| At present,ketosis and fatty liver have become one of the most common and frequently occurring major nutritional metabolic diseases in the perinatal period of high-yield cows in intensive dairy farms in China.Because it causes milk cows to reduce the amount of milk,induce cows to produce true stomach dislocation,no placental clothing or production of sputum and other diseases.It has brought huge economic losses to the dairy farming industry.However,the diagnostic technique of fatty liver in dairy cows still remains to detect the level of hepatic lipid infiltration through the liver puncture of live cows,and it is difficult to be accepted by farms because of the difficulties of liver protection,liver damage,and high detection costs.To date,there is a lack of a rapid,practical and accurate method for monitoring fatty liver in dairy cattle at home and abroad.In view of the development and application of paraoxonase-1(PON-1)enzyme-linked immunosorbent assay(ELISA)to monitor the fatty liver and ketosis in dairy cows in foreign countries,the price is expensive,and similar products with independent research and development are lacking in China.To this end,this study developed bovine PON-1monoclonal antibodies,based on which established PON-1 double anti-sandwich ELISA detection method,and the implementation of clinical trials,in order to provide a new for the rapid monitoring of domestic cow ketosis and fatty liver in the future technology.In this study,the prokaryotic expression technology was used to clone the optimized PON-1 protein gene of bovine to the prokaryotic expression vector pET-28 a,and the recombinant expression plasmid PET28a-PON-1 was constructed.The strains transformed with PET28a-PON-1 were amplified and cultured,expressed under induction of IPTG,and purified to yield a recombinant bovine PON-1 protein of approximately 37 KD.Then,Balb/c mice were immunized with the recombinant bovine PON-1 protein to obtain a hybridoma cell line 4K2 D that stably secretes specific antibodies and has high titer.4K2 D was injected into the abdominal cavity of the produced Balb/c mice,and the collected ascites was purified by AKTA protein purification system to obtain bovine PON-1 monoclonal antibody.In addition,rabbits were immunized with recombinant cow PON-1 protein as an antigen,serum was purified by AKTA protein purification system,and a polyclonal antibody to bovine PON-1 protein was obtained.Finally,the purified monoclonal antibody was used as the detection antibody,the purified polyclonal antibody was capture antibody,and horseradish peroxidase-labeled goat anti-mouse IgG was used as the enzyme-conjugated secondary antibody to successfully construct the bovine PON-1 protein double antibody sandwich ELISA assay.In this study,a self-created bovine PON-1 double antibody sandwich ELISA was used for clinical trials.Forty-six days after the calving,the serum of the cows was randomly collected and the levels of BHBA,GLU,AST,and NEFA in the serum were measured by biochemical analyzer.According to the content of BHBA,they were divided into the ketosis group and thehealthy control group.The cow PON-1 double antibody sandwich ELISA was developed to detect the serum PON-1 level in the test cows.The results showed that the serum PON-1 level in the ketosis group was significantly lower than that in the control group(P<0.05),and there was no significant difference from the commercial bovine PON-1 enzyme-linked immunosorbent assay kit.Moreover,based on the significance analysis,correlation analysis,and binary regression and receiver operating characteristic(ROC)analysis,it was determined that bovine serum PON-1 can be used as an early warning indicator for the risk of ketosis in cows,with an early warning value of <62.37 nmol./L.Conclusion: This study independently established a cow PON-1 double-antibody sandwich ELISA method to establish PON-1 based cows ketosis and risk warning indicators and their early warning values,providing new technologies for the monitoring of cow ketosis and fatty liver in the future. |
PDF Full Text Request