| Trichinellosis is a serious zoonotic nematode disease caused by Trichinella spiralis.Trichinellosis causes serious harm to animal husbandry and human health.It is one of the most expensive food-borne zoonotic parasites in the world.Trichinellosis infection in humans and animals is mainly due to its ingestion of meat rich in muscle larval cysts.Under the action of gastric acid,the cysts release infective muscle larvae.After several hours,the muscle larvae enter the small intestine and produce new larvae in the small intestine to continue their next stage of life cycle.The pathogenic process of T.spiralis is similar to that of Escherichia coli entering the body through the extremely acidic environment of the stomach.In recent years,the research on acid resistance system of pathogenic Escherichia coli is particularly remarkable.Strengthening the examination and diagnosis of the disease is the only effective way to control the dise ase.In this study,si RNA interference technique was used to inhibit the expression of TsGAD gene.Furthermore,the role of glutamic dependented acid-resistant system in the mechanism of acid resistance of Trichinella spiralis was studied.At the same time,two indirect ELISA methods were successfully established by using the TsGAD,TsGLS of T.spiralis as the coated antigen.Based on the published TsGAD mRNA sequences of NCBI,three interference sequences of targeted silencing TsGAD si RNAs: GAD-si RNA1,GAD-si RNA2 and GAD-si RNA3,as well as a negative control: control si RNA were designed in this study.The si RNAs was transfected into T.spiralis by lipofectin 2000.The transfection time was determined to be 12 h by immunofluorescence technique.The effect of RNA interference on the expression of TsGAD gene was detected by real-time quantitative PCR(q PCR),western blot(WB)technique.The results of q PCR and WB showed that all three si RNAs sequences could interfere with the expression of TsGAD gene,and the inhibition rate of GAD-si RNA1 sequence to TsGAD gene was 51.7%~56.7%.At the same time,the optimal transfection concentration of si RNA was determined to be 2 ?M by q PCR and WB experiments.TsGAD gene silenced T.spiralis was cultured in acidic environment in vitro.The mechanism of acid resistance of T.spiralis was studied in terms of the expression of TsGAD gene,the activity of TsGAD enzyme,the survival rate of T.spiralis and the change of p H of culture medium.The results showed that acidic environment could induce the expression of TsGAD gene.Under the same conditions of p H,the enzyme activity of interference group was lower than that of PBS group,and high activity of enzyme in acidic condition.In the same p H culture medium,the survival rate of T.spiralis decreased with the increase of culture time;and the survival rate of T.spiralis in p H 6.6 groups was the highest.Under the same culture time and the same p H,the survival rate of T.spiralis in GAD-si RNA1 group was significantly lower than that in PBS group.The p H value of culture medium in GAD-si RNA1 group was not significantly higher than that in PBS group.After TsGAD gene silencing in T.spiralis,the parasites was used to infect mice,and worm reduction rate of adult T.spiralis,worm reduction rate of RCI,LPG,muscle larvae and thickness of nanny cell wall of muscle larvae were evaluated.The results showed that compared with PBS group,the worm reduction rate of GAD-si RNA1 composition was 31.50%,RCI was 62.51±7.32,LPG was 1250.22±146.48,the worm reduction rate of muscle larvae was 49.15% and nanny cell wall thickened and there were a lot of inflammatory substances around it.These results suggested that TsGAD gene played an important role in acid resistance of T.spiralis.After silencing TsGAD gene,the F1 muscle larvae of T.spiralis were cultured in acidic condition.The results showed that the survival rate of F1 generation larvae was 5 7% after 2 h culture in p H 2.5 medium.The survival rate of T.spiralis F1 was significantly different from that of F0,which indicated that the interference effect of si RNA could not be extended to the next generation.In this study,the recombinant TsGAD,TsGLS protein of T.spiralis was used as coating antigen to establish two indirect ELISA methods for detection of T.spiralis serum antibody.The conditions such as the quantity of antigen