CN-119709683-B - M-MLV reverse transcriptase mutant and preparation method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to an M-MLV reverse transcriptase mutant, a preparation method and application thereof. The invention provides an M-MLV reverse transcriptase mutant, a preparation method and application thereof. The M-MLV reverse transcriptase mutant fuses the structural characteristics of a plurality of mutant variants, and has better performance in the aspects of reverse transcription activity and thermal stability. In particular to an M-MLV reverse transcriptase mutant Super RT with an amino acid sequence shown as SEQ ID NO.01, which has higher continuous synthesis capability, can perform good reverse transcription reaction on a long-chain RNA template, has strong inhibition resistance, can ensure cDNA synthesis in a complex reaction environment, and can complete full-length cDNA synthesis in a shorter time even in the presence of common inhibitors such as guanidine salt, heparin and salt.

Inventors

• ZHENG HONGKUN
• LIU MIN
• ZHANG MENGLONG

Assignees

• 青岛百创智能制造技术有限公司

Dates

Publication Date

20260512

Application Date

20241115

Claims (6)

1. An M-MLV reverse transcriptase mutant, characterized in that the amino acid sequence of said M-MLV reverse transcriptase mutant is shown in SEQ ID No. 01.
2. 2. A coding gene, wherein the coding gene encodes the nucleotide sequence of the M-MLV reverse transcriptase mutant according to claim 1.
3. 3. A biological material, characterized in that it is a recombinant plasmid or a recombinant bacterium, said biological material having the coding gene of claim 2.
4. 4. A method for producing an M-MLV reverse transcriptase mutant according to claim 1, comprising the step of expressing said M-MLV reverse transcriptase mutant using the biological material according to claim 3.
5. 5. The use of the M-MLV reverse transcriptase mutant according to claim 1 for the preparation of a thermostable reagent, said thermostable reagent having biological activity at 65 ℃.
6. 6. Use of the M-MLV reverse transcriptase mutant according to claim 1 in a reverse transcription reaction for the diagnostic and therapeutic purposes of a non-disease.

Description

M-MLV reverse transcriptase mutant and preparation method and application thereof Technical Field The invention relates to the technical field of biology, in particular to an M-MLV reverse transcriptase mutant, a preparation method and application thereof. Background Reverse transcriptase findings demonstrate that genetic information is not unidirectionally transferred from DNA to RNA to protein, but it is also possible to transfer from RNA to DNA in the opposite direction. The ability to reverse transcribe mature mRNA into cDNA is critical for information acquisition in a variety of biomedical environments, including diagnostics, prognosis, biotechnology, forensic biology, and the like. Therefore, reverse transcriptase is a ubiquitous tool in molecular biology. Reverse transcriptase was originally found in Moloney Mouse Leukemia Virus (MMLV), a DNA polymerase with RNA as template, consisting of 671 amino acids, four subdomains (finger, palm, thumb and linker) and a ribonuclease H (RNase H) active domain, with RNase H activity, without 3'-5' exonuclease activity, and can be used for reverse transcription to synthesize cDNA. One of the major challenges in the synthesis of cDNA by reverse transcription is interference of RNA secondary structure, although higher reaction temperatures can remove the secondary structure of the template RNA, excessive temperatures often result in a dramatic decrease in wild-type (WT) MMLV reverse transcription activity. Reverse transcriptase activity is also affected by a number of other factors, such as the presence of inhibitors (e.g., cell lysates and related reagents) in the reaction system, e.g., lower volume reaction systems, which negatively affect WT RT reverse transcription activity. Current studies demonstrate that E69K、E302R、T306K、W313F、L435G/K、N454K（Arezi et al (2009) Nucleic Acids Res. 37(2)：473-481, U.S. Pat. No. 7,078,208, and Baranauskas et al 2012 Prot Engineering 25(10)： 657-668）、L52P、Y64R、K152M、H204R、M289L、T306K isosite changes can improve thermostability (CN 1430670), L139P, D200N, T330P, P448A, D449G, L603W, E K isosite changes can improve reverse transcription full length and anti-cell lysate inhibition and higher thermostability, and still higher reactivity with reaction systems less than 1 nanoliter, furthermore, D524N/a allele changes can reduce or eliminate rnase H activity (CN 110637084 a), Y64W/R, K152R, Q190F, T a/E, V223H/L/F, Y133A/H, F309N/R allele changes can improve reverse transcription fidelity, and Y133A, T197E, F309N isosite changes also demonstrate reduced terminal transferase activity (CN 1430670; U.S. application No. 60/454; 09/808,124). Reverse transcriptase that has achieved various performance improvements in existing studies is mainly through two ways, random mutation and site-directed mutation. Random mutagenesis, i.e., random mutagenesis of amino acids of the domain of M-MLV, is followed by screening by various methods. The method can screen MMLV mutants with improved performances, but has the defects of complex technical process, huge capacity of mutation library (up to 107) and great screening difficulty, and a large number of ineffective mutations can be obtained. The method has stronger purposive property by carrying out amino acid mutation on key active site amino acids such as a nucleic acid binding site, a metal ion binding site and the like so as to change the activity of enzyme. However, this method lacks analysis of the overall structure of the enzyme and does not improve the performance of the enzyme in various aspects. In general, there is a balance between the activity and stability of reverse transcriptase, i.e., mutation of the amino acid site that increases the activity of the enzyme may result in a decrease in the stability of the protein, while mutation of the amino acid site that increases the stability of the protein may decrease the activity of the enzyme. Therefore, it is important to provide a reverse transcriptase having both high temperature stability and high activity. In view of this, the present invention has been made. Disclosure of Invention In order to solve the technical problems, the invention provides an M-MLV reverse transcriptase mutant with high reverse transcription efficiency, high temperature resistance and strong inhibition resistance, and a preparation method and application thereof. Specifically, the technical scheme of the invention is as follows: In a first aspect, the invention provides an M-MLV reverse transcriptase mutant having one or more of the following mutations Y133K, T306H, T330G, L435R in the amino acid sequence compared to the amino acid sequence of a wild-type M-MLV reverse transcriptase. Preferably, the amino acid sequence of the reverse transcriptase mutant has one of the following combinations of mutations compared to the amino acid sequence of the wild type M-MLV reverse transcriptase: Mutation combination 2:y133k, T330G; Mutation combination 3:t306h, T330G; Mutation combi