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Method for preparation of transgenic rice preparation and use thereof

A kind of transgenic rice, transgenic technology

Inactive Publication Date: 2008-09-03
WUHAN HEALTHGEN BIOTECHNOLOGY CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, in developed countries, insulin analogs tend to gradually replace insulin; in China, there are many types of insulin products, including regular human insulin (R), intermediate-acting human insulin (low protamine zinc insulin), protamine zinc insulin (PZI ) and premixed insulin (30R, 50R), etc. In addition, some traditional Chinese medicine preparations are also used for the treatment of diabetes. However, due to the rapid development of diabetic patients in China, the supply of drugs is in short supply. At the same time, insulin needs to be injected, which is very inconvenient to use. Use is prone to resistance and side effects; especially in China, there is no drug with independent intellectual property rights that has specific effects on hyperglycemia patients for treatment and prevention

Method used

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  • Method for preparation of transgenic rice preparation and use thereof
  • Method for preparation of transgenic rice preparation and use thereof
  • Method for preparation of transgenic rice preparation and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Cloning of the Gt13a promoter and signal peptide.

[0031] In order to clone the promoter and signal peptide of the prolamin Gt13a gene from the rice genome sequence, use the primers of sequence 1 and 2 (patent application number: 200610019285.9) and standard polymerase chain reaction (PCR), from Taipei 309 variety A DNA fragment of 1284 bases was amplified from the genomic DNA of the plant, and the amplified fragment was digested with restriction endonucleases NaeI and XhoI, and cloned into the vector plasmid pBI221 to produce an expression vector pOsPMP2 specifically expressed in rice endosperm cells. DNA sequence analysis, this DNA fragment has obvious promoter characteristics and sequence of signal peptide.

Embodiment 2

[0032] Example 2: Artificially synthesizing a fusion protein containing optimized rice genetic codes and a target gene.

[0033] The amino acid sequences of the rice Bip gene (registration number AAB63469) and human insulin-like growth factor-1 (registration number CAA01955) of the target gene were obtained from the NIBC database, and the fusion protein gene was extracted using the DNA analysis software Mark vector MacVector. The nucleotide sequence converted into the genetic code of rice is completely identical to the amino acid sequence. These recombinant fusion proteins and target genes were artificially synthesized by Blue Heron Biotechnology Company in the United States. During gene synthesis, Myyl and XhoI restriction sites were added to both ends of the gene, and cloned into the pUC119 (Blue Heron Biotechnology cloning vector, USA) plasmid vector to generate pOsBipC containing rice codon optimization.

Embodiment 3

[0034] Example 3: Construction of vectors for expressing fusion proteins specifically in rice.

[0035] First, the codon-optimized human insulin-like growth factor (human IGF-1) gene was amplified by PCR, cloned into pOsPMP2 plasmid DNA digested with restriction enzymes MscI and XhoI, and transformed into E.coli host cell DH10B, An intermediate plasmid, pOsPMP3, was generated. The pOsPMP3 plasmid DNA was digested with restriction endonucleases NaeI and NcoI, and pOsPMP2 and pOsBipC plasmid DNA were digested with suitable endonucleases at the same time, the fusion protein gene was connected to the expression vector plasmid pOsPMP2, and then transformed into E. coli strain DH10B to generate the expression vector plasmid is pOsPMP26. For the restriction enzyme map of its plasmid, see figure 1 .

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Abstract

The invention discloses preparation for a genetic modified rice formulation and application thereof. The preparation includes steps of A. acquiring specific promoter for rice endosperm and semaphore peptides thereof; B. constructing special expressing carrier and of rice albuminous cell; C. fusing protein with codon of target gene, optimizing, synthesizing with gene; D. fusing with construction of protein expressing carrier pOsPMP26; E genetically transforming rice gene; F. highly expressing selection of genetic modified plant; G. processing seeds of genetic modified rice, and producing into oral tablets and oral granules by pellet mill. And an application of genetic modified rice formulation in preparing medicament for treating or preventing sugar diabete and hyperglycaemia is also disclosed. The inventive method is easy to implement, convenient to operate, low in preparation cost, safe, non-toxic, and has great efficacy.

Description

technical field [0001] The invention belongs to genetic engineering products as oral preparations, and more specifically relates to a method for preparing transgenic rice preparations, obtaining transgenic rice expressing human insulin-like growth factor (IGF-1) in rice seeds, and directly using the transgenic seeds for oral administration Or an oral preparation, the application of the preparation in the treatment or prevention of diabetes and hyperglycemia. technical background [0002] Insulin-like growth factor (Insulin-like growth factor, IGF) is one of the important growth factors involved in many proliferation and metabolic processes, plays an important role in human bone growth, can also promote the maturation of corresponding cells and participate in human trauma of healing. Insulin-like growth factor-1 (Insulin-like growth Factor-I, IGF-I) is a single-chain polypeptide molecule containing 70 amino acids, 3 disulfide bonds, and no glycosylation sites (De Bree, et al...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/62C12N15/82A01H1/00A01H5/12A61K36/899A61P3/10
Inventor 杨代常
Owner WUHAN HEALTHGEN BIOTECHNOLOGY CORP
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