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Completely humanized anti-dengue virus monoclonal antibody and preparation and application methods thereof

A monoclonal antibody and dengue virus technology, applied in antiviral agents, antiviral immunoglobulins, botanical equipment and methods, etc., can solve problems such as fully human monoclonal antibodies that do not have dengue virus infection diseases

Active Publication Date: 2019-03-12
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no fully human monoclonal antibody for dengue virus infection

Method used

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  • Completely humanized anti-dengue virus monoclonal antibody and preparation and application methods thereof
  • Completely humanized anti-dengue virus monoclonal antibody and preparation and application methods thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1. Construction and expression of fully human monoclonal antibody AD847

[0043] Using a large-capacity fully human phage library (Qiao Yuanyuan, Wang Yan, Chen Xiaosui, etc. Construction and identification of a large-capacity phage antibody library [J]. Chinese Journal of Microbiology and Immunology, 2004, 24(3):194-197.) , and according to the literature "Hu Shi, Shen Yafeng, Li Tian, ​​etc. Screening, preparation and biological activity research of fully human monoclonal antibodies against short-chain neurotoxins from flat-chin sea snakes [J]. PLA Medical Journal, 2017, 42(7): 612-616.)", multiple rounds of screening of monoclonal antibodies against dengue virus ED3 protein were carried out, and finally a fully human anti-dengue virus ED3 protein antibody was obtained, named AD847.

[0044] After sequencing, analyze the sequence and synthesize the full-length heavy and light chains of the full-length human monoclonal antibody. The heavy and light chains are r...

Embodiment 2

[0047] Embodiment 2, Biacore analysis

[0048] The polyclonal anti-human Fc antibody (Jackson ImmunoResearch Company) was coated on the CM5M5 chip (GE Company), and after the detected antibody was captured, the affinity of each fusion protein was detected with Biacore T100 (GE Healthcare). The specific detection affinity values ​​are shown in Table 1 .

[0049] Table 1 Biacore analysis results

[0050]

Embodiment 3

[0051] Embodiment 3, in vitro neutralization test

[0052] The plaque reduction neutralization test was carried out by using the method of fixing the virus and diluting the antibody: the different concentrations of the antibody were mixed with the dengue type 4 virus suspension containing 50-100 PFU in equal amounts, and the BHK21 cells were cultured in a 6-well plate in a 37°C water bath for 1 hour. Incubate at 37°C for 1 h, discard the mixture, and wash the cells with PBS buffer. Add the nutrient agar cover, continue to culture for 5 fills, fix the staining, count the number of plaques, and calculate the neutralizing activity of the antibody.

[0053] Such as figure 1 As shown, the monoclonal antibody has dengue type 4 virus-specific neutralizing activity, and the virus neutralizing activity has a concentration-dependent relationship. As the antibody concentration increases, the virus infection rate decreases significantly.

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Abstract

The invention provides a completely humanized anti-dengue virus monoclonal antibody and preparation and application methods thereof. The completely humanized anti-dengue virus monoclonal antibody is composed of two identical heavy chains and two identical light chains, wherein the amino acid sequence of the heavy chains is shown as SEQ ID NO.1, the amino acid sequence of the light chains is shownas SEQ ID NO.2, the nucleotide sequence for coding the heavy chains is shown as SEQ ID NO.3, and the nucleotide sequence for coding the light chains is shown as SEQ ID NO.4. In vitro neutralization tests verify that the completely humanized anti-dengue virus monoclonal antibody can achieve effective protection to achieve preventing effects before virus infection as well as still achieve virus infection treating effects within a period after a patient is infected, and when combined with other anti-dengue virus drugs, can achieve a broad clinical application prospect.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, in particular to a fully human anti-dengue virus monoclonal antibody capable of specifically binding to the ED3 domain of the dengue virus envelope protein (E protein), a preparation method and an application. Background technique [0002] Dengue fever is caused by dengue virus infection. It is one of the most popular arbovirus infectious diseases. Its vectors are Aedes aegypti and Aedes albopictus. It is mainly prevalent in tropical and subtropical regions. Nearly half of the world's population has Risk of getting dengue fever. The latest research results show that about 100 million people in the world will have overt infection every year, and nearly 300 million people will have recessive infection (BhattS, Gething P W, Brady O J, et al. The global distribution and burden of dengue[J].Nature , 2013, 496(7446):504-507.). [0003] In 2009, WHO reclassified the clinical types of de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/85A61K39/42A61K45/06A61P31/14
CPCA61K39/42A61K45/06A61K2039/505A61P31/14C07K16/1081C12N15/85A61K2300/00Y02A50/30
Inventor 胡适崔瀛书雷长海傅文燕李天
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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