other stages (P < 0.05). No transcription signal of Ts-sp-ZH68 gene was detected in NBL. The minimum appeared in inML24, significantly different with other stages (P < 0.05). There was no significant difference within the three stages (P > 0.05), but the levels of the three stages were significantly different with any stage of the low transcription periods (P < 0.05).Secondly, the expression traits of the four antigen genes of developmental stages were systematically identified. Paraffin sections from parasites and infected tissues were prepared, and the immunofluorescence and histochemistry technique were utilized localize products of the four genes by using the specific polyclonal antibody. The results showed that space-time specificity exits in the expression of the genes. (1) Ts-clp protein was mainly localized inβ-stichocytes of Ad and ML. Six hours after orally infection, stong red signal was detected on the stichosome and surface of the worms. Five days after orally infection, it was detected on the stichosome and surface of the adults. It was worthy of noting that immunofluorescence was barely observed in the early and late embryos in the adults. While in released NBL, a slight signal for expression was detected. Seven days post tail intravenous injection, the signal was faint in worms, while at 18 dpi the red signal was obviously observed in stichosome. At 60 dpi, it enhanced. (2) Ts-serpin protein was mainly localized in bodies of post-encystation worms, and specificly localized in the nucleoplasm of the enlarged nuclei. At 3 days post tail intravenous injection, positive signal was detected inside the worms, restrict to the stichosome. It gradually enhanced from 18 dpi to 28 dpi. From 30 dpi to 50 dpi, stronger signal was detected in worm's bodies. Interestingly, the positive signal appeared specificly in the the nucleoplasm of the enlarged nuclei from 18 dpi to 28 dpi, while there was no detectable fluorescence in nucleoli of the enlarged myocardial nuclei. Especially, at 30 dpi, the intensity of the fluorescence in nucleoli of the enlarged myocardial nuclei reached the peak, and dispersively distributed; at 40 dpi, it diminished, and finally disappeared. (3) In early stage (16 day post tail intravenous injection) the localization signal of Ts-sp-ZH68 was in nucleoli of the enlarged nuclei of the host's muscle cells. Positive fluorescence was detectable at 8 day post tail intravenous injection, but it was faint. At 16 dpi, the signal became slightly strong than that of 8 dpi, the red fluorescence appeared outside the worms, but it only localized in nucleoli of intumescent muscle cells and there was no positive red signal in nucleoplasm. From 26-40 dpi, stronger signal was localized inside but not outside the worms. After 60 dpi, no positive signal was found, suggesting that Ts-sp-ZH68 was expressed in ML stage but the duration was relatively short.Thirdly, the hydrophilia area on C-terminal of Ts-sp-T668 was subcloned into pGEX-4T-1 vector, and then the recombinant protein Ts-sp-T668H was prepared after optimization of expression. Western-blotting results indicated that the reaction activity of Ts-sp-T668H was extremely strong that was better than Ts-sp-T668. Therefore, it could be conclude that the reactionogenicity of the gene was mainly derived from this area. Furthermore, when utilized for immunodiagnosis it could increase the epitopes in unit volume thus enhanced reaction signal.Finally, the mixture of purified recombinant protein of Ts-sp-T668H, Ts-clp and Ts-serpin was utilized as antigen to detect positive pig sera at different dpi after optimization of reaction conditions, meanwhile, an ELISA method for detection of anti-Ts antibody using ES antigen from ML was made use of as control. An overcome-blind-area ELISA method in diagnosis of trichinellosis of pigs was established. The positive result was detected as early as 13 dpi by the mixture of antigens and last until the end of the assay (147 dpi). Primary tests indicated that the established ELISA diagnostic method had good sensitivity and specificity. It overwhelmed the blind area of immunodiagnosis and overcame the weakness of the previously used antigens. It's significant for control of zoonotic trichinellosis in our country.