MMLV enzyme combination mutant

A technology of mutants and site mutations, applied in the field of combined mutants of MMLV enzymes, can solve problems such as 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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  • MMLV enzyme combination mutant
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  • MMLV enzyme combination mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0120] Embodiment 1 Calculation and screening of DDG value of each point

[0121] The MMLV protein sequence was input into the Rosetta algorithm software Cyrus Bench (Cyrus Biotechnology), and the 0-100, 101-200, 201-300, 301-400, 401-500, 501-600, and 601-671 amino acids were segmented for all positions. The DDG value of the point full mutation is calculated, and the information of the mutation site with a significantly reduced DDG value (DDG value <-2) is obtained 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] Gene synthesis was carried out by Suzhou Jinweizhi Biotechnology Co., Ltd. according to the above DNA sequence, 5'(NheI) and 3'(XhoI) restriction enzyme sites were added, and the gene was cloned into the vector pET28a through 5'NheI and 3'XhoI, The plasmid WT-pET28a was constructed, the recombinant plasmid DNA and the glycerol bacteria containing the recombinant plasmid were prepared, and the plasmid WT-pET28a was site-directed mutagenesis according to the mutation sites involved in Example 1 to construct a mutation library Mu1-pET28a~Mu40-pET28a .

Embodiment 3

[0128] Example 3 Expression and purification of MMLV mutants

[0129] The WT-pET28a, Mu1~40-pET28a plasmids were transformed into BL21(DE3) competent cells to obtain 37 expression host bacteria, and then transferred to 3ml LB medium, shaken at 37°C for 5 hours, and then added 0.1Mm IPTG at 18°C Induction culture overnight. The induced cells were collected, lysing solution (50Mm Tris, 50Mm NaCl, pH7.5) was added, lysed by ultrasonic, and the supernatant was separated by centrifugation. The supernatant was taken and purified by Ni NTA metal ion chelating packing to obtain wild-type and 40 mutant MMLV proteins

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Abstract

The invention provides an MMLV enzyme combination mutant, and particularly, a reverse transcriptase (M-MLV) mutation library is constructed, the MMLV enzyme combination mutant with improved thermal stability and higher amplification efficiency is finally screened out through mass screening, and the MMLV enzyme combination mutant disclosed by the invention can be used for efficiently detecting viral nucleic acid.

Description

technical field [0001] The present invention belongs to the field of biotechnology, in particular, the present invention relates to an MMLV enzyme combination mutant for nucleic acid detection. 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 no 3'-5' exonuclease activity, and can be used to synthesize cDNA by reverse transcription. Since RNA has a more complex secondary structure, it has a greater impact on the reverse transcription efficiency of M-MLV enzymes. The easiest way to open the RNA secondary structure is to open the hydrogen bonds of the secondary structure by high temperature, and the RNA becomes a linear single strand. However, the optimal reaction temperature of wild-type M-MLV enzyme was 37℃, and the stability and activity decreased at high temperature. Therefore, it is the main transformation direction of M-MLV enzyme to improve the thermostability of M-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/70C12N1/21C12Q1/70C12Q1/6851C12R1/19C12R1/93
CPCC12N9/1276C12Y207/07049C12N15/70C12Q1/701C12Q1/6851C12Q2521/107C12Q2531/113C12Q2563/107C12Q2545/113
Inventor 蒋析文刘霭珊赵继光王周全
Owner DAAN GENE CO LTD
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