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M<6>A recombinant rabbit monoclonal antibody and preparation method thereof

A monoclonal antibody and heavy chain technology, applied in the field of molecular biology, can solve problems such as low sensitivity, difficult IF experiments, and difficult enrichment, and achieve the effects of improving screening efficiency, easy screening, and high gene diversity

Active Publication Date: 2021-11-30
WUHAN AIBO TAIKE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The enrichment of specific antibodies is a commonly used method, however, the currently used m 6 Antibody A still has the defect of low sensitivity, and it is difficult to perform IF experiments; at the same time, in the presence of a large amount of interfering RNA, m 6 Enrichment of A-modified RNAs is very difficult

Method used

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  • M&lt;6&gt;A recombinant rabbit monoclonal antibody and preparation method thereof
  • M&lt;6&gt;A recombinant rabbit monoclonal antibody and preparation method thereof
  • M&lt;6&gt;A recombinant rabbit monoclonal antibody and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The present invention provides a kind of by m 6 A method for synthesizing an antigen from a hapten, the specific steps are as follows:

[0035] 1. Weigh m 6 A small molecule 40mg, add to a 50ml test tube, then add 0.1M NaIO drop by drop 4 Solution until 5ml, shake while adding, react at room temperature for 30 minutes. Add 1ml ethylene glycol to neutralize excess NaIO 4 , react at room temperature for 5 minutes.

[0036] 2. Take 10ml of KLH carrier protein (Merck, product number: 374805) with a concentration of 6.15mg / ml, add it to a new 50ml test tube, and use 2M K 2 CO 3 The pH value of the solution was adjusted to be between pH 9.0 and 9.5.

[0037] 3. Then slowly add the carrier protein in step 2 to the solution in step 1 and react for 45 minutes. 5% K during 2 CO 3 The pH of the solution was adjusted to maintain the pH between 9 and 9.5.

[0038] 4. Weigh 37.5mg NaBH 4 Dissolve in 2.5ml of water, add dropwise to the reaction solution in step 3, shake whi...

Embodiment 2

[0042] This example provides a method for immunizing animals with the antigen synthesized in Example 1 and screening for positive clones. The specific steps are as follows:

[0043] 1. Multi-point injections on the back and abdomen of rabbits (750ug antigen per rabbit for the first time, 350ug antigen per rabbit afterwards), repeat immunization every 2 weeks, repeat 3 times, the rabbit used in this embodiment is New Zealand white rabbit, for the first time Complete adjuvant (Sigma, product number: F5881) was used for immunization, and incomplete adjuvant (Beijing Boaolong, product number: KX0210047Q-10) was used for subsequent immunization.

[0044] 2. One week after the end of immunization, take rabbit serum for ELASA for polyantibody detection, and synthesize the following primers:

[0045] M6A-1: m 6A-CUGGUAACGAAUGGCUG-Biotin

[0046] M6A-2: A-CUGGUAACGAAUGGCUG-Biotin

[0047] M6A-3: AAAAAAAAAAAAA-Biotin

[0048] The M6A-1 primer is a positive control containing m 6 A ...

Embodiment 3

[0053] This example provides a method for expressing and purifying the antibody from the B cells screened in Example 2. The specific steps are as follows:

[0054] 1. After the cells of positive clones were collected and lysed, RNA was extracted by conventional methods and reverse transcribed into cDNA. The PCR method is adopted, and the heavy chain amplification primers are shown in SEQ.NO.13-SEQ.NO.14.

[0055] Light chain amplification primers are shown in SEQ.NO.15-SEQ.NO.16.

[0056] In SEQ.NO.15, Y is C or T; in SEQ.NO.16, R is G or A). The PCR reaction system is: 95°C for 2min, then 25 cycles of reaction according to the following conditions: 95°C for 1min, 56°C for 1min, 72°C for 0.5min; finally 95°C for 10min.

[0057] The heavy and light chain genes (VH and VL) of the natural paired rabbit monoclonal antibody were amplified from the cDNA of the corresponding positive clone, and the sequence was determined by sequencing. The gene sequence of the variable region of t...

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Abstract

The invention provides an m<6>A recombinant rabbit monoclonal antibody and a preparation method thereof. According to the m<6>A recombinant rabbit monoclonal antibody and the preparation method thereof, the amino acid sequence of a heavy chain CDR1 of the m<6>A recombinant rabbit monoclonal antibody is shown as SEQ.NO.1 in the specification, the amino acid sequence of a CDR2 of the m<6>A recombinant rabbit monoclonal antibody is shown as SEQ.NO.2 in the specification, the amino acid sequence of a heavy chain CDR3 of the m<6>A recombinant rabbit monoclonal antibody is shown as SEQ.NO.3 in the specification, the amino acid sequence of a light chain CDR1 of the m<6>A recombinant rabbit monoclonal antibody is shown as SEQ. NO.4 in the specification, the amino acid sequence of a CDR2 of the m<6>A recombinant rabbit monoclonal antibody is shown as SEQ. NO.5 in the specification, and the amino acid sequence of a CDR3 of the m<6>A recombinant rabbit monoclonal antibody is shown as SEQ. NO.6 in the specification. The m<6>A recombinant rabbit monoclonal antibody has good enrichment capacity for m<6>A modified RNA, and has good sensitivity for detecting the m<6>A modified RNA in a DNA damage repair living cell model.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to m 6 A recombinant rabbit monoclonal antibody and its preparation method. Background technique [0002] N6-Methyladenine (m 6 A) is the most abundant chemical modification found on eukaryotic mRNA, and it is also widely present in various bacteria and RNA viruses. In animals or plants, m 6 A can be modified, demodified and recognized by a series of methyltransferases, demethylases and binding proteins. m 6 A can regulate a variety of biological functions by affecting RNA processing and metabolism. Related studies have also found that RNA m6A modification plays an important role in UV-induced DNA damage response, and RNA m6A 6 A modification is mainly to guide cells to quickly and accurately recruit Polκ to the damage site and accelerate DNA damage repair to promote cell survival. Therefore, the m 6 A modification is an important reference factor for related disease mechani...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/00C12N15/13C12N15/10
Inventor 陈亮张云程瑶李博吴知才
Owner WUHAN AIBO TAIKE BIOTECH CO LTD
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