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A kind of human cytomegalovirus ul49 gene antigen peptide, its antibody and application

A technology of human cytomegalovirus and antigenic peptides, which is applied in the field of immunobiology, can solve problems such as difficulty in obtaining titers, uncertain functions, and complicated immunogen acquisition process, and achieves easy chemical synthesis, high product purity, and strong specificity Effect

Inactive Publication Date: 2011-11-30
JINAN UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the biological function of HCMV UL49 has not yet been determined, and there is no direct evidence to indicate whether the UL49 gene expresses protein. One of the main reasons is that it is difficult to obtain antibodies with high titer and strong specificity
[0004] The traditional preparation of antibodies against HCMV UL49 gene is to use the amino acid sequence encoded by HCMV UL49 to design and prepare prokaryotic expression proteins of full length and truncated fragments, and then use them as immunogens to prepare antibodies. The immunogen acquisition process of this method is complicated and the obtained The purity of the immunogen cannot be guaranteed, and the titer and specificity of the final prepared antibody cannot meet the requirements of the research

Method used

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  • A kind of human cytomegalovirus ul49 gene antigen peptide, its antibody and application
  • A kind of human cytomegalovirus ul49 gene antigen peptide, its antibody and application
  • A kind of human cytomegalovirus ul49 gene antigen peptide, its antibody and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Human cytomegalovirus UL49 gene antigen peptide

[0027] In this example, first input the amino acid sequence of pUL49 protein through the professional online software Genebank, select the secondary structure, transmembrane region, hydrophilicity, accessibility, polarity, flexibility and antigenicity parameter modes, and predict the pUL49 protein under each mode respectively Antigen sequence (as shown in Table 1), after summarizing the above data, and Wu Yuzhang et al. ~22279.) The established B cell epitope prediction method obtained the sequence of high score index (as shown in Table 2) for comprehensive judgment of overlap, and finally designed the human cytomegalovirus UL49 gene antigen peptide, its amino acid sequence As shown in SEQ ID No.1, its nucleotide sequence is shown in SEQ ID No.2.

[0028] Table 1 Prediction of cell epitopes of pUL49 protein by different methods

[0029]

[0030] Table 2 predicts the average antigenicity index of pUL49 prot...

Embodiment 2

[0032] Example 2 Preparation of Antibody

[0033] In this example, the synthetic parameters are set according to the human cytomegalovirus UL49 gene antigenic peptide sequence (its amino acid sequence is shown in SEQ ID No.1) designed in Example 1, and the antigenic peptide resin is synthesized by the Fmoc solid-phase method, and then trifluoro Acetic acid (TFA) cuts the antigenic peptide from the resin, and after preliminary purification of the product peptide, it is then purified by high performance liquid chromatography (HPLC), and finally the purified peptide is freeze-dried.

[0034] Select pathogen-free first-class New Zealand white rabbits, use the above-mentioned purified freeze-dried human cytomegalovirus UL49 gene antigen peptide for antigen, take blood, separate and purify, and prepare antibodies to human cytomegalovirus UL49 gene antigen peptide. Store the antibody at -20°C to -70°C.

[0035] The preparation of the antibody in this example, the various steps invol...

Embodiment 3

[0036] Embodiment 3 ELISA method detects the titer of antibody

[0037] This example uses the ELISA method to detect the titer of the antibody prepared in Example 2. The ELISA detection method used adopts the routine operation of those skilled in the art: the antigen (2 μg / μl) is diluted 2000 times with the coating buffer, coated and blocked, Add the antiserum to coated wells after 10-fold serial dilution, set up a control, incubate, incubate with the working concentration of goat anti-rabbit IgG-HRP, develop color, and measure the OD of each well on an enzyme-linked detector after the reaction is terminated 450 Value, with the corresponding negative control, the ratio of the measured value > 2.1 is a positive reaction.

[0038] The potency curve results are as follows figure 1 As shown, it can be seen from the figure that the titer of the antibody prepared in Example 2 can reach more than 1:8000.

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Abstract

The present invention discloses a human cytomegalovirus UL49 gene antigenic peptide, its antibody and application. The antigenic peptide has the amino acid sequence shown in SEQ ID No.1, and its nucleotide sequence is shown in SEQ ID No.2. The antigenic peptide of the present invention is a 12-short peptide, which is easy to chemically synthesize and has high product purity. The antibody prepared by using the antigenic peptide of the present invention has a titer of more than 1:8000, has high specificity to UL49 prokaryotic expression protein, eukaryotic expression protein and HCMV particles, and can be used to prepare in vitro diagnosis or treatment of human cytomegalovirus medicine. The antibody prepared by the antigen peptide of the present invention is the basic element of HCMV UL49 biological function research, and the HCMV infection can be detected qualitatively or quantitatively by using the antibody.

Description

technical field [0001] The invention relates to the field of immunobiology, in particular to the immunology research of human cytomegalovirus UL49 gene, in particular to a human cytomegalovirus UL49 gene antigen peptide, an antibody and application thereof. Background technique [0002] Human cytomegalovirus (HCMV) is a pathogen that spreads widely and has a high infection rate among different ethnic groups all over the world. Current studies have found that the main hazards caused by HCMV infection are transplant infection and intrauterine infection, resulting in fetal miscarriage, bone marrow dysplasia, deformity, mental retardation, etc. [0003] Virus culture experiments in vitro found that the deletion of the UL49 gene is lethal to HCMV, indicating that the gene may express a functional protein. Therefore, the UL49 gene is expected to become a breakthrough in the research and treatment of HCMV. However, the biological function of HCMV UL49 is still uncertain, and there...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C12N15/38C12N15/63C12N1/00G01N33/569C07K16/08A61K39/42A61P31/20
Inventor 周天鸿朱峰李月琴曾志峰李弘剑
Owner JINAN UNIVERSITY
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