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Anti-human tissue factor single-chain antibody and preparation method thereof

A single-chain antibody and tissue factor technology, which is applied in the fields of botanical equipment and methods, biochemical equipment and methods, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve clinical application difficulties, human Anti-mouse antibody reduces the curative effect and other problems, achieves good application prospects, realizes targeted and selective treatment, and has the effect of small molecular weight

Active Publication Date: 2014-07-09
NANOLATTIX BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the mouse monoclonal antibody has strong immunogenicity, and it is easy to produce human anti-mouse antibody (HAMA) to react and reduce the curative effect; however, there are still many technical obstacles in the preparation of human antibody, which brings difficulties to its clinical application.

Method used

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  • Anti-human tissue factor single-chain antibody and preparation method thereof
  • Anti-human tissue factor single-chain antibody and preparation method thereof
  • Anti-human tissue factor single-chain antibody and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1: Gene cloning of anti-human tissue factor single-chain antibody

[0034] The present invention constructs an anti-human tissue factor single-chain antibody gene, which adopts reverse transcription polymerase chain reaction technology, starting from the anti-tissue factor hybridoma cell line prepared by patent CN 200910227865.0, amplifying and obtaining the light chain of the antibody can be The variable region gene VL and the heavy chain variable region gene VH are connected with a linker sequence Linker to form a single-chain antibody, the nucleotide sequence of which is SEQ ID NO: 1; the single-chain antibody gene contains 354 nucleotides The composed heavy chain variable region gene has a sequence of SEQ ID NO:2; the light chain variable region gene comprising 321 nucleotides has a sequence of SEQ ID NO:3.

[0035] Its preparation method is as follows:

[0036] (1) Extraction of total RNA and synthesis of cDNA first strand

[0037] Select the anti-human tis...

Embodiment 2

[0051] Example 2: Construction of prokaryotic soluble expression vector for anti-human tissue factor single chain antibody

[0052] The cloned anti-human tissue factor single-chain antibody gene was connected to the prokaryotic expression vector phoA plasmid (3076bp) to construct the recombinant expression plasmid phoA-VH-Linker-VL-His. The recombinant plasmid has ampicillin resistance, is about 3811bp, is activated by alkaline phosphatase, and can secrete and express anti-human tissue factor single-chain antibody under the induction of low phosphorus culture.

[0053] Its preparation method is as follows:

[0054] After the VH-Linker-VL PCR product was digested with NcoI and BamHI, the digested product was recovered by agarose gel electrophoresis and a gel extraction kit. The phoA-His expression vector (preserved in our laboratory) was also digested with NcoI and BamHI, and the large fragment was recovered by agarose gel electrophoresis. The fragment of the digested product...

Embodiment 3

[0056] Example 3: Expression and purification of anti-human tissue factor single-chain antibody in MM294 bacteria

[0057] After the prokaryotic soluble expression vector of anti-human tissue factor single-chain antibody is transformed into Escherichia coli, induced to express and purified, the anti-human tissue factor single-chain antibody protein containing 246 amino acids can be obtained. The protein contains 118 amino acids encoded by the heavy chain variable region of the anti-human tissue factor monoclonal antibody, 107 amino acids encoded by the light chain variable region, 15 amino acids encoded by Linker and 6 amino acids of his tag. Its molecular weight is about 32KD.

[0058] Its preparation method is as follows:

[0059] (1) Construction of recombinant engineered bacteria

[0060] The expression vector was transformed into competent MM294 Escherichia coli, and the screened positive clones were inoculated in LB medium and cultured overnight at 37 °C. The next d...

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Abstract

The invention provides an anti-human tissue factor single-chain antibody and a preparation method thereof. The antibody has characteristics of small molecular weight, strong penetrating force for tumor tissues and low immunogenicity. The single-chain antibody is prepared by using a reverse transcription polymerase chain reaction technology, comprising starting from anti-tissue factor hybridoma cell lines prepared in a Chinese patent CN200910227865.0, amplifying to obtain a light chain variable range gene VL and a heavy chain variable range gene VH of the antibody, and using a linker sequence to link, thereby obtaining the single-chain antibody. A nucleotide sequence of the single-chain antibody is as shown in a SEQIDNO: 1. The preparation method of the single-chain antibody comprises: (1) gene clone of the anti-human tissue factor single-chain antibody; (2) construction of a soluble prokaryotic expression vector for the anti-human tissue factor single-chain antibody; (3) expression and purification of the anti-human tissue factor single-chain antibody in MM294 bacteria; and (4) active identification of the anti-human tissue factor single-chain antibody.

Description

technical field [0001] The invention belongs to the field of genetic engineering antibodies, and in particular relates to the preparation and application of an anti-human tissue factor single-chain antibody. Background technique [0002] Tissue factor (tissue factor, TF) is a transmembrane glycoprotein, which is the initial protein that causes blood coagulation reaction, can activate and start the extrinsic blood coagulation pathway, and plays an important role in physiological hemostasis. Studies in recent years have found that tissue factor not only plays an important role in the coagulation pathway, but also is often abnormally expressed in various malignant tumors. Clinical studies have shown that a variety of tumor cells overexpress TF to varying degrees, and the expression of TF is highly positively correlated with the metastasis ability of tumor cells. TF affects tumor invasion and metastasis by forming TF / VIIa complex, activating or upregulating the expression of cy...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/70
Inventor 何永吉赵邑赵峰梅张全爱梁雅丽潘薇薇赵亚蕊曹鹏程卢晓霞
Owner NANOLATTIX BIOTECH CO LTD
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