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Anti-flavivirus envelope E protein monoclonal antibody and application thereof

A monoclonal antibody, flavivirus technology, applied in viral peptides, resistance to vector-borne diseases, microorganism-based methods, etc. The effect of broad-spectrum anti-flavivirus action

Active Publication Date: 2013-07-10
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned monoclonal antibodies are mainly directed against a specific virus, and antibodies specific to flaviviruses are rare. In addition, due to the cross-cutting of antigens between members of the flavivirus genus, especially dengue 1-4 viruses, these There are differences in the neutralizing activity and protective efficiency of genus-specific antibodies against flavivirus members, and some have no cross-protection activity, and may even have negative effects, leading to antibody-dependent infection enhancement and exacerbation of flavivirus
Therefore, most of these flavivirus-specific antibodies are not suitable or require further modification before they can be used in clinical treatment research

Method used

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  • Anti-flavivirus envelope E protein monoclonal antibody and application thereof
  • Anti-flavivirus envelope E protein monoclonal antibody and application thereof
  • Anti-flavivirus envelope E protein monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1. Preparation of dengue type 2 virus antigen

[0079] The 43 strains of dengue type 2 virus (GeneBank No. AF204178) preserved in this laboratory were used as a cell maintenance solution (containing 2% (volume percentage) calf serum, RPMI-1640 medium of 100 U / mL penicillin and streptomycin, pH 7 .0~7.6) After 5-fold or 10-fold dilution, 1-3 day-old Kunming or Balb / C suckling mice were inoculated into the brain, each inoculated with 0.02 mL. Those that died within 24 hours after inoculation were regarded as non-specific death. Then observe the sickness of the suckling mice every day, and the symptoms are encephalitis symptoms such as not feeding, lethargy, convulsions or numbness of the limbs, and bowed back. During the period of 5-9 days after virus inoculation, if there are infected suckling mice, they will be killed by pulling their necks when they are in a dying state, and the brains will be dissected under sterile conditions. Each brain weighs about 0.2g. A...

Embodiment 2

[0080] Example 2. Screening and preparation of monoclonal antibodies

[0081] 10% suckling mouse brain suspension was emulsified with an equal volume of Freund's complete adjuvant (Sigma company product) or Freund's incomplete adjuvant (Sigma company product) to obtain Freund's complete adjuvant antigen and Freund's incomplete adjuvant Antigens were used as antigens to immunize 9-week-old female healthy BALB / C mice. Through intraperitoneal, intramuscular and subcutaneous multi-point injection, immunization was boosted with the same dose of Freund's incomplete adjuvant antigen at intervals of 3-4 weeks. A total of 3 times of immunization, select mice with higher anti-dengue type 2 virus antibody titers in the serum, and inject the brain suspension of suckling mice infected with dengue type 2 virus through the intraperitoneal route, and take the spleen lymphocytes of the immunized mice 3 days later, Using the classic PEG method, the mouse spleen lymphocytes were fused with SP2 / ...

Embodiment 3

[0083] Example 3. Determination of D2-2A10G6 antibody subtype

[0084] The subtype and light chain of the D2-2A10G6 antibody were determined according to the steps in the operation manual of the IsoStripMouseMonoclonalAntibodyIsotypingKit (product of Roche). The results showed that the D2-2A10G6 antibody belonged to IgG1 ( figure 1 In A), the light chain is κ chain ( figure 1 Middle B).

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Abstract

The invention discloses an anti-flavivirus envelope E protein monoclonal antibody and application thereof. The anti-flavivirus envelope E protein monoclonal antibody of the invention is secreted by a mouse source hybrid tumour cell strain D2-2A10G6 with the preservation number of CGMCC No. 3292. The anti-flavivirus monoclonal antibody of the invention can be specifically combined with the function epitope of flavivirus envelope E protein. The amino acid sequence of the functional epitope of envelope E protein is SEQ ID NO: 17. The monoclonal antibody of the invention is subject to screening by indirect immunofluorescent method, and indirect enzyme-linked immunization is used for evaluating the specificity and affinity when being combined with antigen. By adopting the monoclonal antibody or immunoconjugate of the invention, flavivirus infection cell can be blocked and suckling mouse can be protected from virus attack, thus achieving the effect of inhibiting virus infection. The monoclonal antibody or immunoconjugate of the invention also can be used for flavivirus envelope E protein detection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention discloses a functional epitope of flavivirus envelope E protein, a monoclonal antibody specifically binding to the epitope and its use in the preparation of anti-flavivirus drugs . Background technique [0002] Flaviviridae (Flaviviridae) Flaviviruses (flaviviruses) include more than 70 kinds of viruses, 40 of which are related to human diseases, such as Dengue virus (DENV), Japanese Encephalitis Virus (JEV), forest encephalitis Virus (Tick-borneEncephalitisVirus, TBEV), Yellow Fever Virus (YellowFeverVirus, YFV) and West Nile Virus (WestNileVirus, WNV) and so on. These viruses often cause zoonotic diseases, and most of them are mainly transmitted through arthropod vectors, which often lead to outbreaks of diseases and are one of the major public health problems in the world. More than 6 billion people are currently at risk of infection with flaviviruses, includ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/18C12R1/91
CPCY02A50/30
Inventor 秦成峰祝庆余邓永强郭亚军戴建新秦鄂德姬广辉姜涛
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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