RAAV vector for expressing antibody IgG1 and application of rAAV vector

A technology of IgG1-raav and vector, which is applied in the field of rAAV vector, can solve the problem that the cutting efficiency of the linking polypeptide is not very high

Pending Publication Date: 2022-03-01
INNOVATION ACAD FOR PRECISION MEASUREMENT SCI & TECH CAS
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cleavage efficiency of the above-mentioned linked polypeptides is not very high, and the expression level of IgG1 still needs t...

Method used

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  • RAAV vector for expressing antibody IgG1 and application of rAAV vector
  • RAAV vector for expressing antibody IgG1 and application of rAAV vector
  • RAAV vector for expressing antibody IgG1 and application of rAAV vector

Examples

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Embodiment 1

[0016] Example 1. Construction of antibody IgG1-rAAV vector plasmid and verification of its function

[0017] We chose the strategy of utilizing a single rAAV vector to express IgG1 antibody for optimal design. An antibody expression cassette was constructed on the basis of the pAAV-ITR-MCS plasmid backbone. Wherein, the rAAV terminal repeat sequence (ITR) adopts the ITR sequence of type 2 AAV, as shown in SEQ ID NO.1. Between the two ITR sequences is the expression box for antibody IgG1. We chose the human cytomegalovirus (CMV) promoter with a broad-spectrum strong promoter, followed by the IgG1 antibody heavy chain (Heavy Chain, HC) coding region, and then connected to the IgG1 antibody light chain (Light Chain) through a linker sequence (linker) Chain, LC) coding region, and finally the human serum albumin polyadenylate (HGHpA) sequence.

[0018] Since the differences between different IgG1 antibodies are mainly in the heavy chain HC variable region VH and heavy chain LC...

Embodiment 2

[0022] Example 2. Packaging and purifying antibody IgG1-rAAV virus

[0023] We prepared antibody IgG1-rAAV by transfecting HEK 293T cells with three plasmids. In order to facilitate packaging and in vivo testing, we selected the serotype plasmid pAAV-RC9 of type 9 AAV for experiments. First, spread HEK293T cells on a 10 cm culture dish, and when the cells grow to a confluence of about 80%, the pAAV-ITR-CMV-IgG1-HGHpA core plasmid, pAAV-RC9 plasmid and pAAV-Ad-helper plasmid are separated by PEI transfection reagent HEK293T cells were transfected at an equimolar ratio. Three days after transfection, the cells and culture supernatant were collected, in which the packaged IgG1-rAAV9 was present. Then, we used iodixanol (idox) density gradient ultracentrifugation to purify rAAV (refer to Grieger et al., Nat Protoc. 2006; 1(3):1412-28.). The titer of purified IgG1-rAAV9 was detected by fluorescent quantitative PCR to be about 6.5E+12VG / ml. Further, using the method of silver st...

Embodiment 3

[0024] Example 3. Detection of expression of antibody IgG1-rAAV infection in HEK293T cells and C57 mice

[0025] We used the antibody IgG1-rAAV9 virus obtained in Example 2 to infect HEK293T cells cultured in vitro. Three days after infection, cells and culture supernatants were collected. Then, the cells and supernatant samples were processed and subjected to SDS-PAGE electrophoresis and Western blot detection, and HRP-labeled goat anti-human IgG antibody was used to recognize the expressed product IgG1. The experimental results showed that there were obvious specific detection signals of antibody IgG1 heavy chain and light chain in HEK293T cells and supernatant (such as image 3 A).

[0026] We used the antibody IgG1-rAAV9 virus obtained in Example 2 to infect C57 mice by tail vein injection. Blood was taken from the tail vein at 14 days (2 weeks), 21 days (3 weeks) and 28 days (4 weeks) after infection. We placed the blood samples at 37°C for 1 hour, then at 4°C for 12 ...

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Abstract

The invention discloses an rAAV vector for expressing an antibody IgG1 and application of the rAAV vector. The ITR core expression element of the rAAV vector is composed of a broad-spectrum strong promoter CMV, an IgG1 heavy chain secretion signal peptide sequence, an IgG1 heavy chain coding region, a connecting polypeptide coding sequence, an IgG1 light chain secretion signal peptide sequence, an IgG1 light chain coding region and a human serum albumin polyadenylic acid sequence. The heavy chain variable region (VH) and the light chain variable region (VL) can be conveniently and quickly operated through the restriction enzyme cutting sites on the rAAV vector. The antibody IgG1-rAAV obtained by packaging is used for infecting HEK293T cells or C57 mice and the like cultured in vitro, and various antibodies IgG1 with different antigen recognition activities can be expressed.

Description

technical field [0001] The invention belongs to the technical field of viral vectors, and more specifically relates to an rAAV vector capable of long-term expression of antibodies and its application. Background technique [0002] Monoclonal antibody (mAb) therapy is one of the well-established strategies for the treatment of immune diseases, cancer and infectious diseases. The majority of currently licensed therapeutic mAbs are of the IgG1 type, formed as a tetramer composed of two dimers, each dimer comprising a light chain assembled by disulfide bonds (Light Chain, LC ) and heavy chain (Heavy Chain, HC). MAbs have a variable fragment (Fab) and a constant fragment (Fc) that confer antigen-binding properties. In addition to binding, mAbs can also neutralize viruses through Fab interactions and can also participate in immune responses against specific antigens. Interaction of Fc with Fc cell receptors can trigger different mechanisms including programmed cell death, immun...

Claims

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Application Information

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IPC IPC(8): C12N15/864C07K16/32C07K16/28C07K16/40C07K16/06
CPCC12N15/86C07K16/32C07K16/2863C07K16/40C07K16/065C07K2317/56C12N2750/14143C12N2800/60
Inventor 吴阳徐富强王杰王起恬金鼎瑜
Owner INNOVATION ACAD FOR PRECISION MEASUREMENT SCI & TECH CAS
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