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A group of antibodies that recognize pyrimidine dimers caused by ultraviolet irradiation of DNA

A technology of pyrimidine dimer and ultraviolet rays, applied in the field of medicine and biology, can solve the problems of inaccuracy

Active Publication Date: 2021-08-13
北京博雅捷康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, almost all previous studies irradiated cells or tissues with high-dose UV and then performed subsequent detection, which is not accurate enough for studying 6-4PP produced at the physiological level of the body and understanding its repair process

Method used

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  • A group of antibodies that recognize pyrimidine dimers caused by ultraviolet irradiation of DNA
  • A group of antibodies that recognize pyrimidine dimers caused by ultraviolet irradiation of DNA
  • A group of antibodies that recognize pyrimidine dimers caused by ultraviolet irradiation of DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, construction of related plasmids:

[0043] The variable region gene sequences (specific sequences) of the antibodies 64M-2 and 64M-5 against (6-4) photoproducts (6-4PPs) were obtained from the RCSB protein database (http: / / www.rcsb.org) and provided by Jin Synthesized by Sri Company as the target fragment. On the basis of the plasmid previously constructed in our laboratory, conventional cloning methods were used to replace the VH and VL regions in the plasmid with the heavy and light chain variable regions of the 64M-2 antibody by PCR or overlap PCR.

[0044] Among them, 64M-2VH was inserted into the sites of EcoRI and ClaI, 64M-2VL was inserted into the sites of EcoRI and KpnI, and finally two plasmids capable of displaying 64M-2ccFv on the inner membrane of E. coli were obtained. For detailed plasmid construction methods, please refer to the paper "Efficient Escherichia coli Antibody Display and Establishment of Artificial Evolution Platform" published...

Embodiment 2

[0047] Example 2, preparation of photoproduct (6-4PP) probe and purification of probe containing 6-4PP by HPLC:

[0048] 1. Probe preparation

[0049] In order to meet the requirements of flow sorting and detection, we prepared two kinds of probes containing 6-4PP: a long-chain probe (L-probe) containing 36 bases and a long-chain probe (L-probe) containing 10 bases Short chain probe (Sh-probe). The sequences of all probes can be found in Table 1. Because flow cytometry requires the detection of fluorescent signals, we conjugated biotin to the 3' end of all flow sorting and detection probes, which can be labeled with fluorescent molecularly labeled probes with streptavidin .

[0050] In Table 1, except that Sh-probe5 was purchased from TriLink BioTechnologies, all other probe sequences were synthesized by Shanghai Boshang Biotechnology Co., Ltd. The long-chain probe sequence L-probe1-8 was dissolved in double-distilled water to a final concentration of 50 μM. Except for L-p...

Embodiment 3

[0060] Example 3. Based on the 64M-2 antibody, artificial evolution of high-quality antibodies against photoproducts (6-4PP)

[0061] 1. Construction and evolution of 64M-2 antibody library

[0062] Select the V region gene sequence of the 64M-2 antibody as a template, first clone the VH and VL of 64M-2, and then use error-prone PCR to obtain an original antibody library with an average of 4-6 mutations in each gene . The primer pairs we used here are:

[0063] 5'-CGGACTACAAAGATGAATTCGAAGTTCAGCTGCAGCAGAG and

[0064] 3'-CCGCCGCCCTCGAGGTCGACTTTAATTTCCAGTTTGGTGCC.

[0065] We made some adjustments according to the manufacturer's instructions (Takara, Japan):

[0066] PCR buffer (50ul) contains:

[0067] 1ul low-fidelity polymerase (rTaq, Takara),

[0068] 40ng 64M-2 gene template,

[0069] Primer (10mM),

[0070] MnCl 2 (5mM), MgCl 2 (25mM), dNTP (2.5mM) and 20mM each of dCTP and dTTP.

[0071] The PCR program is:

[0072] (1) 94°C: 30s, (2) 55°C: 30s, (3) 72°C, 45s,...

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Abstract

The present invention relates to a group of antibodies that specifically recognize pyrimidine dimers caused by ultraviolet radiation on DNA chains. The basic amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO.1. The heavy chain of the antibody is The basic amino acid sequence of the chain variable region is shown in SEQ ID NO.2, and the light chain variable region of the antibody is: on the basic amino acid sequence of the light chain variable region, it contains at least one of the following mutations: S26N, L88Q, Q95L; the heavy chain variable region of the antibody is: the basic amino acid sequence of the heavy chain variable region contains at least one of the following mutations: V73D, T74P, T97A.

Description

technical field [0001] The invention belongs to the technical field of medicine and biology, and specifically relates to a group of antibodies for recognizing pyrimidine dimers caused by ultraviolet radiation of DNA. Background technique [0002] DNA contains the genetic information that organisms rely on to survive and reproduce. In the process of cell division, DNA transmits genetic information to the next generation through precise replication, so maintaining the integrity of DNA molecules and high fidelity of replication is crucial for cells. However, the DNA in the cells of the organism is being damaged all the time, and the external environment and factors from within the organism may cause damage and changes to the DNA molecule. If these damages cannot be repaired in time, it will affect the function and even the survival of cells. If the damage occurs in the germ cells of the organism, it may also affect the offspring. Therefore, in the process of evolution, organ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/44G01N33/53G01N33/574A61K39/395A61P39/00A61P39/06A61P35/00A61P27/02
CPCA61P27/02A61P35/00A61P39/00A61P39/06C07K16/44C07K2317/56C07K2317/76C07K2317/92G01N33/5308G01N33/57407G01N33/5743G01N2800/164G01N2800/20
Inventor 杭海英汪海林孔冰洁冉凡磊
Owner 北京博雅捷康生物科技有限公司
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