Molecular Modification Of KOD DNA Polymerase And Its Application In Food Allergen Detection

Polymerase chain reaction(PCR)is an important technology in modern molecular biology,which could achieve rapid amplification of trace amounts of DNA.With the expansion of the application field of PCR,the technology is developing in the direction of high specificity,high elongation rate,and high fidelity.The core raw material of PCR reagents-DNA polymerase has also become increasingly demanding.In this paper,for the better PCR performance and higher inhibitor tolerance,molecular modification of wild-type KOD DNA polymerase was carried out,and the reaction conditions and enzymatic properties of KOD DNA polymerase mutant(KOFU-S)were studied.Based on exploring the performance of KOFU-S in PCR reaction,it was applied to food allergen detection.The main research contents and results were as follows:(1)The recombinant expression of KOFU-S.Through technical means such as site-directed mutagenesis,structural domain replacement and double-stranded DNA binding protein Sso7d fusion,the expression vector of recombinant heat-resistant polymerase KOFU-S was successfully constructed.The recombinant expression of KOFU-S in Escherichia coli was constructed.The induction temperature was 18?,the induction time was 16 h,and the IPTG concentration was 0.1 mmol/L.The induced expression yield of KOFU-S in E.coli was 6.35mg/L.After Ni²⁺affinity chromatography,the electrophoretic pure KOFU-S DNA polymerase storage solution was obtained,and its enzyme activity concentration was 4.69×10⁴U/m L.(2)Enzymatic properties of KOFU-S.By comparing and studying the DNA polymerase activity of KOFU-S under different conditions,the optimized reaction 2×buffer for KOFU-S polymerase was obtained:120 mmol/L Tris-HCl,260 mmol/L KCl,30 mmol/L(NH₄)₂SO₄,3.6mmol/L Mg Cl₂,12%glycerol,p H 9.6.KOFU-S still exhibited more than 50%relative activity after heating at 95?for 4 h;after incubating at 0-95?for 2 h,the relative activities of KOFU-S polymerase were not significantly affected.The extension temperature of KOFU-S polymerase involved in the PCR process was 68?.(3)The PCR performance of KOFU-S.The performance of KOFU-S polymerase in PCR reaction was studied by electrophoresis analysis of PCR products and blue-white spot method.KOFU-S polymerase could achieve high fidelity(7.4×10^-6),high speed(5 s/kb),high sensitivity(1?g/L),long fragment(10 kb)PCR amplification.At the same time,the KOFU-S polymerase also has a strong resistance to PCR inhibitors,at least 0.01%(w/v)SDS,100 mmol/L Na Cl,10%(v/v)ethanol,160 mmol/L urea,80 ng/?L of humic acid,0.03 U/m L of heparin and 0.8g/L of bile salt.(4)Application of KOFU-S in food allergen detection.In this paper,using soybean endogenous gene Lectin and wheat endogenous gene Gliadin as targeted genes,a method for detecting soybean and wheat allergens in food based on KOFU-S direct amplification PCR was constructed.,and its detection limit was 0.0001%(w/w),which was much higher than the corporate standard detection limit of 0.01%(w/w).The sensitivity was 0.4 mg/L.The direct amplification PCR method established in this article is suitable for the detection of soybean and wheat allergens in commercial foods.