M-MLV reverse transcriptase mutant and application thereof

A technology of reverse transcriptase and mutants, which is applied in the field of M-MLV reverse transcriptase mutants, can solve the problems of reduced catalytic activity and reduced tolerance to inhibitors, and achieve reduction of additional interference, strong inhibitor tolerance, The effect of high response sensitivity and accuracy

Active Publication Date: 2021-09-07
ZYBIO INC
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  • Description
  • Claims
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Problems solved by technology

However, reverse transcriptase with improved thermostability may be accompanied b

Method used

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  • M-MLV reverse transcriptase mutant and application thereof
  • M-MLV reverse transcriptase mutant and application thereof
  • M-MLV reverse transcriptase mutant and application thereof

Examples

Experimental program
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Embodiment 1

[0020] The construction of embodiment 1M-MLV reverse transcriptase mutant

[0021] 1. Plasmid construction of reverse transcriptase mutant strains

[0022] According to the wild-type MMLV reverse transcriptase protein sequence (the gene sequence of the MMLV reverse transcriptase mother is shown in SEQ ID NO.1), after performing codon optimization suitable for Escherichia coli, insert it into the vector pBAD-hisA as the wild-type reverse transcriptase expression plasmid. According to the structural characteristics of reverse transcriptase, the mutation site is mainly selected:

[0023] (1) The structural domain involved in substrate binding and cDNA synthesis, mutating amino acids can improve the substrate binding ability;

[0024] (2) amino acids connecting the polymerase domain and the RNaseH domain, and mutating amino acids to improve its stability;

[0025] (3) RNase H domain, mutating amino acids to improve thermal stability or inhibitor tolerance and the ability to syn...

Embodiment 2

[0045] Embodiment 2M-MLV reverse transcriptase mutant performance verification

[0046] Perform performance evaluation on the obtained mutant reverse transcriptase, select clinical samples for detection, and set up 3 sets of reaction systems in total, and the 3 sets of reaction systems are all configured according to the reaction system described in Example 1, the difference is only in the reverse transcriptase, All the other components and consumption are exactly the same.

[0047] Group A: the reverse transcriptase is the reverse transcriptase described in the present invention;

[0048] Group B: the reverse transcriptase is Thermo Fisher's SuperScriptⅢ (SSⅢ);

[0049] Group C: the reverse transcriptase was Thermo Fisher's SuperScript IV (SS IV).

[0050] (1) Thermal stability verification of M-MLV reverse transcriptase mutants

[0051] In order to detect the thermal stability of reverse transcriptase, the above three groups of reaction systems were subjected to reverse t...

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Abstract

The invention discloses an M-MLV reverse transcriptase mutant and application thereof. The amino acid sequence of the M-MLV reverse transcriptase mutant is shown as SEQ ID NO.2 in a sequence table. The reverse transcriptase provided by the invention is obtained through site-specific mutagenesis rational design screening on the basis of wild type M-MLV reverse transcriptase in combination with the existing structural characteristics of reverse transcriptase structure research progress and fusion of a plurality of mutants, the reverse transcriptase mutant has very strong inhibitor tolerance, the interference of an inhibitor on cDNA synthesis is reduced. In the reaction process of a sample added with whole blood and sputum, the activity of the mutant reverse transcriptase is not influenced or is slightly influenced. Meanwhile, the reverse transcriptase mutant provided by the invention can tolerate a high temperature of 70 DEG C, has relatively high reaction sensitivity and accuracy while having relatively good thermal stability, and has a very strong application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an M-MLV reverse transcriptase mutant and application thereof. Background technique [0002] Reverse transcriptase (Reverse transcriptase) generally has three activities: DNA polymerase activity using RNA as a template, DNA polymerase activity using DNA as a template, and RNase H activity to degrade RNA in the RNA-DNA hybrid strand. Therefore, reverse transcriptase is often used in cDNA library construction, mRNA sequencing, RT-PCR quantification, etc., and is widely used in research and medical molecular diagnosis. Moloney Murine Leukemia Virus (MMLV) or avian myeloblastosis virus (Avian Myeloblastosis Virus, AMV) pol gene produced reverse transcriptase are the two most commonly used reverse transcriptase, RNase of AMV reverse transcriptase H has high activity and high thermal stability, but the yield of the product is low; MMLV reverse transcriptase has a high yield, but poor ther...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12N15/70C12N1/21C12Q1/686C12R1/19
CPCC12N9/1241C12N15/70C12Y207/07049C12Q1/686C12Q2521/107Y02A50/30
Inventor 宋丹凤赵伟夏娟严子成汪瑶
Owner ZYBIO INC
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