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Novel M-MLV reverse transcriptase and application thereof

A reverse transcriptase and expression vector technology, applied in the field of MMLV reverse transcriptase with high continuous synthesis ability and high heat resistance, can solve the problems of expensive imported reagents and lack of reverse transcription reagents, and improve detection sensitivity and experimental efficiency. , High continuous synthesis ability, the effect of avoiding pollution

Active Publication Date: 2021-01-22
北京健为医学检验实验室有限公司 +1
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0009] At present, the most important product of reverse transcription reagents is Invitrogen’s SuperScript series products, which almost monopolize the main domestic scientific research and diagnostic reagent markets, but imported reagents are expensive, and the cost problem has become an important factor for many domestic laboratories to choose testing raw material reagents.
In recent years, with the development of molecular biology, various domestic manufacturers have also launched their own reverse transcription reagents, but most of the domestic reagents can only meet the two-step RT-PCR at the same time, and can meet the two-step RT-PCR and one-step RT-PCR at the same time. RT-PCR, and reverse transcription reagents that are compatible with RNA crude extracts or RNA virus lysates are still relatively lacking

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: SuperRT reverse transcriptase cloning, expression purification and detection

[0038] All DNA manipulations were performed using standard techniques, particularly cloning using PCR, PCR-based mutagenesis procedures, and standard restriction digestion and ligation. All proteins were expressed in Escherichia coli, Ni-NTA resin was purchased from Qiagen, and CM resin was purchased from QE Company. The SuperRT reverse transcriptase sequence of the present invention is shown in SEQ ID No.1, by cloning it into a modified pET21 + plasmid to construct a recombinant expression plasmid, and transforming the host bacteria, purifying, identifying and storing the host bacteria after culturing; the specific method is as follows :

[0039] (1) Plasmid construction: construct the expression plasmid pET-SRTHis of the Super RT, using primer 5'-ATATA CATATG CTAAAT ATAGAAGATGAG-3' and 5'-ATATC CTCGAG TATGAGGAGGGTAGA -3'Amplified a 2035bp DNA fragment encoding SuperRT reverse...

Embodiment 2

[0045] Example 2: Detection of reverse transcription performance of purified SuperRT reverse transcriptase

[0046] The reverse transcription performance of the purified protein was tested using the extracted human total RNA as a template. After the human RNA template RT is reverse-transcribed into cDNA, use the primers ACTB primers indicating the sequence to configure the PCR reaction. The specific experimental operation is as follows:

[0047] Prepare cDNA first-strand synthesis reaction solution, each reaction includes 7 μl nuclease-free water, Super RT reaction buffer, 4 μl 2.5mM dNTP, 2 μl random primers, 1 μl SuperRT to be tested or purchased superscrip III; the prepared Mix the cDNA first-strand synthesis reaction solution, centrifuge briefly, and divide into PCR reaction tubes, 18 μl per tube; add 2 μl human RNA template to each of the above-mentioned reaction tubes, mix well, and put it into the PCR machine for reaction at 50°C 10min, 5min at 85°C to stop the revers...

Embodiment 3

[0050] Embodiment 3: Detection of poultry IBV virus with SuperRT reverse transcription reaction system

[0051] Using the SuperRT reverse transcription reagent and reaction system provided by the present invention, two avian IBV coronavirus samples (RNA virus, provided by Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd., belong to attenuated strains, are not infectious and pathogenic to humans. sex) for detection, specifically including the following steps:

[0052] 1. RNA sample preparation

[0053] 1.1 Roughly handle RNA samples: Dilute 2 poultry IBV stock solutions to 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 and 10 -8 Dilution gradients are crude RNA samples for subsequent RT-PCR detection operations.

[0054] 1.2 Extract RNA samples: Take 2 poultry IBV virus stocks for RNA extraction. It is recommended to use the virus nucleic acid extraction kit CWY071 of Kangwei Century Biotechnology Co., Ltd., and operate according to the instructions. The extracted RNA...

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Abstract

The invention relates to an M-MLV reverse transcriptase and an application thereof. The novel M-MLV reverse transcriptase designed by the inventor of the invention is a novel M-MLV reverse transcriptase with relatively low RNase H activity, relatively high continuous synthesis capability and relatively strong anti-inhibition capability, and has high continuous synthesis capability and high heat resistance. A reverse transcription reagent system containing the M-MLV reverse transcriptase can be compatible with various RNA sample types, is especially suitable for one-step RTPCR, and has wide application prospect and market promotion value.

Description

technical field [0001] The invention mainly relates to the field of biotechnology, in particular to a MMLV reverse transcriptase with high continuous synthesis ability and high heat resistance and application thereof. Background technique [0002] Reverse transcription, also known as reverse transcription, refers to the process of synthesizing complementary cDNA using RNA as a template. In this process, the process of nucleic acid synthesis and transcription (DNA to RNA) is opposite to the flow direction of genetic information (RNA to DNA), so it is called reverse transcription. The reverse transcription process is one of the replication forms of viruses, such as retroviruses in RNA viruses, hepadnaviruses in DNA viruses, and cauliflower mosaic virus. Replication requires reverse transcription, and the reverse transcription process requires reverse transcription The enzyme (reversetranscriptase) catalyzes the completion. Reverse transcriptase is an RNA-dependent DNA polyme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/70C12N1/21C12Q1/686C12Q1/70C12R1/19C12R1/93
CPCC12N9/1276C12N15/70C12Q1/686C12Q1/701C12Y207/07049C12Q2521/107C12Q2527/125
Inventor 高明严兵刘淑君庄志华王春香
Owner 北京健为医学检验实验室有限公司
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