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4 connecting body thymosin alpha 1 gene order and preparation method of transgene tomato

A technology for gene sequence and thymosin, which is applied in the field of genetic engineering, can solve the problems of difficult to remove endotoxin contamination of yeast system protein modification, affecting the safety and biological activity of thymosin α1, and increasing the production cost of thymosin α1.

Inactive Publication Date: 2009-06-10
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Biological products derived from Escherichia coli and yeast systems need to be purified and processed before they can be used clinically, and the purification process requires necessary equipment support and expensive media, which increases the production cost of thymosin α1
At the same time, the problems of secondary pollution and purification reagents affecting the safety and biological activity of Thymosin α1 during processing are difficult to overcome
In particular, the recombinant protein produced by the microbial system is difficult to remove endotoxin contamination and the defects of protein modification in the yeast system are not yet overcome

Method used

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  • 4 connecting body thymosin alpha 1 gene order and preparation method of transgene tomato
  • 4 connecting body thymosin alpha 1 gene order and preparation method of transgene tomato
  • 4 connecting body thymosin alpha 1 gene order and preparation method of transgene tomato

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] 4×Tα1 gene synthesis and vector construction:

[0113] 1. Gene synthesis

[0114] The total length of the thymosin α1 (Tα1) gene synthesized according to plant-biased codon design and synthesis of the present invention is 124bp (including the linker consisting of protective bases and restriction sites), and the detailed sequence is shown in SEQ ID NO.1; after synthesis, it is connected pUC18 vector, namely pUC18-Tα1. According to the properties of XbaI and SpeI homologous enzymes, a linear quadruplex thymosin α1 gene (4×Tα1) was constructed by means of DNA recombination, and then ligated into pCAMBIA2300, namely pCA2300-4×Tα1.

[0115] 2. Digestion of the restriction enzyme

[0116] Double-cut pCA2300-4×Tα1 with Bam HI and Sac I to obtain a 4×Tα1 gene DNA fragment with Bam HI and Sac I cohesive ends (same as the expression vector);

[0117] 3. Connection (ligation)

[0118] Connect the cut 4×Tα1 gene into the large fragment of the plant binary expression vector pRD1...

Embodiment 2

[0127] 4×Tα1 gene sequence information and homology analysis:

[0128] The full length of the 4×Tα1 gene of the present invention is 417bp (including the linker consisting of protective bases and restriction sites), and the detailed sequence is shown in SEQ ID NO.2; wherein the open reading frame is located at nucleotides 16-408. The deduced amino acid sequence of the full-length thymosin α1 has a total of 131 amino acid residues, a molecular weight Mw=14,595.12, and an isoelectric point pI=4.43. See SEQ ID NO.3 for the detailed sequence.

[0129] The full-length sequence of the 4×Tα1 gene and its encoded protein, designed and synthesized according to the plant-biased codons, were stored in the Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR databases using the BLAST program Nucleotide and protein homology searches were carried out, and it was found that it was similar to bovine synthetic thymosin α1 nucleotide sequen...

Embodiment 3

[0131] Eukaryotic expression of thymosin Tα1 in tomato cells:

[0132] 1. Construction of expression vector containing target gene (4×Tα1)

[0133] The cloning vector plasmid pCA2300-4×Tα1 containing the 4×Tα1 gene was digested with Bam HI and Sac I, and the target DNA fragment was recovered with a gel recovery kit (Huasun Biotechnology Co., Ltd., Shanghai) to obtain the corresponding The target gene 4×Tα1 at the cohesive end is then cloned into the plant binary expression vector PG-pRD12 (not limited to this, but also includes plant expression vectors such as pBI121 or transformed pCAMBIA2300), and identified by sequencing or enzyme digestion, On the premise of ensuring that the reading frame of the target gene in the expression vector is correct, the expression vector plasmid is transformed into Agrobacterium (such as EHA105) by a freeze-thaw method, and tomato is transformed by the Agrobacterium-mediated method.

[0134] 2. Tomato Genetic Transformation

[0135] ① Sow the...

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Abstract

The invention discloses a 4*thymosin alpha1 gene sequence and a method for preparing transgenic tomato, which belongs to the field of gene engineering. The method comprises the following steps: a nucleotide sequence with bioactive thymosin alpha1 polypeptide is encoded according to a plant preferential codon, and has at least 75 percent of homology with a nucleotide sequence containing 16th to 408th nucleotides in SEQ ID NO.2; or the nucleotide sequence can be hybridized with the nucleotide sequence containing 16th to 408th nucleotides in the SEQ ID NO.2 under moderate stringent conditions. The invention also provides a method for expressing thymosin alpha1 functional protein by utilizing a plant system. A 4*T alpha1 gene expresses the important effect and application value of a product in improving the immunity of human bodies and preventing and treating human major diseases such as hepatitis, HIV, cancer and diabetes in eukaryotic cells.

Description

technical field [0001] The invention relates to a plant gene sequence and a transgenic method in the field of genetic engineering, in particular to a 4-connected thymosin α1 gene sequence and a method for preparing a transgenic tomato. Background technique [0002] Thymosin α1 (Thymosin α1, Tα1) is widely used clinically in the treatment of immunodeficiency, viral infection and autoimmune diseases, and has achieved great success in the treatment of hepatitis (HBV and HCV), AIDS (HIV), cancer (cancer) and diabetes (diabetes) a satisfactory effect. At present, the production of thymosin α1 mainly adopts tissue extraction, chemical synthesis and genetic engineering. Due to the limited source of tissue and the high cost of chemical synthesis, the clinical application of thymosin α1 is limited. The production of thymosin α1 by genetic engineering has potential, especially the production of thymosin α1 by plant system has obvious advantages in terms of safety and cost reduction....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/16C12N15/82A01H1/00A01H4/00A01H5/00
Inventor 赵凌侠芦丽亚杨宁张昱张冉
Owner SHANGHAI JIAO TONG UNIV
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