Prokaryotic Expression Of Milk Serum Amyloid A,preparation Of Monoclonal Antibody And Establishment Of Electrochemical Sensor Detection Method

Dairy cow mastitis is one of the most important diseases that affect the health of dairy cows and the development of the dairy industry.The causes are complex,which may be caused by bacteria,viruses,mycoplasma,and poor management methods.The main types are clinical and subclinical.Clinical mastitis can be judged by intuitive methods such as eye observation and palpation.Subclinical mastitis often fails to be diagnosed in time and causes mastitis outbreaks,causing huge losses.Therefore,the establishment a method for early diagnosis of subclinical mastitis is particularly critical.Serum amyloid A(MAA)in milk has been confirmed by multiple studies as a marker of mastitis in dairy cows,and the concentration of MAA in dairy cows increases sharply during the subclinical mastitis stage.Unlike other mastitis markers such as enzymes,conductivity,and p H,MAA is less affected by the environment,seasons,and cow health.It can be intuitively used as a standard for diagnosing mastitis in dairy cows,and its concentration in milk is positively correlated with SCC and CMT.Researching and establishing a method for detecting MAA is of great significance for the detection and diagnosis of dairy cow mastitis,especially subclinical mastitis.As a kind of "artificial antibody" technology,molecular imprinting not only has the specificity of natural antibodies,but also is cheaper,simpler and more sensitive.The molecularly imprinted electrochemical sensor(MIECS)combines artificial antibody technology with convenient electrochemical technology,and the modification of nanoparticles makes its detection performance better.In this study,the successful expression of soluble MAA was mainly obtained by synthesizing MAA gene,constructing plasmid and transforming,inducing expression,and finally through a series of purification steps.Part of the obtained MMA was used to immunize mice to prepare monoclonal antibodies to prepare for the development of related immunological detection methods,and the other part is used to construct molecular imprinting electrochemical sensors for detecting MAA in milk.1.Soluble Expression of Milk Amyloid A and Preparation of Monoclonal AntibodyThe E.coli prokaryotic expression system was used to express MAA.The selected solubilizing expression vector is p ET28a-SUMO,and the target gene(396bp)is synthesized according to the MAA sequence searched on Gen Bank.The MAA gene was introduced into the vector by homologous recombination,and the recombined plasmid was transformed into E.coli BL21(DE3)p Ly Ss competent.The expression temperature was optimized at 16℃,28℃,37℃,and the expression time of 3,5,7,10,12 h was selected,and the inducer concentration was 0.3,0.5,0.7,1.0,2.0mmo L/L,respectively.Large-scale expression under optimal expression conditions,and purification of the target protein with a nickel column and a protein purifier.The following results were obtained.The SUMO-MAA protein was expressed at 16°C and 0.5mmo L/L IPTG for 7 hours to obtain the best supernatant expression.The recombinant protein was successfully purified with a size of 35 k Da.The SUMO-MAA is cut by the SUMO enzyme that specifically recognizes the SUMO tag,and then purified again to obtain the complete MAA protein.The MAA protein immunized BALB/c female mice were screened to secrete the hybridoma cell lines of the monoclonal antibody,the ascites fluid was prepared and purified to obtain the monoclonal antibody,and its titer and specificity were measured.The results showed that two strains of antibodies 3E11 and 8G10 were screened,and the antibody titers were1:51200 and 1:102400,respectively.In summary,this study successfully expressed the soluble MAA protein and its two monoclonal antibodies.2.Preparation and preliminary application of a molecularly imprinted electrochemical sensor for the detection of MAASuccessfully constructed a molecularly imprinted electrochemical sensor with superior performance for detecting MAA.First,the Au NPs@r GO nanocomposite materials used to modify the electrode were prepared: Au NPs were prepared by the trisodium citrate method,then r GO was modified by chit,and the Au NPs were loaded on the r GO by ultrasound.Then,the material was modified to the GCE electrode by the dropping method.Using MAA as the template molecule and pyrrole as the functional monomer,a molecularly imprinted polymer is formed by electropolymerization.The template protein was eluted with 10% acetic acid(V/V)containing 10% SDS as the eluent to make a molecularly imprinted electrochemical sensor.The polymerization conditions including Au NPs@r GO,number of polymerization cycles,pyrrole concentration,polymerization rate,elution time and adsorption time were optimized to prepare a sensor for MAA detection.The results showed that the molecularly imprinted electrochemical sensor showed two good linear relationships in the MAA concentration range of 0.01 to 200 ng/m L.The lower limit of detection is about 5 pg/m L(S/N = 3).Other parameters include selectivity,reproducibility(RSD 3.2%)and recovery rate(96.1-103%)are all excellent.In the simulation of the actual sample detection,the accuracy is equivalent to that of the ELISA method,but the detection efficiency is higher.Compared with traditional methods,the sensor method established in this research can detect dairy cow mastitis more quickly and sensitively,especially subclinical mastitis.