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Humanized single-chain variable fragments of targeted B lymphoma cells

A single-chain antibody, antibody light chain technology, applied in the fields of genetic engineering and protein engineering, can solve the problems of insufficient binding activity of single-chain antibodies, and achieve the effect of good application prospects

Inactive Publication Date: 2015-04-29
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, the binding activity of most single-chain antibodies is insufficient and often requires further optimization, so it is more challenging to obtain single-chain antibodies with sufficient binding activity

Method used

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  • Humanized single-chain variable fragments of targeted B lymphoma cells
  • Humanized single-chain variable fragments of targeted B lymphoma cells
  • Humanized single-chain variable fragments of targeted B lymphoma cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 The DNA sequence of the single-chain antibody of the present invention and the construction of the recombinant vector

[0062] The gene fragment encoding the single-chain antibody in the present invention can be obtained by classical molecular biology techniques, and the gene sequence can be optimized for the mammalian expression system to obtain better expression. The recombinant vector can be obtained by reconnecting the single-chain antibody gene fragment with the corresponding expression vector, which is suitable for the expression and screening of mammalian cells.

[0063] The heavy chain variable region (VH) and light chain variable region (VL) genes of the human CD19 single chain antibody variable region of the present invention are derived from human antibody germline genes (see figure 1 ). The nucleotide sequence encoded by VH is shown in SEQ ID NO.1 in the sequence listing, and the amino acid sequence is shown in SEQ ID NO.2 in the sequence listing....

Embodiment 2

[0066]Example 2 Expression and purification of the single-chain antibody of the present invention

[0067] In this example, the single-chain antibody was expressed in CHO cells and secreted into the culture medium, and purified by nickel column affinity chromatography.

[0068] 1. Expression of single chain antibody in CHO cells

[0069] After obtaining the above-mentioned high-purity recombinant plasmid encoding CD19 single-chain antibody, use the Lipofectamine 2000 plasmid transfection kit (Invitrogen Company) to operate according to its instructions, transfect the recombinant plasmid into CHO cells, and culture in serum-free medium for three years. CHO cell supernatants were collected 1 day later, and the expression of single-chain antibodies could be detected by Western blotting (see Figure 4 ), the detection antibody used was anti-His6 antibody.

[0070] The above transient expression method can be used to quickly obtain a small amount of antibody protein, and if it is...

Embodiment 3

[0073] Example 3 Detection of the binding activity of the single-chain antibody of the present invention to CD19 positive cells

[0074] The single chain antibody of the present invention can bind to the corresponding target cells in vitro.

[0075] In this example, B-cell tumor cells Raji cells were used as CD19-positive cells, and the prepared single-chain antibody CD19ScFv-VHVL was used to detect cell binding activity.

[0076] The single chain antibody was mixed with Raji cells in PBS buffer containing 0.02% sodium azide, and incubated on ice for 1 hour. Cells were washed once with PBS buffer, and then incubated with fluorescein isothiocyanate (FITC)-labeled mouse anti-His6 antibody on ice for 30 minutes. A commercial FITC-labeled CD19 antibody was used as a positive control. As a negative control, on the one hand, Raji cells were incubated with FITC-labeled mouse anti-His6 antibody; on the other hand, CD19-negative HepG2 cells were incubated with the antibody of the pre...

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Abstract

The invention relates to the technical field of genetic engineering and protein engineering, in particular to DNA for encoding a recombinant fusion protein of a variable fragment which contains a humanized CD19 (Cluster of Differentiation 19) antibody, the fusion protein encoded by the DNA, a production method for the fusion protein, and the application of the fusion protein. The invention provides the protein which comprises a humanized CD19 single-chain variable fragment, which can combine targeted B lymphoma cells with the CD19, cannot combine the targeted B lymphoma cells without the CD19, shows specific targeted binding activity, and has a very good application prospect.

Description

technical field [0001] The present invention relates to the technical fields of genetic engineering and protein engineering, in particular to a DNA encoding a variable region fragment of a human CD19 antibody that can target B lymphoma cells, a recombinant protein encoded by it, a production method of the recombinant protein, the Use of recombinant proteins. Background technique [0002] B lymphocytes are a kind of immune cells widely distributed in the human body and play an important role in humoral immunity. If B lymphocytes undergo malignant transformation, it can lead to the occurrence of lymphoma. Studies have shown that CD19 (cluster of differentiation 3) is highly expressed on the surface of a variety of B cell-related malignant tumor cells, including acute non-Hodgkin's lymphoma, B lymphocytic leukemia, etc., and CD19 is expressed in the early stages of B lymphocyte development. It begins to be expressed and covers almost all stages of B cell development, so it is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/30C12N15/13C12N15/85C12N5/10A61K39/395A61P35/00
Inventor 勾蓝图杨金亮魏于全
Owner SICHUAN UNIV
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