Preparation And Immune Analysis Of Recombinant Hunman Serum Amyloid Protein A1

Human serum amyloid A1 is a member of the human serum amyloid A(SAA)family.At present,bacterial,viral infection,coronary heart disease,atherosclerosis,cancer and other diseases were detected in serum SAA increased.As a new type of inflammatory marker,SAA has important guiding and application significance in the monitoring of clinical disease.In this paper,human serum amyloid protein A1(SAA1)was expressed in Escherichia coli BL21(DE3)prokaryotic system.According to the BL21(DE3)expression preference,we designed and synthesized the SAA1 protein gene fragment,constructed the expression vector pET-28a-SAA1,and transformed into the expression host BL21(DE3).Through the optimization of expression conditions of recombinant target protein were obtained the best induced condition: the final concentration of IPTG was 0.6 mM,temperature was 34?,time was 8 h,pH was 7 and the speed was 180 rpm.The protein was purified and concentrated by Ni column,and the purity was up to 95% and the final concentration was 8.09 mg/mL of SAA1 protein.Based on the specific binding of antigen and antibody,this paper established the enzyme-linked immunosorbent assay(ELISA)and turbidimetric assays of SAA1.Through the experiment to the reaction conditions and experimental consumables and reagents to optimize in order to achieve the best detection results.The sensitivity,reproducibility and specificity of the detection reagents were evaluated by the experiment.ELISA method was established to determine the critical value of the positive samples OD450 nm was 0.222,linear range 100-2400 ng/mL,the minimum detection limit was 50 ng/mL.The results showed that the specificity of the detection reagent was good,the intra-assay and inter-assay coefficients of variation were less than 7%,the recovery was within the allowable range and the average recovery was 99.91%.At the same time,the turbidimetric detection method was successfully established.In the UV spectrophotometer at 560 nm detected the absorbance with the addition of SAA1 protein content showed a linear correlation,the standard equation was Y = 0.0698ln(X)+ 0.255,and the linear range was 0.075-2.4 ?g/mL,and the correlation coefficient R2 was 0.99287,and the recovery rate in 93.6%-106.4% in the allowable range.