阅读说明：本技术 与禽法氏囊病毒vp2蛋白特异性结合的多肽序列及其应用 (Polypeptide sequence specifically combined with avian bursal disease virus VP2 protein and application thereof ) 是由 刘运超 张改平 陈玉梅 王方雨 尚延丽 魏蔷 冯华 刘东民 邢广旭 邓瑞广 于 2019-10-08 设计创作，主要内容包括：本发明涉及与禽法氏囊病毒VP2蛋白特异性结合的多肽序列及其应用,属于分子生物学领域。本发明借助于计算机辅助设计,在禽法氏囊病毒VP2蛋白晶体结构的基础上,通过分子对接虚拟筛选技术,搜寻虚拟多肽数据库中与靶标蛋白结合模式与亲和力最佳的多肽配体,其多肽序列为SERWDN,即IBDVL9-15。本发明利用表达纯化的IBDV VP2蛋白分别进行ELISA和局域表面等离子体共振试验,结果表明,IBDVL9-15多肽能够与IBDV VP2蛋白有良好的结合能力,借助本发明设计而成的多肽,可用于对IBDV VP2抗原进行快速检测和分离纯化。(The invention relates to a polypeptide sequence specifically combined with avian bursal disease virus VP2 protein and application thereof, belonging to the field of molecular biology. The invention searches a polypeptide ligand with the best binding mode and affinity with a target protein in a virtual polypeptide database by a molecular docking virtual screening technology on the basis of the crystal structure of the avian bursal disease virus VP2 protein by means of computer-aided design, wherein the polypeptide sequence is SERWDN, namely IBDVL 9-15. The invention respectively carries out ELISA and local surface plasma resonance tests by utilizing the expressed and purified IBDV VP2 protein, and the result shows that the IBDV L9-15 polypeptide can have good binding capacity with IBDV VP2 protein, and the polypeptide designed by the invention can be used for carrying out rapid detection, separation and purification on IBDV VP2 antigen.)

1. The polypeptide sequence specifically combined with the avian bursal disease virus VP2 protein is characterized in that the polypeptide sequence is SEQ ID NO. 1: and (4) SERWDN.

2. The polypeptide sequence of claim 1, comprising the core polypeptide sequence, any corresponding modifications or alterations to the polypeptide sequence; modifying materials include, but are not limited to, nanomaterials, fluorescent materials, enzymes, biotin, and specific proteins.

3. The use of the polypeptide sequence according to claim 1 or 2 for the rapid detection, purification and vaccination of the avian bursal disease virus VP2 protein.

4. The polypeptide sequence of claim 1, wherein the polypeptide sequence is used for identification of avian bursal disease virus VP2 protein, including but not limited to enzyme-linked immunosorbent assay (ELISA) detection.

5. Use of a polypeptide sequence according to claim 1 or 2 for the quantitative and qualitative detection of the avian bursal disease virus VP2 protein antigen.

Technical Field

The invention relates to a polypeptide sequence specifically combined with avian bursal disease virus VP2 protein and application thereof, belonging to the field of molecular biology.

Background

Based on the three-dimensional structure and charge distribution characteristics of protein, the screening of affinity peptide by adopting a molecular virtual docking technology becomes an important technical means for polypeptide ligand and pharmacological design. The method mainly comprises the steps of realizing one-by-one butt joint of small molecular polypeptides on active sites of target proteins by means of quick operation of computer software, wherein the small molecular polypeptides are from a virtual peptide library prepared in advance, finding the optimal binding conformation of the small molecular polypeptides and the target proteins by continuously optimizing the aspects of space conformation, amino acid residue side chains and the like of the target proteins, calculating the binding mode and affinity of the small molecular polypeptides and the biomacromolecule proteins, scoring results, selecting the optimal ligand according to the scoring results, and then carrying out in-vitro experimental verification and screening on the optimal ligand.

Infectious Bursal Disease (IBD) is a high-incidence and high-contact viral Infectious Disease which is caused by Infectious Bursal Disease Virus (IBDV) and seriously harms immune organs of chickens, so that the chickens fail to immunize, and B lymphocytes are seriously damaged. The disease is widely popularized in China in the eighties of the last century, and brings huge economic loss to poultry industry in China. The method is mainly characterized in that IBDV attacks the bursa of Fabricius of central immune organs of chickens, and the IBDV is proliferated in B lymphocytes of the bursa of Fabricius in a large amount to cause severe killing on the B lymphocytes of the bursa of Fabricius, so that severe immunosuppression is caused, the immunity of organisms of chicken flocks is reduced, the immunity of vaccines is disabled, and great challenge is brought to epidemic disease prevention and control of poultry breeding industry. The VP2 protein is located on the outer surface of the virion, is the main structural protein of IBDV, and has a molecular weight of about 37-40 kDa, accounting for 51% of the total structural protein. VP2 is not only the main host immunoprotective antigen of IBDV, but also the main region of antigenic variation of segment A of the IBDV genome. Therefore, the VP2 protein has an important role in the variation of the antigenicity and virulence of IBDV, and is closely related to the cell tropism, pathogenicity, apoptosis and the like of viruses. The VP2 protein is the only capsid protein, contains neutralizing antigen epitope, and can stimulate the body to produce neutralizing antibody. Therefore, the subunit vaccine developed at present mainly takes the VP2 protein as a main antigen gene.

