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hhscfv gene sequence by plant-preference codon

A technique for favoring codons and gene sequences, applied in the field of hHscFv gene sequences

Inactive Publication Date: 2006-08-16
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, prokaryotic expression systems including bacteria and eukaryotic expression systems such as yeast, animal cells and transgenic animals have obvious defects compared with plants in terms of large-scale production, product safety and cost (Fischer et al. Transgenic Research, 2000 , 9:279-299; Ma et al.Nature reviews genetics, 2003, 4:794-805), but there is no report of expressing hHscFv in plant system

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Design and synthesis of hHscFv gene, sequence information and homology analysis

[0033] 1. hHscFv gene synthesis (gene synthesis)

[0034] The hHscFv gene was designed by our laboratory according to the principle of plant preferred codons and commissioned by Shanghai Sangong to synthesize it and then ligated into the pUC18 vector, namely pUC18-hHscFv

[0035] 2. hHscFv gene sequence information

[0036] The hHscFv gene designed and synthesized according to plant preferred codons of the present invention has a full length of 826bp (including a linker consisting of protective bases and restriction sites), and the detailed sequence is shown in SEQID NO.1. The open reading frame is located at 51-812 nucleotides, which is composed of heavy chain variable region (VH) (51-428), flexible peptide 3 × (Gly 4 Ser 1 ) coding region (position 429-473) and light chain variable region (VL) (position 474-812) constitute the open reading frame sequence. The deduced amino acid seque...

Embodiment 2

[0081] Construction of hHscFV gene expression vector

[0082] 1. Restriction enzyme cutting

[0083] The pUC-18 cloning vector (pUC18-hHscFv) containing the hHscFv gene was double digested with Bam H I and Sac I to obtain a DNA fragment of the hHscFv gene with Bam H I and Sac I sticky ends (same as the expression vector);

[0084] 2. Connection (ligation)

[0085] Connect the excised hHscFv 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 detect the transformed Escherichia coli by blue and white screening or PCR to obtain positive results. clone.

[0086] 3. PCR detection of the target gene

[0087] According to the nucleotide sequence of the gene, design primers: hHscFvF: 5'-atg gag gtt caactt gtt gag-3' (forward primer), hHscFvR: 5'-tct ctt aat ctc aac ctt agt-3' (reverse primer) To the primer (SEQ ID NO.4), carry out PCR amplification using the vector o...

Embodiment 3

[0090] Expression of hHscFv in tomato and tobacco eukaryotic cells

[0091] 1. Preparation of engineering bacteria containing hHscFv gene plant expression vector

[0092] The plant expression vector containing the hHscFv gene obtained in Example 2 was identified and sequenced by enzyme digestion, and under the premise of ensuring that the hHscFv gene connection and reading frame in the expression vector were correct, the constructed vector was then transformed into Agrobacterium ( As in EHA105), the engineering bacteria used for plant genetic transformation were obtained, and tomato and tobacco were transformed using the Agrobacterium-mediated method.

[0093] 2. Agrobacterium-mediated transformation of tomato

[0094] 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 are unfolded, the coty...

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Abstract

The hHscFv gene sequences encode the plants. The synthetic DNA includes: the nucleic acid sequence which is 70% same to the 51-812bp nucleic acids of the SEQ IDNO.1 encodes the multi peptides with the hHscFv activity. The sequence encodes the multi peptides with the amino acid sequence of the SEQ ID NO.2. The peptides have the important value for curing the hepatocellular carcinoma.

Description

technical field [0001] The present invention relates to a gene sequence in the field of biotechnology, more specifically, relates to a hHscFv gene sequence according to plant preferred codons. Background technique [0002] Hepatocellular carcinoma (HCC) is highly malignant, with insidious onset, early metastasis, and high mortality, which seriously endangers human health. It ranks fourth among many lethal tumors and ranks second in my country. Traditional surgery, radiotherapy, and chemotherapy have the defects of easy recurrence and poor targeting of tumor cells in the treatment of liver cancer. Antibodies are currently the most specific targeted drugs in tumor treatment. However, the clinical application of intact antibodies is limited due to factors such as large molecular weight (difficult to enter tumor tissue), immunogenicity (current antibodies are mostly of mouse origin), large dosage and high price. Therefore, the production of humanized single-chain antibodies wit...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/13C07K14/435
Inventor 赵凌侠崔丽洁唐克轩开国银钱虹妹陈玉辉张慧
Owner SHANGHAI JIAO TONG UNIV
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