Method for analyzing epitope of monoclonal antibody by using yeast surface display system and application of method in vaccine development

An antigenic epitope, yeast technology, applied in the preparation methods of peptides, chemical instruments and methods, and the introduction of foreign genetic material using vectors, can solve the problems of easy airborne phage transmission, small phage individuals, cross-contamination, etc. The effect of accuracy and screening throughput, ease of operation, and few interference factors

Inactive Publication Date: 2012-01-18
TSINGHUA UNIV
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  • Claims
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Problems solved by technology

However, the phage display peptide library technology also has great limitations: (1) Most of the short peptide libraries that can be generated are linear epitopes, which cannot well mimic the conformational epitopes that account for more than 80% of antigenic epitopes; (2) The foreign molecules that can be accommodated by the capsid protein of phage are small and simple in structure, and lack of modification of foreign proteins. Many cytotoxic molecules and eukaryotic proteins in this system are difficult to express a

Method used

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  • Method for analyzing epitope of monoclonal antibody by using yeast surface display system and application of method in vaccine development
  • Method for analyzing epitope of monoclonal antibody by using yeast surface display system and application of method in vaccine development
  • Method for analyzing epitope of monoclonal antibody by using yeast surface display system and application of method in vaccine development

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Embodiment 1

[0068] Embodiment 1, the construction of pCTCON2-T plasmid (recombinant yeast display vector)

[0069] In order to facilitate library construction, the pCTCON2 plasmid needs to be transformed into a T vector, and the steps are as follows:

[0070] 1. Synthesize the DNA (double strand) shown in sequence 5 of the sequence listing.

[0071] 5'-AGTC GCTAGC GGATCC GGGT-3' (SEQ ID NO: 5).

[0072] The Nhe I recognition sequence is underlined on the left, the BamH I recognition sequence is marked on the right underline, and the two Xcm I recognition sequences are marked in bold.

[0073] 2. Digest the DNA synthesized in step 1 with restriction endonucleases Nhe I and BamH I, and recover the digested product.

[0074] 3. Digest the pCTCON2 plasmid with restriction endonucleases Nhe I and BamH I, and recover the vector backbone.

[0075] 4. Ligate the digested product of step 2 with the vector backbone of step 3 to obtain the pCTCON2-T plasmid (the backbone vector is a pCTCON2...

Embodiment 2

[0076] Embodiment 2, the preparation of AVFluIgG03 monoclonal antibody

[0077] 1. Synthesize the light chain variable region gene shown in sequence 6 in the sequence listing, and clone it into the Pac-L-Fc vector (PROGEN PR3003, Germany) between the Xba I and Sac I restriction sites to obtain recombinant plasmid A.

[0078] 2. Synthesize the heavy chain variable region gene shown in sequence 7 in the sequence listing, and clone it into the space between the Xho I and SpeI restriction sites of the recombinant plasmid A to obtain the recombinant plasmid B.

[0079]3. Using the BaculoGold co-transfection kit from Pharmogen, USA, the recombinant plasmid B was introduced into Sf9 cells, and the culture supernatant was collected to obtain AVFluIgG03 monoclonal antibody.

Embodiment 3

[0080] Embodiment 3, application method of the present invention analyzes the epitope of gp160 protein

[0081] 1. Construction of DNA library of gp160 protein (random cutting fragment library)

[0082] To construct the DNA library of the gp160 protein, first the coding gene of the gp160 protein is randomly cut by DNase I to obtain small fragments of about 50-100bp, and then a random fragment DNA library with a specific length distribution is obtained by random recombination (Lin, Z., S. Li and Y. Chen, Identification of Viral Peptide Fragments for Vaccine Development. Viral Applications of Green Fluorescent Protein: Methods and Protocols, 2009: p.261-274.; Chen, Y., et al., Random dissection to select for protein split sites and its application in protein fragment complementation. PROTEIN SCIENCE, 2009.18(2): p.399-409.). The above method is very similar to the program of DNA shuffling (Lorimer, I.and I.Pastan, Random recombination of antibody single-chain fv sequences after...

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Abstract

The invention discloses a method for analyzing epitope of monoclonal antibody by using a yeast surface display system and application of the method in vaccine development. The method comprises the following steps of: (1) constructing a DNA library of target protein; (2) displaying the DNA library on the surface of yeast to obtain a yeast display library; (3) mixing the monoclonal antibody of the target protein and a yeast display library A, and screening yeast combined with the monoclonal antibody; and (4) extracting plasmid of the screened yeast, sequencing, and analyzing the epitope of the target protein. Compared with the traditional method, the method has the advantages that: (1) ways of modifying the protein by yeast cells on are more, and high molecular weight and complicated protein can be displayed; (2) the yeast can be screened one by one by fluorescence-activated cell sorting (FACS), and the accuracy and screening flux are improved; (3) the yeast can independently complete self growing and reproducing process, the operation is simple and convenient and interference factors are a few; and (4) the yeast is an immunologic adjuvant, and can check whether the epitope can induce the generation of antibody with similar activity again.

Description

technical field [0001] The invention relates to a method for analyzing the antigenic epitope of monoclonal antibody by using yeast surface display system and its application in vaccine development. Background technique [0002] The host's antibody response plays an important role in the process of resisting pathogenic infection, and the polyclonal antibody response in the body is composed of monoclonal antibodies against different antigens or different epitopes of the same antigen. A deep understanding of this complex process can provide key information for the study of disease pathogenesis, and can also provide an important theoretical basis for the development of efficient vaccines and disease treatment. [0003] Among the many research methods of antigenic epitopes, the phage display peptide library technology emerged in 1990 (Scott, J.K. and G.P.Smith, Searching for peptide ligands with an epitope library. Science, 1990.249(4967): p.386-90. ), that is, using a randomly ...

Claims

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Application Information

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IPC IPC(8): C07K1/00C12N15/11C12N15/81C12N1/19C40B50/06
Inventor 张林琦史宣玲左腾汪桦姚辰
Owner TSINGHUA UNIV
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