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A kind of mutant reverse transcriptase and its preparation method and application

A technology of reverse transcriptase and mutant type, which is applied in the field of mutant reverse transcriptase and its preparation, can solve problems such as limited heat resistance, and achieve the effects of fast reverse transcription, reduced interference, and high-efficiency reverse transcription reaction

Active Publication Date: 2022-02-15
广州英赞生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing reverse transcriptases have limited resistance to inhibitors and heat resistance, and improving the resistance to inhibitors and heat resistance is also crucial for the application of reverse transcriptase

Method used

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  • A kind of mutant reverse transcriptase and its preparation method and application
  • A kind of mutant reverse transcriptase and its preparation method and application
  • A kind of mutant reverse transcriptase and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Embodiment 1 constructs the recombinant vector containing the nucleotide sequence of coding mutant type reverse transcriptase

[0068] (1) According to the amino acid sequence (SEQ ID NO.1) of the mutant reverse transcriptase, after codon optimization of the expression system of Escherichia coli, a DNA molecule capable of high-efficiency expression in Escherichia coli is obtained, and the splicing PCR method is used, The DNA molecule encoding the mutant reverse transcriptase is artificially synthesized, specifically as shown in SEQ ID NO.2.

[0069] SEQ ID NO.1 (mutated amino acid in the box):

[0070]

[0071]

[0072] SEQ ID NO.2:

[0073]

[0074] (2) Recombining the DNA molecule encoding the mutant reverse transcriptase with the expression vector pET-28a. The synthetic primer sequences are as follows:

[0075] FP1: 5'-ATCCATGGAGAAATATACAGAA-3' (SEQ ID NO.3);

[0076] RP1: 5'-GTACCGAATTCCTTCAAGCC-3' (SEQ ID NO.4);

[0077] FP2: 5'-GGCTTGAAGGAATTCGGTAC-3...

Embodiment 2

[0081] The preparation of the transformant of embodiment 2 expressing mutant reverse transcriptase

[0082] Transform the recombinant vector obtained in Example 1 into the host cell E.coliBL21(DE3), pick a single colony, inoculate into liquid LB medium (containing 50 μg / mL of kanamycin) and cultivate to OD 600 was 0.8, added IPTG to a final concentration of 0.1mmol / L, induced at 18°C ​​for 16h, collected the bacteria and sonicated, and detected the expression of the target protein by SDS-PAGE electrophoresis. It was found that the prepared transformant could highly express the mutant reverse transcriptase.

Embodiment 3

[0083] Expression and Identification of Embodiment 3 Mutant Reverse Transcriptase in Recombinant Escherichia coli

[0084] The positive transformant strains capable of expressing mutant reverse transcriptase obtained in Example 2 were inoculated into 60 mL LB medium containing 50 μg / mL kanamycin, and placed in a shaker at 37° C. for overnight culture; The cultured seed liquid was inoculated into 500 mL of LB medium containing 50 μg / mL kanamycin at a volume ratio of 1:100, and continued shaking culture in a shaker at 37 ° C for 3 h; IPTG was added to each bottle of bacterial liquid to a final concentration of 0.1 mmol / L, continue shaking induction at 18°C ​​for 16 hours; collect and weigh the cells after induction by centrifugation, and record the wet weight of the cells; take 1 mL of each of the bacterial liquids before and after induction, centrifuge at 12000 r / min for 1 min, take the precipitate, add 150 μL to lyse Buffer solution (50mmol / L Tris, 300mmol / L NaCl, 15mmol / L imi...

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Abstract

The invention discloses a mutant reverse transcriptase, its preparation method and application. The mutant type reverse transcriptase disclosed by the invention is transformed from wild-type Moloney murine leukemia virus reverse transcriptase through site-directed mutation. The mutant type reverse transcriptase not only completely retains the wild protein structure, but also has the polymerase activity of the wild type reverse transcriptase, and its RNase H hydrolysis activity is lost. The mutant reverse transcriptase is resistant to inhibitors and has strong heat resistance, and has high efficiency of reverse transcription and synthesis of cDNA using RNA as a template. The mutant reverse transcriptase has high tolerance to various inhibitors in the application system, and can effectively prevent false negatives caused by inhibitors interfering with cDNA synthesis in gene detection, thereby improving the accuracy of detection. The mutant reverse transcriptase can be used for RNA amplification and detection, rapid synthesis of common and long-chain cDNA, and reverse transcription of RNA templates with complex secondary structures.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a mutant reverse transcriptase and its preparation method and application. Background technique [0002] Polymerase Chain Reaction (PCR) is an indispensable experimental technique in modern molecular biology. This technology is widely used in many fields such as medical testing, pathogenic microorganism detection, forensic identification and food safety testing. The template used in PCR technology is single-stranded cDNA or double-stranded DNA. When the detection template is RNA, the RNA needs to be reverse-transcribed into cDNA first, and then PCR amplification is performed using the cDNA as a template. Reverse transcriptase is a key enzyme that converts single-stranded RNA into cDNA. Reverse transcriptase generally has three enzymatic activities: RNA-dependent DNA polymerase activity, DNA-dependent DNA polymerase activity and RNase H activity, wherein, RNase H activit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/70C12N1/21C12Q1/6806C12R1/19
CPCC12N9/1276C12Y207/07049C12N15/70C12Q1/6806C12Q2521/107
Inventor 胡松青陈雪梅赵劲松刘光毅侯轶苑歆
Owner 广州英赞生物科技有限公司
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