Plant seed expression system for single-chain antibody against CD20

A single-chain antibody and plant expression vector technology, applied in the field of genetic engineering, can solve the problems of increased production costs, high prices, and high costs, and achieve the effects of avoiding pollution, reducing production costs, and facilitating industrialization

Inactive Publication Date: 2014-08-13
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Rituxan produced by mammalian cells (CHO) requires expensive equipment and culture media, which increases its production costs and leads to high prices (the cost of a course of treatment is about 200,000 yuan).

Method used

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  • Plant seed expression system for single-chain antibody against CD20
  • Plant seed expression system for single-chain antibody against CD20
  • Plant seed expression system for single-chain antibody against CD20

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Obtaining of intermediate vector p1301b

[0032] According to the nucleotide sequence published by TC.Hall and McBride, Summerfelt et al., the promoter and terminator sequences of β-phaseolin were artificially synthesized, and Xho I and Nco I restriction sites were added at both ends of the promoter respectively. Add Hind III and Kpn I restriction sites at both ends of the terminator respectively; and obtain the 35S+Bar gene from the pBASTA plasmid by KpnI and Bst EII double restriction method; simultaneously use Kpn I and Bst EII double restriction restriction pCambia1301, The 35S+Bar original was connected to construct the intermediate vector p1301Bas. Then, the original promoter and terminator of β-phaseolin were connected to p1301Bas by enzyme digestion and ligation reaction, and the intermediate vector p1301b was constructed.

Embodiment 2

[0033] Example 2 The artificial synthesis of anti-CD20 single chain antibody, i.e. VL-VH-Linker-hIgG1Fc

[0034] Entrusted Jiangsu Jinweizhi Biotechnology Co., Ltd. to artificially synthesize the gene sequence of plant-preferred codons, that is, the nucleotide sequence encoding the barley α-Amylase signal peptide sequence, the light chain variable region of the monoclonal antibody C2B8, the connecting peptide, and the single Cloning the heavy chain variable region of antibody C2B8, Hinge and CH2 and CH3 sequences of human IgG1. In addition, Nco I and Hind III restriction sites were added at both ends of the synthetic sequence. Its base sequence is shown in SEQ ID NO.1.

Embodiment 3

[0035] Example 3 Construction of anti-CD20 single-chain antibody seed expression vector

[0036] Nco I and Hind III were used to double-digest p1301b and the plasmid pUC57-Ri containing the above-mentioned single-chain antibody gene, and the obtained fragments of about 12000bp and 1500bp were ligated to obtain the plant expression vector pAmy-scFv- Fc.

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Abstract

The invention discloses a plant seed expression system for a single-chain antibody against CD20. According to the invention, a single-chain antibody gene against CD20 is designed and artificially synthesized according to the sequence of VL-VH-Linker-hIgG1Fc by adding the nucleotide sequence of a signal peptide sequence coding barley alpha-Amylase at the 5' end of the gene, the gene is inserted into a plant seed specific expression vector p1301b, and an Agrobacterium-mediated method is employed for transformation of plants; an expression frame with a seed specific promoter, the single-chain antibody gene against CD20 and a terminator is integrated into the genomes of the plants; plants with the gene are screened and cultured, screening and propagation are further carried out so as to obtain a homozygous plant, and seeds produced by the homozygous plant contain the single-chain antibody against CD20; recombinant protein is directionally expressed in vacuole. The expressed single-chain antibody against CD20 has biological activity, and ADCC killing capability of the antibody on Daudi cells is not significantly different from that of Rituxan.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an anti-CD20 single-chain antibody gene and a method for highly expressing an anti-CD20 single-chain antibody in plant seeds by utilizing a plant seed-specific expression promoter. Background technique [0002] According to the "2012 China Cancer Registration Annual Report", there are about 3.12 million new cancer cases in my country every year, and more than 2 million cancer deaths. Malignant tumors have become the number one cause of death among our urban and rural residents. Non-Hodgkin's lymphoma (NHL) is the most common malignant tumor of the lymphatic system, most of which are B-cell lymphomas. CD20 antigen is a B cell differentiation antigen, located on the surface of B cells, only present in mature B cells and B cell precursors, not expressed in normal tissues, but over 95% in B cell lymphomas Express. This also makes CD20 an ideal target for t...

Claims

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Application Information

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IPC IPC(8): C12N15/13C12N15/84C07K16/28A01H5/00A61K39/395A61P35/00A61P37/00
Inventor 姜潮李校堃王德仲李海燕刘秀明姚娜金立波
Owner JILIN AGRICULTURAL UNIV
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