coating,the dilution of serum,the condition of antigen coating,the selection and time of blocking solution,the dilution of enzyme-labeled antibody,incubation time and TMB action time were optimized in this study.The results showed that the optimal antigen coating quantity of TsGAD recombinant antigen indirect ELISA method was 50 ng,the optimal serum dilution was 1:200,the optimal antigen coating condition was 4 ? overnight,and the optimal blocking solution was 5% skim milk,the optimum blocking time was 37 ? for 2 h,the optimum dilution of enzyme labeled antibody was 1:5000,the optimum incubation time was 1 h at 37 ?,and the reaction time of TMB was 15 min.The optimal antigenic coating quantity of TsGLS recombinant antigen indirect ELISA method was 100 ng,the optimal antigenic coating condition was 4 ? overnight,the optimal blocking solution was 5% defatted milk,and the optimal blocking time was 37 ? for 2 h,and the optimal antigenic coating condition was 4 ? for overnight coating,and the optimal blocking time was 37 ? for 2 h,and the optimal dilution of enzyme-labeled secondary antibody was 1:5000,the optimal incubation time was 37 ? for 1 h,and the reaction time of TMB was 15 min.The negative critical value of indirect ELISA method of TsGAD was 0.848.The negative critical value of indirect ELISA method of TsGLS was 0.963.The accuracy of the two indirect ELISA methods established in this study was t ested by repeatability test,sensitivity test,specificity test and contrast test.The results showed that the maximum coefficient of variation was 5.647% for TsGAD indirect ELISA assay and 6.049% for inter-assay repeatability test,all of which were less than 10%,and the repeatability was good.When the positive serum of T.spiralis was diluted 3200 times,the P/N value was more than 2.1,which indicated the sensitivity of TsGAD indirect ELISA method was high.The positive sera of Ascaris lumbricoides,Clonorchis sinensis,Schistosome and Toxoplasma gondii were detected by TsGAD indirect ELISA method,and no cross-reaction was found that showed that the method had good stability and specificity.The maximum coefficient of variation of the inter-assay and inter-assay reproducibility of TsGLS indirect ELISA method was 7.318% and 8.536% repeatability,all of which were less than 10%,and the repeatability was good.When the positive serum of T.spiralis was diluted 3200 times,the P/N value was more than 2.1,which indicated the sensitivity of TsGLS indirect ELISA method was high.The positive sera of Ascaris lumbricoides,Clonorchis sinensis,Schistosome and Toxoplasma gondii were detected by TsGLS indirect ELISA method,and no cross-reaction was found that showed that the method had good stability and specificity.At the same time,mice infected with T.spiralis were detected by the method of pressing diaphragm,two indirect ELISA methods and rapid detection card for T.spiralis antibody.The results showed that the more dose of T.spiralis infection,the sooner the trichinellosis was detected and the higher the detection rate was.T.spiralis can be detected by the method of pressing diaphragm as early as 21 days after T.spiralis infection,no matter how much dose of infection,which was related to the life history of T.spiralis.TsGAD indirect ELISA method and TsGLS indirect ELISA method detected trichinellosis with 30 doses as early as 7 days after infection.T.spiralis rapid detection card detected trichinellosis with 30 doses as early as 14 days after infection.The results of TsGAD indirect ELISA method and TsGLS indirect ELISA method for heart,liver,spleen,lung and kidney were similar.The infection dose of T.spiralis was the first to be detected from the grinding fluid of the heart,and the detection rate of the grinding fluid of the same organ increased with the increase of the infection dose and the infection time of T.spiralis.In this study,the expression of TsGAD gene was successfully inhibited by RNA interference technique,and the results showed that TsGAD played an important role in gastric acid tolerance,which provided a basis for further research.At the same time,two indirect ELISA methods developed in this study provide necessary test means for early diagnosis and epidemiological investigation of trichinellosis. |
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