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Reverse transcriptase mutant capable of improving thermal stability and inhibitor tolerance and application of reverse transcriptase mutant

A technology of reverse transcriptase and thermal stability, applied in the biological field, can solve problems such as high price and achieve the effects of improving thermal stability, high thermal stability, and improving inhibitor tolerance

Active Publication Date: 2022-06-24
江苏百时美生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the high price of imported reagents, the market still lacks domestic reverse transcription reagents with low cost and excellent performance; so it is very important to develop a reverse transcriptase mutant with high thermal stability, good synthesis performance and good inhibitor tolerance

Method used

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  • Reverse transcriptase mutant capable of improving thermal stability and inhibitor tolerance and application of reverse transcriptase mutant
  • Reverse transcriptase mutant capable of improving thermal stability and inhibitor tolerance and application of reverse transcriptase mutant
  • Reverse transcriptase mutant capable of improving thermal stability and inhibitor tolerance and application of reverse transcriptase mutant

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Mutant thermostability identification

[0028] Using SEQ ID NO. 1 as a template, using conventional site-directed mutagenesis or gene synthesis, and protein recombinant expression and purification methods, two M-MLV reverse transcriptase mutants containing different combinations of mutation sites were obtained respectively. One contains 5 mutation sites including L139P, T197A, G308C, D524G and L603W and is named Mut1; the other contains 6 mutation sites including L139P, T197A, D209A, G308C, D524G and L603W and is named Mut2.

[0029] The above mutants Mut1 and Mut2 were selected respectively. At the same time, wild-type M-MLV reverse transcriptase was used as the control, 1 μg of extracted human total RNA was used as the template, and Oligo(dT) was used as the template. 20 For the reverse transcription primers, the reverse transcription reaction system was prepared according to Table 1, and the reaction was carried out at 37°C, 50°C, and 60°C for 30 min respectively. T...

Embodiment 2

[0031] Identification of mutant processivity

[0032] Mutants Mut1 and Mut2 were taken respectively, 1 μg of human total RNA was used as the template, and Oligo(dT) was used as the template. 20 For the reverse transcription primers, the reverse transcription reaction system was prepared according to Table 1, and the reaction was carried out at 50 °C for 30 min; at the same time, the wild-type M-MLV reverse transcriptase was used as a control, and the reaction was carried out at 37 °C for 30 min. Then take about 100ng of cDNA products each, take the human HERC1 gene cDNA as a template, and use three pairs of primers (SEQ ID No. 4-9) with a distance of 6kb, 9kb and 12kb from the reverse transcription start site to carry out PCR amplification. The program is: pre-denaturation at 94°C for 3 min, followed by 35 cycles of 94°C for 30s, 60°C for 30s, and 72°C for 20s ( figure 2 A), use agarose gel electrophoresis to judge whether the length of the synthesized cDNA reaches 6kb, 9kb ...

Embodiment 3

[0034] Identification of mutant inhibitor tolerance

[0035] Take mutant Mut1 and Mut2 respectively (optimum reaction temperature 50 ℃), at the same time wild-type M-MLV reverse transcriptase (optimum reaction temperature 37 ℃) and purchased commercial HiScript II Reverse Transcriptase (competing product 1, the most The optimum reaction temperature was 50°C) and TransScript Uni Reverse Transcriptase (competing product 2, the optimum reaction temperature was 50°C) as the control, 1 μg of human total RNA was used as the template, and Oligo(dT) 20For reverse transcription primers, prepare a reverse transcription reaction system according to Table 1, in which Trizol (Merck, Item No. T9424) was additionally added to the reaction system, with a final concentration of 0.03%; or SDS (Aladdin, Item No. S108349), with a final concentration of 0.004%, Then react at the respective optimum temperature for 30 min. cDNA products were detected by real-time quantitative PCR using GAPDH gene-s...

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Abstract

The invention discloses a reverse transcriptase mutant capable of improving thermal stability and inhibitor tolerance and application of the reverse transcriptase mutant, the reverse transcriptase mutant is obtained by substituting amino acids at the following positions in an amino acid sequence as shown in SEQ ID NO.1, and the amino acids specifically comprise L139P, T197A, G308C, D524G and L603W; the invention also relates to a method for preparing the same, which comprises L139P, T197A, G308C, D524G, L603W and D209A. Compared with a wild type, the two mutants constructed by the invention have the advantages that the thermal stability is obviously improved, and the heat resistance can reach 60 DEG C or above; meanwhile, the tolerance to a plurality of inhibitors is remarkably enhanced, and wide application prospects and market values are achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a reverse transcriptase mutant with improved thermal stability and inhibitor tolerance and application thereof. Background technique [0002] Reverse transcriptase [EC 2.7.7.49] is a DNA polymerase that directs deoxynucleotide triphosphates to synthesize complementary DNA (cDNA) as a template, mostly derived from retroviruses using RNA as genetic material, in eukaryotes There are also discoveries. Reverse transcriptase is a multifunctional enzyme with three enzymatic activities, including RNA- and DNA-dependent DNA polymerase activity, and RNase H activity that catalyzes the degradation of RNA in RNA-DNA hybrid strands. In the field of biomedicine, reverse transcriptase is widely used in viral RNA detection, gene expression and function analysis, cDNA library preparation, RNA sequencing, etc. It is one of the most widely used biomedical tool enzymes. [0003] At present...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12P19/34
CPCC12N9/1276C12Y207/07049C12P19/34
Inventor 张旭宁刘杨邓锐程槐旭高尊昉
Owner 江苏百时美生物科技有限公司
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