Synthetic human insulin gene and use thereof in cultivation of transgenic tomatoes

A human insulin, artificial synthesis technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as limitations, low yield of culture devices, and inability to carry out mammalian protein translation.

Inactive Publication Date: 2010-03-31
林忠平
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, the dendritic cells scattered in the intestinal cells can also take up the antigens in the intestinal lumen and activate the immune response through the circulation of the lymphatic system, but whether this process induces tolerance or immunity is still unclear
[0007] The treatment of oral tolerance has a certain therapeutic effect on the disease because it can induce antigen-specific immune responses, but it is limited by some practical problems, namely This therapy requires large amounts of protein antigens in both humans and

Method used

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  • Synthetic human insulin gene and use thereof in cultivation of transgenic tomatoes
  • Synthetic human insulin gene and use thereof in cultivation of transgenic tomatoes
  • Synthetic human insulin gene and use thereof in cultivation of transgenic tomatoes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] The artificial synthesis of embodiment 1 human insulin:

[0068] According to the situation of codon usage in plants provided by Murray E.E. et al., it is speculated that plants prefer codons. The summary is shown in Table 1.

[0069] Find the sequence of human proinsulin in GENEBANK, get the sequences of human insulin A chain and B chain, and design the replacement bases according to the plant preferred codons. Add the designed connecting short peptide between the B chain and A chain of the designed synthetic human insulin; add the start codon ATG before the B chain, and add the DNA sequence encoding KDEL and the stop codon TAA after the A chain and adding two restriction endonuclease sites BamHI and EcoRI before and after the synthetic gene; the human insulin gene designed and synthesized is obtained.

[0070] Divide the double strands of the synthetic human insulin gene into 8 segments (see Table 2), so that one of the 5' ends of the two strands is slightly shorter...

Embodiment 2

[0115] Example 2 Construction of plant expression vector pCAM35S-InsK:

[0116] For the construction procedure of the plant expression vector pCAM35S-InsK, see figure 1 and Figure 1 continued :

[0117] (1) The pBI221 plasmid and p11Z-InsK plasmid containing the 35S promoter were completely digested with HindIII and XbaI respectively, and the vector and insert were recovered after agarose gel electrophoresis, and the molar ratio of insert:vector was 3:1 Connecting, so that 35S is inserted into the upstream of the synthetic human insulin gene, named p11Z-InsK-35S;

[0118] (2) The constructed p11Z-InsK-35S containing the insulin gene driven by the 35S promoter and plant expression were completely digested with HindIII and EcoRI in the vector pCAMBIA1390, and then ligated to obtain the plant expression vector pCAM35S-InsK.

[0119] HinidIII and BamHI double enzyme digestion cut out a small fragment of 0.8kb, PCR amplification can also obtain a small fragment of 0.8kb, which ...

Embodiment 3

[0120] Example 3 Construction of plant expression vector pCAM2A12-InsK:

[0121] For the construction procedure of the plant expression vector pCAM35S-InsK, see figure 2 and Figure 2 continued :

[0122] (1) The pTM plasmid and p11Z-InsK plasmid containing the 2A12 promoter were completely digested with HindIII and XbaI respectively, and the vector and insert were recovered after agarose gel electrophoresis, and the molar ratio of insert:vector was 3:1 Connecting 2A12 into the upstream of the synthetic human insulin gene and naming it p11Z-InsK-2A12;

[0123] (2) The constructed p11Z-InsK-2A12 containing the insulin gene driven by the 2A12 promoter and plant expression were completely digested with HindIII and EcoRI in the vector pCAMBIA1390, and then ligated to obtain the plant expression vector pCAM2A12-InsK.

[0124] A small fragment of 0.8 kb was cut out by HinidIII and BamHI double enzyme digestion, and a small fragment of 0.8 kb could also be obtained by PCR amplifi...

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Abstract

The invention relates to the field of plant gene engineering and provides a sequence of a human insulin gene designed and synthesized according to plant preferred codons. A plant expression vector isbuilt for the synthetic human insulin gene, so that the gene can be transferred into tomatoes by an agrobacterium rhizogenes-mediated method under the drive of a CaMV 35S promoter and a fruit-specificpromoter 2A12 to express human insulin in tomato fruit. The tomatoes capable of making human insulin can be used as a convenient oral product for preventing and treating insulin-dependent diabetes mellitus and autoimmune disorder diabetes mellitus, and human insulin can be extracted from the tomatoes to be made into injection preparations.

Description

[0001] This application is a divisional application of CN03109739.1. The original application title is: Method for Producing Human Insulin Using Transgenic Tomato. The filing date of the original application is: April 14, 2003. Technical field: [0002] The invention relates to a method for producing human insulin by transgenic plants and its product. Background technique: [0003] Bioreactors are an area of ​​great concern to many biotechnology R&D departments in the near future. Human growth factors are produced in mammary bioreactors such as dairy cows and dairy goats. In the research of plant bioreactor, the research of using potato tubers to produce hepatitis B vaccine has received extensive attention. Lettuce and tomato, as vegetables or fruits that can be eaten raw, have great application potential in the production of oral polypeptide drugs. Human insulin is very important for the treatment of insulin-dependent diabetic patients. Metabolic regulation in some diabe...

Claims

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Application Information

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IPC IPC(8): C12N15/17C12N15/82
Inventor 林忠平倪挺陈溪胡鸢雷
Owner 林忠平
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