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Mers-cov membrane protein receptor binding domain dimer and its encoding gene and application

A dimer and protein technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problem of uncertain source and host, and achieve the effect of good immunogenicity and extensive theoretical guidance value

Active Publication Date: 2022-06-07
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Gene sequence comparison shows that MERS-CoV belongs to betacoronavirus, but its source and host have not yet been determined

Method used

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  • Mers-cov membrane protein receptor binding domain dimer and its encoding gene and application
  • Mers-cov membrane protein receptor binding domain dimer and its encoding gene and application
  • Mers-cov membrane protein receptor binding domain dimer and its encoding gene and application

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0054] Example 1. Preparation of MERS-CoV RBD monomer and MERS-CoV RBD dimer

[0055] 1. Construction of recombinant plasmids

[0056] Replace pFastBac with the double-stranded DNA molecule shown in SEQ ID NO: 6 of the Sequence Listing TM A small fragment between the BglII and SalI restriction sites of the dual vector was obtained to obtain the recombinant plasmid A. In Sequence 6 of the sequence listing, the 1-114 nucleotides encode the gp67 signal peptide, the 124-792 nucleotides encode the RBD monomer, and the 793-810 nucleotides encode His 6 Label. The DNA molecule shown in SEQ ID NO: 6 of the Sequence Listing encodes the protein shown in SEQ ID NO: 5 of the Sequence Listing. The gp67 signal peptide is cleaved during the process of insect cell expression and secretion to form a mature protein consisting of amino acid residues 39-270 in SEQ ID NO: 5 of the sequence listing.

[0057]Insert the double-stranded DNA molecule shown in Sequence 8 of the Sequence Listing into...

Embodiment 2

[0090] Example 2. Antigenicity analysis (ELISA test)

[0091] Test antibody: MERS-4 or MERS-27 or 10E8. MERS-4 is the monoclonal antibody MERS-4, and the preparation method is described in the patent "Monoclonal Antibody MERS-4 and Its Encoding Gene and Application" (Application No. 201310566227.8; Authorized Announcement No. CN104628849B). MERS-27 is the monoclonal antibody MERS-27, and the preparation method is described in the patent "Monoclonal Antibody MERS-27 and Its Encoding Gene and Application" (Application No. 201310565893.X; Authorized Announcement No. CN104628848B). Both MERS-4 and MERS-27 are neutralizing antibodies against MERS-CoV RBD. 10E8 is a monoclonal antibody against HIV-1 membrane protein (used as a negative reference).

[0092] Antigen solution: MERS-CoV RBD monomer solution or MERS-CoV RBD dimer solution prepared in Example 1. Blocking solution: PBS buffer containing 1% (v / v) fetal bovine serum. Test antibody solution: Take the test antibody and dil...

Embodiment 3

[0102] Example 3, structural analysis

[0103] The MERS-CoV RBD monomer solution and the MERS-CoV RBD dimer solution prepared in Example 1 were taken respectively, concentrated to 10 mg / ml with a 10 kD ultrafiltration tube, and the crystals were screened by the sitting drop method.

[0104] Spindle-shaped single crystals were grown under the condition of 25% PEG1500.

[0105] Diffraction data collection was performed using a synchrotron X-ray light source.

[0106] Use HKL-2000, DPP4, Coot, phoenix and other software to process the data, use the molecular replacement method to solve the phase, and finally analyze the structure.

[0107] see the results Figure 4 . Figure 4 A is the structure of MERS-CoV RBD monomer, Figure 4 The B is the structure of the MERS-CoV RBDdimer. The MERS-CoV RBD monomer contains 8 cysteines (Cys) and forms 4 pairs of intramolecular disulfide bonds. One monomer of MERS-CoV RBD dimer contains 9 Cys, of which 8 Cys will form 4 pairs of intramol...

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Abstract

The invention discloses a MERS-CoV membrane protein receptor binding domain dimer and its encoding gene and application. The present invention first protects the dimer of MERS-CoV RBD; MERS-CoV RBD is the protein shown in sequence 3 of the sequence listing or the protein shown in sequence 7 of the sequence listing. The present invention determines the structure and immunogenicity of the Middle East respiratory syndrome coronavirus membrane protein receptor binding domain dimer (MERS‑CoV RBD dimer). Further, the present invention finds that MERS-CoV RBD dimer has better immunogenicity than MERS-CoV RBD monomer, and can induce stronger neutralizing antibody responses in animals. The discovery has important theoretical guiding value and broad application prospects for the development of vaccines for the treatment and prevention of MERS.

Description

technical field [0001] The present invention relates to a MERS-CoV membrane protein receptor binding domain dimer and its encoding gene and application. Background technique [0002] Middle East respiratory syndrome coronavirus (MERS-CoV) was first discovered to infect humans in the Middle East in 2012, and this virus infection has subsequently appeared in several European countries and regions. [0003] More than half of MERS-CoV-infected patients develop severe respiratory disease, with clinical symptoms very similar to the SARS-CoV-caused atypical pneumonia in 2003. Since this virus can be transmitted from person to person, it has attracted great attention around the world. [0004] Gene sequence alignment shows that MERS-CoV belongs to beta coronavirus, but its source and host have not yet been determined. Similar to other coronaviruses, MERS-CoV uses the membrane protein S glycoprotein on its surface to enter susceptible cells. SUMMARY OF THE INVENTION [0005] The...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/165C12N15/50C12N15/866A61K39/215A61P31/14A61P11/00
CPCC07K14/005C12N15/86A61K39/12A61P31/14A61P11/00C12N2770/20022C12N2770/20034C12N2710/14043Y02A50/30
Inventor 张林琦周盼盼史宣玲
Owner TSINGHUA UNIV
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