Reverse transcriptase and reverse transcription detection reagent

A reverse transcription and kit technology, applied in the field of reverse transcriptase and reverse transcription detection reagents, can solve problems such as the uncertainty of enzyme transformation

Pending Publication Date: 2022-05-13
DAAN GENE CO LTD
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  • 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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  • Reverse transcriptase and reverse transcription detection reagent
  • Reverse transcriptase and reverse transcription detection reagent
  • Reverse transcriptase and reverse transcription detection reagent

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] Example 3 Expression and purification of MMLV mutants

[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 reverse transcriptase and a reverse transcription detection reagent, specifically, a reverse transcriptase (M-MLV) mutation library is constructed, a reverse transcriptase mutant with improved thermal stability and relatively high amplification efficiency is finally screened out through mass screening, and the reverse transcription detection reagent containing the reverse transcriptase mutant is further provided.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a reverse transcriptase and a reverse transcription detection reagent 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 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 thro...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12N15/70C12N1/21C12N15/10C12R1/19
CPCC12N9/1276C12Y207/07049C12N15/70C12N15/10
Inventor 蒋析文刘霭珊连献兰谢晓成
Owner DAAN GENE CO LTD
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