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Middle east and respiratory syndrome coronavirus antibody and preparation method thereof

A respiratory syndrome, coronavirus technology, applied in respiratory diseases, antibodies, antiviral immunoglobulins, etc., can solve problems such as deficiency

Active Publication Date: 2014-06-18
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Due to the current lack of anti-MERS-CoV virus neutralizing antibodies, and it takes a long time to prepare a large number of antibodies and apply them clinically, it cannot be quickly and effectively tested clinically

Method used

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  • Middle east and respiratory syndrome coronavirus antibody and preparation method thereof
  • Middle east and respiratory syndrome coronavirus antibody and preparation method thereof
  • Middle east and respiratory syndrome coronavirus antibody and preparation method thereof

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0029] Embodiment 1 plasmid construction

[0030]We ligated the DNA fragment encoding MERS RBD protein (amino acid E367-Y606, NCBI accession number JX869059) into the pFastBac1 vector with EcoRI and XhoI restriction sites, and added signal peptide gp67 before the protein coding fragment to facilitate protein secretion For expression, insert the coding sequence of 6 histidine tags (hexa-His-tag) and the translation stop codon after the coding fragment of the protein, so that a recombinant protein with a histidine tag at the C-terminal will be obtained, which will facilitate subsequent The protein was purified and named as pFastBac-MERS RBD. Insert the MERS RBD gene (amino acids E367-Y606, NCBI accession number JX869059) into the pCAGGS vector between EcoR Ⅰ and Xho Ⅰ restriction sites, and add the mouse Fc gene fragment (amino acid 239G- 469K, NCBI accession number AY311599), which is convenient for subsequent protein purification and cell staining, named pCAGGS-MERS RBD mFc. ...

Embodiment 2

[0031] Example 2 MERS RBD protein expression and purification

[0032] First, the recombinant plasmid pFastBac-MERS RBD was transformed into DH10Bac competent cells, cultured overnight at 37°C, positive clones were identified by blue-white screening and PCR, and the recombinant Bacmid (baculovirus plasmid), which already contained the MERSRBD gene sequence, was extracted . The recombinant Bacmid was transfected into sf9 cells, and the culture supernatant was collected 3 days after transfection to obtain the P1 generation baculovirus. A large number of high-titer viruses can be obtained by continuously amplifying three generations of viruses. Then Hi5 cells were infected, and the cell supernatant was collected by centrifugation for 48 hours, and the cell culture supernatant was combined with Ni-NTA Resin (GE) overnight at 4°C. Wash the resin with buffer A (20mM Tris, 150mM NaCl, pH8.0) to remove non-specifically bound proteins. Finally, the target protein was eluted from the...

Embodiment 3

[0033] Example 3 Preparation and Purification of Murine Monoclonal Antibody

[0034] MERS-RBD protein was used as an antigen to immunize 3 Balb / c mice. 100 g protein was dissolved in 50 μl sterile PBS solution, mixed evenly with 50 μl Freund's complete adjuvant, and injected subcutaneously at multiple points. A booster immunization was given after 2 weeks, and tail vein blood was taken 3 days later to detect the serum antibody titer. The splenocytes of the mouse with the highest titer were fused with myeloma cell SP2 / 0 at a ratio of 5:1, and the culture medium was screened by HAT Selection culture was used to obtain hybridoma cells by limiting dilution method. The purified MERS-RBD protein was used as the coating antigen, and detected by ELISA, a monoclonal cell line that efficiently secreted the MERS-RBD protein antibody was obtained. Follow hybridoma cells (1-5) x 10 6 The amount of / mouse was injected into the peritoneal cavity of the mouse to prepare ascites monoclonal a...

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Abstract

The invention discloses a middle east and respiratory syndrome coronavirus antibody and a preparation method thereof, and belongs to the technical field of cell-mediated immunity. RDB protein of an MERS-CoV virus S protein receptor binding domain is obtained by using in-vitro expression and purification of a baculovirus expression vector system, and Bab / c mice are immunized by using high-purity MERS RBD protein, an efficient antiviral neutralizing anti-body is screened by the methods such as an enzyme-linked immuno sorbent assay (ELISA), a false virus neutralization experiment and the like. The antibody and the crystal structure of the MERS RBD protein are analyzed, and a crucial amino acid of the antibody combined with viral proteins is determined from the molecular level. Thus, the antibody is further humanized, and the possibility is provided for clinical detection, prevention and treatment of MERS-CoV virus infection.

Description

technical field [0001] The invention relates to a Middle East respiratory syndrome coronavirus antibody and a preparation method thereof, belonging to the technical field of cellular immunity. Background technique [0002] Middle East respiratory syndrome coronavirus (Middle East Respiratory Syndrome Cononavirus, MERS-CoV) and SARS-CoV belong to the same family of Coronaviridae, betacoronavirus. It is a positive-sense, single-stranded RNA virus. Its surface spike protein, the S protein, plays a key role in the virus infecting humans and animals. Some scientists once speculated that the virus may have originated from bats. Sequence analysis found that the MERS-CoV virus has a very high sequence homology with bat and porcine coronaviruses, up to 60% with bat coronaviruses HKU4 and HKU5. At present, the source and natural host of the MERS-CoV virus have not been determined, and effective measures cannot be taken to prevent and stop the spread of the virus. [0003] Because ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/63A61K39/395A61P11/00
Inventor 高福严景华李燕逯光文齐建勋
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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