Nucleic acid detection kit containing reverse transcriptase mutant

A technology of mutants and reverse transcription, applied in the direction of transferase, microbial determination/testing, enzymes, etc., can solve the problems of uncertainty in enzyme transformation

Pending Publication Date: 2022-05-13
DAAN GENE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In general, due to the complexity of protein structure, not only the amino acids located in the active site but also some amino acids far away from the active site may affect the overall structure and performance of the enzyme, so there is great uncertainty in the transformation of enzymes

Method used

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  • Nucleic acid detection kit containing reverse transcriptase mutant
  • Nucleic acid detection kit containing reverse transcriptase mutant
  • Nucleic acid detection kit containing reverse transcriptase mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0120] Calculation and screening of each site DDG value in embodiment 1

[0121] Input the MMLV protein sequence into the Rosetta algorithm software Cyrus Bench (Cyrus Biotechnology), and carry out the whole position of amino acid segments 0-100, 101-200, 201-300, 301-400, 401-500, 501-600, 601-671 Calculate the DDG value of the point full mutation, and the information of the mutation site with a significantly lower DDG value (DDG value <-2) is as follows:

[0122] Table 1

[0123]

[0124]

Embodiment 2

[0125] Example 2 Construction of MMLV mutation library

[0126] According to the above protein sequence, codon optimization was performed by Suzhou Jinweizhi Biotechnology Co., Ltd., and the DNA sequence (SEQ ID NO.: 2) was compiled.

[0127] Suzhou Jinweizhi Biotechnology Co., Ltd. carried out gene synthesis based on the above DNA sequence, added 5' (NheI) and 3' (XhoI) restriction enzyme sites, and cloned the gene into the vector pET28a through 5'NheI and 3'XhoI, Construct plasmid WT-pET28a, prepare recombinant plasmid DNA and glycerol bacteria containing the recombinant plasmid, and perform site-directed mutation on plasmid WT-pET28a according to the mutation sites involved in Example 1, and construct mutation libraries Mu1-pET28a~Mu40-pET28a .

Embodiment 3

[0128] Expression and purification of embodiment 3MMLV mutant

[0129] Transform WT-pET28a, Mu1~40-pET28a plasmids into BL21(DE3) competent cells to obtain 37 expression host bacteria, then transfer to 3ml LB medium, shake and culture at 37°C for 5 hours, then add 0.1Mm IPTG at 18°C Induce the culture overnight. Collect the induced cells, add lysate (50Mm Tris, 50Mm NaCl, pH7.5), ultrasonically lyse, and centrifuge to separate the supernatant. Take the supernatant and purify it with Ni NTA metal ion chelating filler to obtain wild type and 40 kinds of mutant MMLV proteins

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Abstract

The invention provides a nucleic acid detection kit containing reverse transcriptase mutants, specifically, a reverse transcriptase (M-MLV) mutation library is constructed, and the reverse transcriptase mutants with improved thermal stability and higher amplification efficiency are finally screened out through mass screening. The invention further provides a nucleic acid detection kit containing the reverse transcriptase mutant.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a nucleic acid detection kit containing a reverse transcriptase mutant. Background technique [0002] Murine leukemia reverse transcriptase (M-MLV) is a DNA polymerase that uses RNA as a template. It has RNase H activity and does not have 3'-5' exonuclease activity. It can be used for reverse transcription to synthesize cDNA. Due to the complex secondary structure of RNA, it has a great influence on the reverse transcription efficiency of M-MLV enzyme. The easiest way to open the secondary structure of RNA is to open the hydrogen bonds of the secondary structure through high temperature, and the RNA becomes a linear single strand. However, the optimal reaction temperature of the wild-type M-MLV enzyme is 37°C, and the stability and activity decrease at high temperature. Therefore, improving the thermal stability of M-MLV enzymes through mutations and adapting...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12Q1/48
CPCC12N9/1276C12Q1/48C12Y207/07049G01N2333/9128
Inventor 蒋析文刘霭珊赵继光张伟
Owner DAAN GENE CO LTD
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