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HEscFv gene sequence with plant favored codon

A technology that favors codons and gene sequences, and is used in plant genetic improvement, genetic engineering, and botanical equipment and methods.

Inactive Publication Date: 2006-07-26
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The expression of medicinal proteins and antibodies in plant expression systems is significantly superior to traditional bacteria, yeast, animal cells and transgenic animals in terms of product safety, production cost and large-scale production (Fischer et al. Transgenic Research, 2000, 9: 279-299 ; Ma et al.Naturereviews genetics, 2003, 4:794-805), but there is no report on the use of plant systems to express hEscFv

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] hEscFv Gene Synthesis and Sequence Information and Homology Analysis

[0034] 1. hEscFv gene synthesis (gene synthesis)

[0035] The hEscFv gene was designed by our laboratory according to the principle of plant-preferred codons and synthesized by Shanghai Sangon, and then ligated into the pUC18 vector, pUC18-hEscFv, which was verified by 2 times of sequencing;

[0036] 2. hEscFv gene sequence information

[0037] The hEscFv gene designed and synthesized according to plant preferred codons of the present invention has a full length of 814bp (including protective bases and restriction site linkers), and the detailed sequence is shown in SEQ ID NO.1. The open reading frame is located at 51-800 nucleotides, which is composed of heavy chain variable region (VH) (51-422), flexible peptide 3 × (Gly 4 Ser 1 ) coding region (position 423-467) and light chain variable region (VL) (position 468-800) constitute the open reading frame sequence. The deduced amino acid sequence o...

Embodiment 2

[0086] Construction of hEscFv gene vector

[0087] 1. Restriction enzyme cutting

[0088] The pUC18 cloning vector containing the hEscFv gene (pUC18-hEscFv) was digested with Bam H I and Sac I to obtain a DNA fragment of the hEscFv gene with Bam H I and Sac I sticky ends (the same as the expression vector) (the gene is designed with BamH I and Sac I site linker);

[0089] 2. Connection (ligation)

[0090] Connect the excised gene into the plasmid pBI121 that has been excised with Bam H I and Sac I or the large fragment of the pCAMBIA2300 plant expression vector transformed with pBI121, and use blue and white screening or PCR to detect the transformed Escherichia coli to obtain positive clones .

[0091] 3. PCR detection of the target gene

[0092] According to the nucleotide sequence of the gene, design primers: hEscFv F: 5'-atg gag gtt caactt ctt gag-3' (forward primer) and hEscFv R: 5'-ctt aat ctc aag ctt agt tcc-3' (reverse primer), carry out PCR amplification with the...

Embodiment 3

[0095] Expression of hEscFv Gene in Tomato and Tobacco Eukaryotic Cells

[0096] 1. Preparation of engineering bacteria containing target gene (hEscFv) plant expression vector

[0097] The plant expression vector containing the hEscFv gene obtained in Example 2 was identified or sequenced by enzyme digestion, and under the premise of ensuring that the target gene (hEscFv) in the expression vector was correctly read and connected to the vector, the constructed vector Transformation of Agrobacterium (such as EHA105) by freezing-thawing method to obtain engineering bacteria for plant genetic transformation, transformation of tomato and tobacco by Agrobacterium-mediated method.

[0098] 2. Agrobacterium-mediated transformation of tomato

[0099] ① Sterilize the seeds of tomato (Lycopersicon esculentum.var.cerasiforme cv.Yellow fruit No.22) (22# yellow cherry tomato) with 75% ethanol and 20% sodium hypochlorite and sow them on 1 / 2 MS medium, and wait for 6-8 days After the leaves...

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Abstract

This invention relates to a hEscFv gene sequence that composes by the plant preference codon, which belongs to the gene project field. This invention includes: the code designed by the plant preference codon has the nucleotides sequence which has the biology activity hEscFv polypeptides, and the nucleotides sequence has at least 70% same fountain with the 51-800 nucleotides in the SEQ ID NO. 1; or they can cross under the middle tension condition. The polypeptides are expressed or produced in the eukaryotic, the character of which is that they have the amino acid sequence showed in the SEQ ID NO. 2 of the list, and the eukaryotic product has the target action to the vessel epidermal growth factor (EGF), and it is important for borderline tumour treatment.

Description

technical field [0001] The present invention relates to a gene sequence in the field of biotechnology, more specifically, relates to a hEscFv gene sequence according to plant preferred codons. Background technique [0002] Folkman (Ann Surg, 1972, 175(3): 409-416) believes that "tumor growth depends on angiogenesis", and Ferrara (Endocr Rev, 1997, 18(1): 4-25) believes that the most critical factor for angiogenesis is vascular endothelium Growth factor (vascular endothelial growth factor, VEGF), Brown et al. (Hum Pathol, 1995, 26 (1): 86-91) research results show that common malignant solid tumors have overexpression of VEGF. Antibody is the specific targeted drug with the most definite target in tumor treatment, which overcomes the defects of poor targeting of surgery, radiotherapy and chemotherapy in traditional tumor treatment. However, problems such as the large molecular weight of antibodies (difficult to enter tumor tissue), large dosage, high price, and immunogenicit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/13C07K14/435
Inventor 赵凌侠任薇薇唐克轩开国银钱虹妹陈玉辉张慧
Owner SHANGHAI JIAO TONG UNIV
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