Expression Of Three Diagnostic Antigens Of Fasciola Hepatica And Establishment Of ELISA Diagnostic Method

Fascioliasis hepatica is a globally distributed parasitic disease.The disease is the main cause of morbidity and death of ruminants(such as cattle and sheep),seasonal disease,and serious damage to ruminants.The conventional fecal examination method can only detect the eggs excreted by the mature worms,and cannot play an early diagnosis role.Establishing an early diagnosis method for early diagnosis is an effective means to effectively control the occurrence and spread of the disease.Therefore,this experiment successfully cloned the Fasciola hepatica cathepsin L(Fh Cat L)genes,Fasciola hepatica glutathione S transferase(Fh GST)genes and Fasciola hepatica serine protease inhibitor(Fh SPI)genes,And prepared into recombinant protein,establish an indirect ELISA diagnostic method,laying the foundation for effective control of Fascioliasis Hepatica.This study was designed based on the gene sequence of Fasciola hepatica cathepsin L(Fh Cat L),Fasciola hepatica glutathione S transferase(Fh GST)and Fasciola hepatica serine protease inhibitor(Fh SPI)published in Gen Bank.Primers were synthesized and the total RNA of the adult flukes of Fasciola hepatica was extracted and amplified by PCR.The obtained target gene was ligated with the cloning vector p MD18-T,and after identification by double enzyme digestion,PCR and sequencing,the correct target fragment was ligated to the expression vector p ET-30 a,and after correct identification by double enzyme digestion,IPTG was used.The target protein is induced to express.The results of SDS-PAGE showed that the molecular weight of the target protein was 42 KDa,31KDa and 44.7KDa,and it existed in the form of inclusion bodies.The p ET-30a-Fh Cat L recombinant strain was induced with 0.2mmol/L IPTG for 6h at 37?,and the p ET-30a-Fh GST recombinant strain was induced with 0.2mmol/L IPTG for 3h and 0.6mmol/L IPTG to induce p ET-30a-Fh SPI recombinant strain 4h,recombinant protein was highly expressed.By Western blotting,the recombinant expressed protein can be specifically recognized by the F.hepatica antibody.Purified recombinant Fh Cat L,Fh GST and Fh SPI were used as diagnostic antigens respectively.By optimizing the indirect ELISA conditions,the final detection results were the optimal antigen coating concentration of Fh Cat L was 3.0?g/m L,the optimal serum dilution was 1:100,the optimal working concentration of optimal HRP-goat anti-mouse Ig G was 1:7000,the best antigen package of Fh GST The concentration was 3.0 ?g/m L,the optimal serum dilution was 1:100,The optimal working concentration of optimal HRP-goat anti-mouse Ig G is 1:6000,the optimal antigen coating concentration of Fh SPI is 6.0?g/m L,the optimal serum dilution is 1:100,The optimal working concentration of the best HRP-goat anti-mouse Ig G was 1:6000.In the sensitivity test,the earliest detection time of Fh Cat L,Fh GST and Fh SPI was 14 d in artificially infected sheep.In the specificity test,Fasciola hepatica did not cross-react with Schistosoma japonicum,Clonorchis sinensis and Haemonchus contortus.The repeatability test between the plates and the plates was carried out using the ELISA plate with different production batch numbers.The results showed that the coefficient of variation of the repeatability test of the three methods was less than 10%.he clinical detection of 240 samples showed that the positive detection rate of Fh Cat L was 32.92%(79/240),the positive detection rate of Fh GST was 30.83%(74/240),and the positive detection rate of Fh SPI was 31.67%(76/240),and the corresponding 240 feces were tested,the positive detection rate was 28.75%(69/240).By analyzing the correlation between the three indirect ELISA methods and the stool test,the results showed that the positive detection rate of the indirect ELSIA diagnostic methods of the three proteins was higher than that of the stool test,and the difference was significant(p<0.05).There are some differences among the three methods.The sensitivity and negative predictive rate of Fh Cat L were higher than the other two methods,the specificity and Yoden index of Fh GST were slightly higher than the other two method,and the false positive rate was lower than the other two methods,the false positive rate of Fh SPI was slightly higher than the other two methods.Three indirect ELIS A methods can be used for the early diagnosis of leptin of Fascioliasis Hepatica.