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Antigen polypeptide recognized by lppz antibody and use thereof

An antigen polypeptide and antibody recognition technology, applied in the field of biomedicine, can solve the problems of reduced specificity, increased false positives, and high production costs, and achieves the effects of low cost, simple preparation and high detection efficiency.

Active Publication Date: 2019-11-29
BEIJING CHEST HOSPITAL CAPITAL MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The antigen used by IGRA is the whole protein encoded by the RD (region of difference) gene of Mycobacterium tuberculosis, which is expensive to produce and is not recommended by the World Health Organization as a screening method for low- and middle-income countries.
And some studies have also shown that in countries with high prevalence of tuberculosis, the diagnosis of IGRA is interfered by environmental factors, the specificity is reduced, and the false positives are increased.
[0005] Therefore, the current research on tuberculosis serum immunological diagnosis and detection still needs to be strengthened

Method used

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  • Antigen polypeptide recognized by lppz antibody and use thereof
  • Antigen polypeptide recognized by lppz antibody and use thereof
  • Antigen polypeptide recognized by lppz antibody and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1 Preparation of Polypeptide

[0075] 1. Preparation of Peptide Library

[0076] The amino acid sequence of LppZ (Rv3006) was obtained from the protein public resource database uni-prot, and its sequence from N-terminal to C-terminal is as follows:

[0077] MWTTRLVRSGLAALCAAVLVSSGCARFNDAQSQPFTTEPELRPQPSSTPPPPPPLPPVPFPKECPAPGVMQGCLESTSGLIMGIDSKTALVAERITGAVEEISISAEPKVKTVIPVDPAGDGGLMDIVLSPTYSQDRLMYAYISTPTDNRVVRVADGDIPKDILTGIPKGAAGNTGALIFTSPTTLVVMTGDAGDPALAADPQSLAGKVLRIEQPTTIGQTPPTTALSGIGSGGGLCIDPVDGSLYVADRTPTADRLQRITKNSEVSTVWTWPDKPGVAGCAAMDGTVLVNLINTKLTVAVRLAPSTGAVTGEPDVVRKDTHAHAWALRMSPDGNVWGATVNKTAGDAEKLDDVVFPLFPQGGGFPRNNDDKT(SEQ ID NO:4)。

[0078] The full-length sequence of the LppZ protein was split into a group of 15 amino acid-length polypeptides, and 12 amino acid residues were sequentially overlapped between adjacent polypeptides to form the LppZ peptide library, with a total of 121 peptides.

[0079] 2. In situ synthesis of LppZ protein peptide library to...

Embodiment 2

[0081] Example 2 Screening for polypeptides related to tuberculosis (screening of antigenic epitopes)

[0082] 1. Hydration and Western Blotting of Peptide Chips

[0083] The polypeptide chip prepared in Example 1 (herein referred to as "polypeptide chip 1") was immersed in 40 ml of 100% ethanol and shaken on a shaker for 5 minutes. Then, the polypeptide chip was immersed in 40 ml of 75% ethanol and shaken on a shaker for 5 minutes. Next, immerse the peptide chip in 40ml of 50% ethanol and shake it on a shaker for 5 minutes. Next, add 150ml PBS and soak for 30 minutes. Finally, immerse the peptide chip in 40ml of 5% skim milk powder / PBS-T solution, and incubate at room temperature for 3 hours to block.

[0084] Dilute the serum samples involved in the following primary screening and identification experiments with 5% skimmed milk powder / PBS-T solution 1:1000 to prepare an immune reaction solution, put the peptide chip into the hybridization bag, and add the reaction solutio...

Embodiment 3

[0095] Example 3 ELISA test

[0096] The three polypeptides (shown in Table 1) screened in Example 2 were used as antigens to coat 96-well plates, and the ELISA method was used to detect LppZ antibodies in the following samples: 82 tuberculosis patient sera and 38 healthy control sera.

[0097] The specific detection method is as follows:

[0098] Firstly, the antigenic polypeptide was synthesized according to the sequence of amino acid residues in the sequence listing using the polypeptide synthesizer MultiPep RS.

[0099] Next, use pH 9.6 carbonate buffer (NaCO 3 0.159g, NaHCO 3 0.293g, add water to 100ml, pH value is 9.6) dilute the peptide to 1μg / ml, add 100μl / well to the wells of a 96-well microplate, and seal the plate overnight at 4°C.

[0100] Next, discard the liquid in the well, 0.1% TBS-T washing buffer (the TBS-T solution containing 0.1% Tween-20 is prepared as NaCl 8.7g, Tris 1.21g, add deionized water to 1000ml, pH 7.5, Then add Tween-20 1ml) to wash the mi...

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Abstract

The invention discloses a LppZ antibody identified antigen polypeptide and application thereof. The antigen polypeptide has at least one of the following polypeptide active fragments: (1) SGCARFNDAQSQPFT which is a sequence as shown in SEQ ID NO:1; (2) EPDVVRKDTHAHAWA which is a sequence as shown in SEQ ID NO:2; (3) HAHAWALRMSPDGNV which is a sequence as shown in SEQ ID NO:3; (4) at least two connecting products selected from amino acid sequences as shown in SEQ ID NO:1-3; and (5) amino acid derived sequences formed by modifying, substituting, deleting or adding amino acid residues to the polypeptide active fragment. By utilizing the LppZ antibody identified antigen polypeptide, the LppZ antibody level of a sample (like serum samples), such as blood samples of patients with tuberculosis or suspected tuberculosis can be effectively detected.

Description

technical field [0001] The present invention relates to the field of biomedicine. Specifically, the present invention relates to antigenic polypeptides recognized by LppZ antibodies and uses thereof. More specifically, the present invention relates to the binding site between human serum antibody and LppZ protein, the antigenic polypeptide recognized by LppZ antibody, the use of the antigenic polypeptide in the preparation of a kit for detecting the level of LppZ antibody in serum, and the detection of LppZ antibody in a sample Level kit. Background technique [0002] Tuberculosis is one of the three major infectious diseases in the world. The epidemiological investigation report released by the World Health Organization (WTO) shows that the pathology of Mycobacterium tuberculosis infection in my country accounts for 12% of the total number of cases in the world, ranking second in the world after India . The high incidence and prevalence of tuberculosis have seriously affe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/35G01N33/569
CPCC07K14/35G01N33/5695G01N2333/35G01N2469/20
Inventor 岳文涛谭金晶顾勐张丽娜
Owner BEIJING CHEST HOSPITAL CAPITAL MEDICAL UNIV
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