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Poxvirus human monoclonal antibody and application thereof

A monoclonal antibody, pox virus technology, applied in the field of medicine, can solve the problems of lack of cytotoxicity, reduced efficacy, allergic reactions, etc., and achieve the effects of reducing heterogeneity, universal applicability, and increasing accuracy

Active Publication Date: 2021-12-31
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with mouse monoclonal antibody or chimeric antibody, it lacks the shortcomings of cell-mediated cytotoxicity and complement-dependent cytotoxicity, thus reducing the curative effect and even causing allergic reactions, and using A33 as an antigen to sort specific memory B cells A very effective method for developing humanized neutralizing antibodies against poxviruses

Method used

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  • Poxvirus human monoclonal antibody and application thereof
  • Poxvirus human monoclonal antibody and application thereof
  • Poxvirus human monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Expression and Purification of Vaccinia Outer Membrane Protein A33 Protein in Orthopoxvirus

[0038] In view of the role of A33 in poxvirus infection, we compared the extracellular amino acid sequences of A33 in smallpox virus, mousepox virus, soybean sprout virus, and monkeypox virus. A33 was also demonstrated to be an important target of natural antibody responses against poxviruses.

[0039] Then select pET28a (+) as the expression vector, and select NcoI and BamHI as the insertion site of the A33 protein gene between the two restriction sites to obtain the recombinant plasmid, and transform the recombinant plasmid into BL21 Escherichia coli competent cells, When the OD600 of the bacterial solution is between 0.6-0.8, IPTG is added for induction. After about 3-4 hours, the inclusion bodies are collected, and then the inclusion bodies are refolded by dilution method. Finally, the renatured inclusion bodies are purified with a Ni column and passed through SDS-PAGE ide...

Embodiment 2

[0054] Performance testing of single chain antibodies

[0055] (1) ELISA detection of binding of single chain antibody to A33

[0056] In order to verify whether the screened antibody specifically binds to A33 and whether it is functional, it is verified by ELISA detection and in vitro neutralization test.

[0057] The single-chain antibody plasmid was transfected into 293T cells, and the supernatant was collected after 24 hours, and its expression was detected by Western assay, as shown in Figure 4 . Then dilute the A33 protein with the coating solution, coat the 96-well plate, and detect its binding status through the supernatant of the single-chain antibody, and screen the H2 single-chain antibody from it, such as Figure 5 shown.

[0058] (3) Combination of surface plasmon resonance detection and A33

[0059] Surface plasmon resonance analysis was performed using a Biacore T100 (Biacore Inc).

[0060] The specific steps are as follows: firstly, immobilize the H2 sing...

Embodiment 3

[0066] Expression and purification of H2 IgG antibody

[0067] The signal peptide sequence SEQ ID NO.9 and the light and heavy chain variable region sequence SEQ ID NO.1, SEQ ID NO.3 of H2 and the constant region sequence SEQ ID NO.7, SEQ ID NO.8 of H2 are spliced ​​together to construct Intact H2 IgG form. Transfect the constructed H2 IgG plasmid into 293F cells, collect the cell supernatant after 3 days of culture, purify the H2IgG antibody by protein A, and finally verify the purification by polyacrylamide gel electrophoresis, as shown in Figure 9 shown.

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Abstract

The invention discloses a poxvirus human monoclonal antibody and application thereof, and belongs to the technical field of medicine. According to the invention, vaccinia virus surface film antigen A33 protein expressed by Escherichia coli is taken as an antigen; specific memory B cells of the A33 protein are screened from PBMCs of a volunteer inoculated with a ceiling vaccine; then, the specific B cells are reversed and amplified to obtain a variable region fragment of the antibody; and the variable region fragment and a constant region are further connected into an expression vector to obtain the human monoclonal antibody capable of protecting the pox virus through mammalian cell expression and purification and a series of function detections. Through ELISA detection, the antibody has good fixation capability with the antigen, and in addition, the high protection effect is achieved in the in vitro neutralization test and the mouse infection. The human antibody has an application value in clinical treatment and prevention of the pox virus.

Description

technical field [0001] The invention belongs to the technical field of medicine. Background technique [0002] Smallpox is a severe infectious disease caused by Variola virus, and it is also the only infectious disease that has been eradicated by humans worldwide so far. Smallpox virus belongs to the genus Orthopoxvirus, which also includes vaccinia virus (VACV, used as a smallpox vaccine), monkeypox virus (Monkeypox) and mousepox virus (Ectromelia virus, ECTV), etc. These poxviruses are all highly homologous. Monkeypox virus or other genetically modified poxviruses can infect humans across species. Because mass immunization has stopped for nearly 40 years, most of the population is currently not immune to smallpox. Therefore, it is very necessary to construct a high-affinity, broad-spectrum specific humanized antibody against poxvirus, which can provide a rapid detection method for poxvirus and provide a strategic antibody reserve for the immunotherapy of the disease. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08A61K39/42A61P31/20G01N33/569G01N33/577C12N15/85G01N33/58
CPCC07K16/081A61P31/20G01N33/577G01N33/56983G01N33/582C12N15/85C07K2317/56C07K2317/52C07K2317/622C07K2317/21A61K2039/505G01N2333/07G01N2333/065C12N2800/107C07K2317/92G01N2469/10Y02A50/30
Inventor 方敏顾秀玲张毓凡姜威卢娇顾光磊
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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