Polyclonal antibody for efficiently recognizing proteinserine heptanose glycosylation, and reparation method and application thereof

A technology of serine heptose and purification method, which is applied to peptide preparation methods, chemical instruments and methods, antibodies, etc., can solve the problems of lack of relevant tools and effective means for detection, and achieve good application prospects, good specificity, and production low cost effect

Inactive Publication Date: 2019-01-25
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although there are many means and tools for detecting protein glycosylation (such as protein Ser / Thr-O-GlcNAc modification or Asn-N-glycosylation modification), the detection and exploration of special Ser-O- There is still a lack of relevant tools for Hep modification, and there is still a lack of effective means for anti-adhesion therapy using Ser-O-Hep modification

Method used

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  • Polyclonal antibody for efficiently recognizing proteinserine heptanose glycosylation, and reparation method and application thereof
  • Polyclonal antibody for efficiently recognizing proteinserine heptanose glycosylation, and reparation method and application thereof
  • Polyclonal antibody for efficiently recognizing proteinserine heptanose glycosylation, and reparation method and application thereof

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Experimental program
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Embodiment 1

[0060] The synthesis of embodiment 1 key intermediate 6

[0061] Using mannose 2 as a raw material, under the action of acetic anhydride and iodine, the fully acetyl-protected mannose 3 was obtained. Under the catalysis of boron trifluoride ether, the end of mannose 3 was coupled with p-cresylthiol to obtain compound 4 , followed by deprotection of the hydroxyacetyl group of compound 4 under the action of sodium methoxide, the 6-position primary alcoholic hydroxyl was selectively protected with TBDMS to obtain compound 5, and compound 5 was protected with benzyl to obtain compound 6. In order to selectively expose the 6-position primary alcohol hydroxyl group, the primary alcohol hydroxyl group was selectively removed under the conditions of acetic acid and TBAF, and the compound 7 was obtained by TBDMS protection. Compound 7 was oxidized to Aldehydes can be used to obtain compound 8, and compound 8 undergoes a Wittig reaction to convert the aldehyde group into aglycone 9, and...

Embodiment 2

[0062] The synthesis of embodiment 2 heptose amino acids 16a and 13b

[0063] The 6-position D-type heptose amino acid was synthesized based on the glycosylation reaction of trichloroimidate, and the 6-position L-type heptose amino acid was synthesized based on the glycosylation reaction of glucosinolate.

[0064] Specifically, the synthesis of the heptose amino acid intermediate 16a is as follows: After compound 10a removes the terminal p-cresyl thiol under the action of NBS, the three hydroxyl groups of 1, 6, and 7 are acetylated under the condition of acetic anhydride pyridine Compound 12a was obtained by protection at the position, and compound 12a was selectively removed under the catalysis of hydrazine acetate to protect the acetyl group of terminal hydroxyl to obtain compound 13a, and 13a was converted into activated trichloroimidate under the action of trichloroacetonitrile and DBU, Using the activated trichloroimidate as a sugar donor, under the action of triflate, th...

Embodiment 3

[0066] Synthesis of Example 3 Serine Heptosylated Polypeptides 1a and 1b

[0067] For the synthetic route of hapten 1a, 1b, see Figure 7 .

[0068] First, 2-chlorotrityl resin is used as a solid phase carrier, and Fmoc-Leu-OH, Fmoc-Gly-OH, and 16aFmoc-Gly-OH are connected to the carrier in turn, and the terminal amino group is acetylated under the action of acetic anhydride pyridine. Glycopeptides and protective groups were cut off from the resin with a gentle cutting reagent (acetic acid / TFE / DCM=1:1:8) during cutting to obtain fully protected glycopeptide 18a; with 2-chlorotrityl Base resin as a solid phase carrier, Fmoc-Leu-OH, Fmoc-Gly-OH, 13b, Fmoc-Gly-OH are connected to the carrier in sequence, the terminal amino group is capped with acetyl group under the action of acetic anhydride pyridine, when cutting The glycopeptide together with the protecting group was cleaved from the resin with a mild cleavage reagent (acetic acid / TFE / DCM=1:1:8) to obtain fully protected gly...

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Abstract

The invention relates to a polyclonal antibody for efficiently recognizing proteinserine heptanose glycosylation, and a reparation method and application thereof. The antibody is used for detecting different structures of natural protein modified by proteinserine heptanose glycosylation and screening and discovering novel antibiotics. The preparation process of the antibody comprises the steps offirstly building a C7 sugar framework by Wittig reaction; then, obtaining six-bit heptose of two absolute structures through Sharpless asymmetrical oxidization reaction; under the help of glucosinolates and trichloroacetimidate mediated glycosylation reaction, obtaining proteinserine heptanose with the same glycosylation modification structure in the organism; then, obtaining the corresponding glycopeptide with the sequence being Ac-GS(Hep)GL-OH by using a polypeptide solid phase synthesis method; coupling the obtained semi-antigen onto the carrier protein BSA to obtain antigen; immunizing animals by the antigen; collecting immunized New Zealand rabbit blood to prepare antiserum; obtaining IgG through affinity purification. The prepared antibody has high specificity and high valence.

Description

technical field [0001] The invention relates to the technical field of medicinal chemistry, in particular to a polyclonal antibody for efficiently recognizing protein serine heptosylation, a preparation method and application thereof. Background technique [0002] The ability to adhere to host cells or tissues is a prerequisite for most pathogenic bacteria to achieve effective infection. Pathogenic bacteria usually use a class of proteins called adhesins to specifically recognize receptors on the surface of host cells to efficiently adhere to the cell surface. Diffuse-adhering E.coli (DAEC) and enterotoxigenic E.coli (ETEC) are important pathogenic bacteria that cause diarrhea in humans and young animals. They encode AIDA-I and TibA respectively. an adhesin. Previous studies have shown that these two adhesins are directly related to bacterial pathogenicity, and it has been reported that both AIDA-I and TibA are glycoproteins, but their glycosylation mechanisms and specific...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K9/00C07K1/04C07K1/06C07K16/00A61K39/395A61P31/00
CPCA61K2039/505A61P31/00C07K9/001C07K16/00
Inventor 胡宏岗李翔吴也陈思雷青云张卫东
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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