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C terminal specific human DNA mismatch repair protein MLH1 polypeptide and antibody preparation method

A mismatch repair and species-specific technology, applied in the field of biomedicine, can solve the problems of non-specific antibody binding protein position, high antibody price, low affinity, etc., and achieve the effect of strong affinity, good antibody specificity and low cost.

Inactive Publication Date: 2008-12-10
BEIJING IMMUNOHUNT CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The present invention is to solve the problem of specifically detecting the expression and natural existence of human DNA mismatch repair protein MLH1 through immune experiments, and to establish the basic biological products required for corresponding in vitro immune analysis, and provides a kind of protein that can specifically target human MLH1 C-terminal synthetic polypeptide, corresponding antibody and preparation method thereof
The present invention can also solve the problems of high price, low affinity, non-specific position of antibody-binding protein and poor antigenicity of similar antibodies currently used.

Method used

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  • C terminal specific human DNA mismatch repair protein MLH1 polypeptide and antibody preparation method
  • C terminal specific human DNA mismatch repair protein MLH1 polypeptide and antibody preparation method
  • C terminal specific human DNA mismatch repair protein MLH1 polypeptide and antibody preparation method

Examples

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Effect test

Embodiment 1

[0016] Example 1: Synthesis of human MLH1 antigenic peptide.

[0017] DNAstar, OMIGA, UWGCG and other protein analysis software were used to analyze the hydrophilic structure (hydrophilicity), antigenicity index (antigenicity), surface accessibility (surface probability) and homology (homology) of the amino acid sequence of human MLH1, and then Combined with our verification experience in actual preparation of antibodies, the 446th to 460th positions were finally determined as the target, and its amino acid sequence was SLEGDTTKGTSEMSE. The crude product was obtained after decremental synthesis from the fixed carboxyl group to the amino terminal on an automatic peptide synthesizer. After being dissolved in 30% acetonitrile, it was analyzed by high pressure liquid chromatography (HPLC), the area of ​​the main peak was calculated and collected, and the synthesized peptide was purified and identified by mass spectrometry after refrigerated vacuum drying.

Embodiment 2

[0018] Example 2: Coupling of synthetic polypeptides to carriers.

[0019] Select KLH (Keyhole limpet hemocyanin) as the carrier protein, and use the bifunctional reagent maleamide benzoic acid-N-succinate (MBS) to couple KLH with the synthetic peptide: Take 5 mg (0.11 μmol of KLH, containing lysine Acid 2.2μmol), dissolved in 0.75mL coupling buffer 1 (50mmol / L borate buffer, pH8.5); 3mg MBS (11μmol) was dissolved in 75μL dimethylformamide (DMF). Add the MBS solution to the KLH solution in 3 times, rotate and mix well, and act for 30 minutes at room temperature. After rapid centrifugation, the reaction mixture (about 0.8 mL) was added to the coupling buffer 2 (0.1 mol / L phosphate, 0.15 mol / L NaCl, 0.01 mol / L Na 2 EDTA, pH 7.0) equilibrated PD-10 column. Elute with coupling buffer 2, and collect the eluate (ie MBS-KLH solution). Dissolve 1.5mg of synthetic peptide in 0.15mL of coupling buffer 2, add MBS-KLH buffer 0.56, rotate and mix at room temperature, let it react for ...

Embodiment 3

[0020] Example 3: Preparation of anti-polypeptide antibody.

[0021] Take 500 μg of coupled KLH-polypeptide, dissolve in 500 μL phosphate buffer, add an equal volume of complete Freund's adjuvant. New Zealand rabbits (weight standard: 2-2.5 kg) were selected for immunization at the right age, and after adaptive feeding, they were injected intradermally at no less than 15 points on the back. After 2 weeks, the amount of antigen was halved (250μg), and an equal volume of incomplete Freund's adjuvant was added for the first booster immunization. The second booster immunization was carried out 2 weeks later, and the method was the same as before. Another 2 weeks later, a small amount of blood was collected from the ear vein, and the enzyme plate was coated with synthetic polypeptide (1 μg / mL), and the titer of the immune serum was detected by indirect ELISA. The immunization was repeatedly strengthened, and the immunization was stopped when the antibody titer of the blood test...

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Abstract

The invention discloses C-terminal specific human DNA mismatch repair protein MLH1 polypeptide, as well as an antibody and a preparation method thereof, belonging to the biomedicine technical field. The amino acid sequence of the C-terminal specific human MLH1 polypeptide is SLEGDTTKGTSEMSE. Antihuman MLH1 polypeptide antibody is prepared according to the following method that: (1) human MLH1 epitope is analyzed; (2) human MLH1 C-terminal polypeptide is synthesized; (3) synthesized polypeptide is crosslinked with carrier protein; (4) rabbit anti-human MLH1 polypeptide antibody is prepared; (5) serum containing the antibody is obtained through collection and separation, and the antibody is purified, and then the antihuman MLH1 polypeptide antibody can be obtained. The C-terminal specific antihuman MLH1 synthesized polypeptide antibody is high in potency, strong in affinity, good in specificity, capable of having specific binding reaction with natural human DNA mismatch repair protein MLH1 and low in preparation cost; purified antibody can be completely used for immunoblot and enzyme linked immunosorbent assay, as well as the establishment of in vitro immunization analytical method. The antibody provides a useful tool for the research on human MLH1 biological function as well as the relation between the human MLH1 and related diseases.

Description

technical field [0001] The invention relates to biological products for in vitro tests characterized by antibodies, in particular to a C-terminal-specific human DNA mismatch repair protein MLH1 polypeptide and an antibody preparation method thereof, belonging to the field of biomedical technology. Background technique [0002] MLH1 (MutL protein homolog 1) is a DNA mismatch repair protein. The repair of mismatched DNA is an essential link to maintain the integrity of genetic information in different phases. Slippage of an interstrand mismatch during DNA replication can cause a change in a tiny repeat, which is known as microsatellite instability. Studies have linked defects in this DNA repair function to carcinogenesis in humans. With the elucidation of the genetic basis of hereditary nonpolyposis rectal cancer (HNPCC), the importance of mismatch repair genes has become increasingly apparent. MSHS2 is involved in the initial recognition process of mismatched nucleotides i...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K16/18
Inventor 杜宏武陈光宇吕玉娥
Owner BEIJING IMMUNOHUNT CORP
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