Disclosure of Invention

The invention searches a polypeptide ligand with the best binding mode and affinity with a target protein in a virtual polypeptide database by a molecular docking virtual screening technology on the basis of the crystal structure of the avian bursal disease virus VP2 protein by means of computer-aided design, wherein the polypeptide sequence is SEQ ID NO. 1: SERWDN, IBDVL 9-15. The polypeptide IBDV L9-15 is synthesized by a solid phase synthesis method, ELISA and Local Surface Plasmon Resonance (LSPR) tests are respectively carried out by utilizing the expressed and purified IBDV VP2 protein, and the result shows that the IBDV L9-15 polypeptide can have good binding capacity with IBDV VP2 protein, so that the polypeptide designed by the invention can be used for quickly detecting, separating and purifying IBDV VP2 antigen.

In order to achieve the purpose, the invention adopts the technical scheme that:

a polypeptide sequence specifically binding to the avian bursal disease virus VP2 protein is SERWDN.

The polypeptide sequence comprises the polypeptide sequence as a core, and any corresponding adjustment or modification is carried out on the polypeptide sequence; modifying materials include, but are not limited to, nanomaterials, fluorescent materials, enzymes, biotin, and specific proteins.

The polypeptide sequence is applied to the rapid detection, purification and vaccine of the avian bursal disease virus VP2 protein.

The polypeptide sequence is used for identifying the avian bursal disease virus VP2 protein, including but not limited to enzyme-linked immunosorbent assay (ELISA) detection.

The polypeptide sequence is applied to quantitative and qualitative detection of the avian bursal disease virus VP2 protein antigen.

The invention has the beneficial effects that:

1. the invention obtains a polypeptide sequence specifically combined with the avian bursal disease virus VP2 protein based on the crystal structure (PDB ID:2DF7) of the avian bursal disease virus VP2 protein by a molecular docking virtual screening technology, wherein the polypeptide sequence is SERWDN, namely IBDVL 9-15. Synthesizing polypeptide IBDV L9-15 by solid-phase synthesis method, performing affinity identification with avian bursal disease virus VP2 protein, and determining equilibrium dissociation constant K of interaction between IBDV L9-15 sequence and artificially expressed IBDV VP2 protein_DIs 4.37 multiplied by 10^-6M, i.e.4.37 uM, indicates better affinity.

2. Due to the limitation of IBDV VP2 protein purification technology, an antibody aiming at IBDV VP2 protein is difficult to obtain, and the IBDV L9-15 sequence designed by the invention well solves the problem and realizes rapid artificial synthesis and low-cost detection.

3. The designed IBDV L9-15 sequence can be combined with artificially expressed IBDV VP2 protein and natural IBDV, has no cross reaction with other virus proteins, and has good specificity.

4. Compared with the traditional phage screening polypeptide library, the invention has the characteristics of simplicity, rapidness and low cost; by the molecular docking virtual screening technology, better theoretical guidance can be provided for realizing the IBDV VP2 protein structure function analysis.

5. Compared with the process of obtaining the protein-specific antibody by immunizing the artificially expressed and purified protein, the method has the advantages of simple operation, time and labor saving, low cost and the like; by marking the screened IBDVL9-15 sequence, the qualitative and quantitative rapid detection of the IBDVVP2 protein can be realized.

Drawings

FIG. 1 shows the docking results of the IBDVL9-15 sequence with Cap protein.

FIG. 2 shows the result of the LSPR affinity identification of the sequence IBDV L9-15 and the artificially expressed IBDV VP2 protein.

Wherein the ordinate represents the signal value detected by the sensor; the abscissa represents the time of interaction of the sample in the sensor.

FIG. 3 shows the results of ELISA identification of the sequence of IBDV L9-15 and the artificially expressed IBDV VP2 protein.

FIG. 4 shows the result of ELISA identification of the sequence of IBDV L9-15 and artificially inoculated IBDV.

Detailed Description

The following examples further illustrate the embodiments of the present invention in detail.