Recombinant Expression Of Reverse Transcriptase Mutant And Its Application In RT-qPCR

Extraction-Free Probe One-Step Reverse Transcriptase-Quantitative Real-time Polymerase Chain Reaction(EFPO RT-qPCR)is a time-saving and labor-saving nucleic acid detection technique with wide application prospects in the field of rapid food testing.At present,EFPO RT-qPCR system has problems such as insufficient detection sensitivity and low specificity.Reverse transcriptase is one of the core components of EFPO RT-qPCR,but wild-type reverse transcriptase(W-RT)has poor thermal stability and weak inhibitor tolerance.Therefore,the modification of reverse transcriptase with high inhibitor tolerance is important to promote the progress of EFPO RT-qPCR technology.In this paper,we applied genetic engineering technology to modify wild-type Moloney murine leukemia virus reverse transcriptase(W-MMLV RT),studied the enzymatic properties of mutant Moloney murine leukemia virus reverse transcriptase(CMMLV RT),and established a blood EFPO RT-qPCR assay system based on CMMLV RT.The main studies and conclusions are as follows.(1)Recombinant expression of CMMLV RT.The CMMLV RT gene was designed and synthesized,and the p MAL-c2x recombinant expression vector was constructed to realize the recombinant expression of CMMLV RT in E.coli BL21.The induced expression conditions of CMMLV RT were optimized as follows:induction temperature of 25?,induction time of12 h,and concentration of inducer IPTG of 0.1 m M.The electrophoretic pure CMMLV RT stock solution was prepared by nickel ion affinity chromatography with protein concentration of 0.247 mg/m L and enzyme activity concentration of 1.470×10³ U/?L.(2)CMMLV RT enzymatic properties study.Based on the results of two-step RT-PCR amplification,the composition of CMMLV RT buffer was optimized as follows:50 mmol/L Tris-HCl,3 mmol/L Mg Cl₂,75 mmol/L KCl,10 mmol/L DTT,p H 8.3.The temperature range of reverse transcription action of CMMLV RT was 40-75?.Using 100 ng of Ha Cat cell-derived total RNA as the template,reverse transcription could be completed to generate c DNA within 10 s at 55?.CMMLV RT showed 92%and 79%activity residues after 1 h and 2 h incubation at 50?,respectively;26%activity residues after 1 h incubation at 55?,and activity was lost after 2 h incubation at 55?;moreover,the CMMLV RT had stronger extension ability than W-MMLV RT.(3)CMMLV RT inhibitor resistance study.The Ct value(the number of amplification cycles elapsed when the fluorescence signal reaches a set threshold)in the two-step RT-qPCR was used to calculate the inhibition rate of reverse transcriptase activity by different inhibitors.The inhibition rate of CMMLV RT activity did not exceed 50%when 3.0 mmol/L EDTA,0.525 U/m L sodium heparin,0.75?g/?L astragalus polysaccharide,350 mmol/L urea or 15%formamide were present in the reverse transcription reaction system.(4)An EFPO RT-qPCR reaction system and method were developed for the detection of novel coronaviruses in blood specimens.The amplification efficiencies of the established assays were close to 100%at 2%-8%(v/v)blood addition;The established detection method with high amplification efficiency,well repeatability,and were in the same order of magnitude as that of the commonly used two-step RT-qPCR method.