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M-MLV reverse transcriptase and preparation method thereof

A technology of reverse transcriptase and reverse transcription, which is applied in the field of biological enzymes, can solve the problems of poor thermal stability, weak ability to recognize RNA templates, and low stability, and achieve high yield, high purity, and good thermal stability.

Pending Publication Date: 2022-03-22
苏州译酶生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the M-MLV reverse transcriptase in the industry currently has low activity, weak ability to recognize RNA templates, and low stability when performing reverse transcription system reactions, and poor thermal stability in reverse transcription reactions. The yield of cDNA obtained from the reaction is not high

Method used

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  • M-MLV reverse transcriptase and preparation method thereof
  • M-MLV reverse transcriptase and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] construct vector

[0065] 1) Synthesize the gene sequence according to SEQ ID NO:1 and insert the whole gene fragment into the vector pET-28a (XhoI and XbaI) vector. The pET-28a expression vector was used in this example, but it is not limited to this vector.

[0066] 2) According to the positions of the mutation points D524G, E562Q, D538N, and G638H, modify the DNA sequence so that the corresponding amino acid changes, which can be modified to one or more mutations of D524G, E562Q, D538N, and G638H.

[0067] 3) Those skilled in the art fully understand that since the same amino acid may be determined by multiple different codons, the construction of a synthetic gene sequence corresponding to the mutation of the above amino acid site is not limited to one, but may be multiple cores Nucleotides are synonymously mutated to obtain a nucleotide sequence that can also encode the amino acid sequence of the mutant described in the present invention, or a nucleotide sequence c...

Embodiment 2

[0069] induced expression

[0070] Select the recombinant M-MLV plasmid that has been modified at all four sites D524G, E562Q, D538N, and G638H in Example 1.

[0071] Transformation experiment: Take a C3029 / C2529 competent cell and place it on ice for 20 minutes, and wait until the competent cell is thawed into a liquid state; take 1 μL of 10 ng / μL M-MLV plasmid and add it to the thawed competent cell (50-100 μL), Gently flick twice to mix well, rest on ice for 25 minutes; heat shock in 42°C water bath for 90 seconds, then quickly ice-bath for 5 minutes; add 1000 μL of fresh anti-antibiotic-free LB liquid medium, place on a shaker at 200 rpm and incubate at 37°C for 1 hour;

[0072] Take 200μL and spread evenly on Kan + Incubate the resistant LB plate (0.1%) in a constant temperature incubator at 37°C for 16 hours.

[0073] Induced expression: Pick the monoclonal colonies with normal growth in the plate to 150mL Kan + Resistant LB liquid medium (100μg / mL Kan + , stock solu...

Embodiment 3

[0075] Bacteria treatment

[0076] High-pressure crushing: use an electronic balance to weigh 20g of wet bacteria, add 30mL M-MLV Ni BindingBuffer (20mM Tris-HCl, 500mM NaCl, pH=8.0@25℃), and use a vortex mixer to quickly vortex until No obvious bacterial clumps were observed with the naked eye. Continue to add M-MLV Ni Binding Buffer and vortex until no obvious bacterial clumps were observed with the naked eye. Finally, use M-MLV Ni Binding Buffer to dilute to 200mL and place in an ice bath for later use. The cold trap of the high-pressure cell disruptor was pre-cooled to 4°C in advance, drained 75% ethanol in the pipeline, added 500mL process water to clean the pipeline, then added 200mL M-MLV Ni Binding Buffer to rinse the pipeline, drained it, and poured it into a heavy Suspension liquid, 1200bar, flow rate adjusted to 50, broken 6 times, cell broken liquid placed in ice bath for temporary storage. After pressure relief, add 500mL of process water to flush the pipeline, a...

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Abstract

The invention discloses an M-MLV reverse transcriptase and a preparation method thereof. A reverse transcriptase gene sequence containing M-MLV is constructed, the reverse transcriptase gene sequence is connected to a vector and then is introduced into escherichia coli for expression, and a gene mutation site contains one or more of D524G, E562Q, D538N and G638H. According to the present invention, the amino acid of the mutation key site is charged or the hydrophobicity is changed so as to change the space structure of the M-MLV reverse transcriptase, weaken the M-MLV RNase H Minus, and improve the activity and the stability of the M-MLV reverse transcriptase;

Description

technical field [0001] The invention relates to the technical field of biological enzymes, in particular to a reverse transcriptase, especially an M-MLV reverse transcriptase and a preparation method thereof. Background technique [0002] M-MLV transcriptase has extremely low RNase H activity, which not only improves the elongation ability, but also ensures the stability of the product, which is conducive to the development of downstream experiments. M-MLV reverse transcriptase mutant has higher elongation and reaction efficiency, and supports high temperature reaction at the same time. It also has good compatibility with RNA templates with high GC content or secondary structure. If the obtained cDNA needs to be preserved, it needs to be divided and stored at -20°C or -80°C, avoid repeated freezing and thawing, and try to use it within half a year. According to needs, Oligo dT, random primers or specific primers can be selected as reverse transcription primers, and the obt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/70C12N15/54
CPCC12N9/1276C12N15/70C12Y207/07049C12N2800/101C12N2800/22
Inventor 吴宁张惠丹
Owner 苏州译酶生物科技有限公司
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