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Minicircle DNA carrier expressing IgG antibody, preparation method and application thereof

An antibody and carrier technology, applied in the field of biomedicine, can solve the problems of inconvenient use, high cost, hidden safety hazards, etc., and achieve the effects of convenient use, low cost and good safety.

Pending Publication Date: 2018-11-02
SYNO MINICIRCLE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the existing AAV expression antibody technology has disadvantages, including high cost, safety concerns
Specifically include: (a) the packaging and purification process of the AAV virus vector is complex, requiring a large number of cell cultures, which is time-consuming and labor-intensive; (b) the preservation and transportation of the AAV virus requires a low-temperature environment, resulting in increased costs and inconvenient use; (c) The AAV virus shell is easy to provoke a strong immune response, and the random insertion of the DNA sequence carried in the vector can easily lead to gene mutation and even cancer risk
[0006] Similarly, the disadvantages of expressing purified HIV antibodies or other IgG antibodies in vitro by cell culture are mainly high cost and inconvenience
Specifically include: (a) the in vitro expression and purification process of antibodies is complex and costly; (b) protein storage and transportation usually require low temperature environments, which are relatively demanding; (c) antibodies need to be injected intravenously or subcutaneously, with a limited half-life and long-term use , increasing the burden on patients

Method used

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  • Minicircle DNA carrier expressing IgG antibody, preparation method and application thereof
  • Minicircle DNA carrier expressing IgG antibody, preparation method and application thereof
  • Minicircle DNA carrier expressing IgG antibody, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, the construction of anti-HIV IgG antibody (Anti-HIV IgG antibody)

[0043] (a) Construction of Anti-HIV IgG antibody that neutralizes human immunodeficiency virus (HIV).

[0044] (b) The IgG antibody comprises a heavy chain variable region VH with the amino acid sequence shown in SEQ ID NO:1, and a light chain variable region VL with the amino acid sequence shown in SEQ ID NO:2.

[0045] (c) Constructing a minicircle DNA vector expressing the Anti-HIV IgG antibody, including the coding gene of the IgG antibody; and further comprising appropriate expression elements in the minicircle DNA vector.

[0046] (d) expression cassette comprising Anti-HIV IgG antibody in the minicircle DNA vector (see figure 1 shown), including the following structure:

[0047] Kozak-SP-AA-HIV.VH-CH1-hinge-CH2-CH3-Furin-GSG-T2A-SP-AA-HIV.VL-HIV.CL-His6

[0048] Among them, Kozak is the Kozak sequence, SP is the signal peptide (Signal Peptide); hinge is the antibody hinge region;...

Embodiment 2

[0051] Embodiment 2, the construction of microcircle DNA carrier and the preparation of microcircle (MC)

[0052] (a) Genes encoding synthetic IgG antibodies.

[0053] (b) Select an appropriate site to double-enzyme digest the empty minicircle DNA vector pMC.BESPX.

[0054] (c) Use seamless cloning or traditional cloning method (enzyme digestion-ligation) to insert the coding gene of the IgG antibody into the empty vector pMC.BESPX after double enzyme digestion to construct an IgG antibody minicircle DNA vector (pMC.IgG antibody).

[0055] (d) Transform E. coli E. coli.ZYCY10P3S2T with pMC.IgG antibody, and obtain microcircle (MC.IgG antibody) according to the standard microcircle preparation method

[0056] Among them, the microcircle DNA empty vector pMC.BESPX, the engineering bacteria E coli.ZYCY10P3S2T, and the microcircle preparation method refer to the literature Nat Biotechnol.2010,28(12):1287-9.

Embodiment 3

[0057] Embodiment 3, expression and purification of Anti-HIV IgG antibody

[0058] 1. Expression of IgG antibody in 293T cells

[0059] The superfect plasmid transfection kit (Invitrogen Company) was used to transfect 293T cells with the above-mentioned minicircle DNA, and the supernatants of 293T cells were collected after culturing in serum-free medium for three days, and the expression of Anti-HIVIgG antibody protein was detected by Western Blot.

[0060] 2. IgG antibody purification

[0061] The 293 cell culture supernatant was subjected to low-temperature centrifugation or depth filtration, and the supernatant was collected, and then purified by Protein A affinity chromatography. The purified protein was qualitatively detected by Western Blot, and the protein concentration was quantitatively detected by ELISA. The purified product should be stored below -20°C for long-term storage.

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Abstract

The invention relates to a minicircle DNA carrier expressing IgG antibody, a preparation method and application thereof, in particular to a minicircle DNA carrier for in vivo expression of IgG antibody with complete functions. The IgG antibody neutralizes human immunodeficiency virus (HIV), other pathogene antigen or cancer cell antigen. The minicircle DNA carrier and the IgG antibody can be usedfor preventing and treating diseases, such as acquired immune deficiency syndrome (or HIV infection), other pathogen infection or cancers.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a recombinant gene carrier, in particular to a microcircle DNA carrier used for expressing fully functioning IgG antibodies in vivo. Background technique [0002] Since monoclonal antibody drugs were approved for marketing in the 1980s, they have been infused into the body to bind, neutralize or clear specific antigens in the body, including cytokines, cell membrane surface receptors, and pathogen antigens. Monoclonal antibody drugs are widely used in the prevention and treatment of immune-related, cancer and infectious diseases, and have achieved very good clinical results. [0003] AIDS is mainly a fatal infectious disease caused by human immunodeficiency virus (HIV) infection, and has been widely prevalent in the world. Currently clinically used anti-HIV drugs, supplemented by highly effective antiretroviral therapy, can prolong the survival time and improve the quality of life of HIV...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/13C07K16/10A61K39/42A61P31/18A61P31/00A61P35/00A61P37/02
CPCC12N15/63C07K16/1045A61K39/00C07K2317/76C07K2317/56
Inventor 谢亦武
Owner SYNO MINICIRCLE BIOTECH CO LTD
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