Method for preparation of transgenic rice preparation and use thereof

A technology for transgenic rice and rice, which is applied in the fields of botanical equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve the problems of low expression, inconvenient use, high price, etc., and achieves low cost, convenient use and practicality. strong effect

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

Due to the use of recombinant DNA technology to express recombinant human granulocyte macrophage colony-stimulating factor in bacteria and yeast, there are consistent problems of low expression levels, and the need for purification and other steps, so the

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

[0020] Example 1: Cloning of rice endosperm-specific promoter and signal peptide

[0021] In order to obtain a strong promoter and signal peptide for the specific expression of rice endosperm, according to the research of proteomics, it was found that the protein expression level of Gt13a gene, a member of the prolamin gene family cluster of rice storage protein, was expressed in rice endosperm cells. As the highest, it is deduced that this gene member has a strong promoter activity. In order to obtain the Gt13a promoter and its signal peptide sequence, a pair of nucleotide primers 5'-ccaagcttcaacctgctgagaagaacaactgac-3' and 5'-cggtgccggc tagagagccattgcacaagag-3' were synthesized according to Gt13a in the gene database (GenBankAccession No. AP003256) for PCR Amplification. In order to facilitate gene cloning, a HindIII restriction site was added to the 5' end of the forward primer, and a NaeI restriction site was added to the 5' end of the reverse primer. Genomic DNA was ext...

Embodiment 2

[0022] Example 2: Synthesis of human GM-CSF gene with rice genetic code optimization.

[0023]The amino acid sequence of the mature GM-CSF gene (GenBank accession No.CAA01491) was obtained from the gene bank of the American Center for Biotechnology Information (NCBI), and the GM-CSF (GenBank accession No. The amino acid sequence of the CAA01491) gene was converted into a nucleotide sequence containing optimized rice genetic codes, and a human GM-CSF gene (SEQ ID NO: 1) was artificially synthesized. The nucleotide sequence of human GM-CSF gene changed by 25.5% after codon optimization. The genetic code changed by 71.1%. Restriction endonuclease Myyl and XhoI were respectively added to the two ends of the artificially synthesized GM-CSF gene, and then cloned into the pUC19 vector (ClontechInc company in the United States), resulting in a carrier with the recombinant GM-CSF gene; Digested with endonuclease Myyl and XhoI, a blunt end was produced at the 5' end and a sticky end w...

Embodiment 3

[0024] Example 3 Constructing a rice-specific expression vector of GM-CSF.

[0025] The vector of the recombinant GM-CSF gene; digested with restriction endonuclease Myyl and XhoI, a blunt end and a cohesive end were produced at the 5' end, and the vector plasmid was digested with NaeI and XhoI at the same time pOsPMP1 (patent application number: 200610019285.9), also produced a blunt end at the 5' end and a sticky end at the 3' end, separated by agarose gel electrophoresis, recovered the GM-CSF gene fragment, and recombined The fragment of the GM-CSF gene was connected to the vector plasmid pOsPMP2 digested by NaeI and XhoI, and then transformed into Escherichia coli strain DH10B to produce the vector plasmid pOsPMP7 ( figure 1 and sequence 1). The pOsPMP1 plasmid DNA was digested with restriction endonucleases Myyl and XhoI, while the pOsPMP1 plasmid DNA was digested with NaeI and XhoI, the GM-CSF gene fragment was ligated with the vector plasmid pOsPMP1 digested by NaeI an...

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Abstract

The invention discloses preparation for a genetic modified rice formulation and usage thereof. The preparation includes steps of: A. acquiring specific promoter for rice and semaphore peptides thereof; B. obtaining human GM-CSF gene of rice's genetic codon; C. genetically transforming rice gene and highly expressing selection of genetic modified plant; D. processing seeds of genetic modified rice. An application of genetic modified rice seed as medicament for treating or preventing leucocytopenia is also disclosed. The invention is simple in process, low in cost, good in safety property and curative effect, and has no side effect. Because recombinant rhGM-CSF is stored in rice flour, and rice is main food of human, any purification steps are not needed in tablet preparation, and the tablet contains no pathogenic bacteria and virus of human, the invention is safe and reliable. The tablet has functions to stimulate proliferation of leukocyte, improve immunologic competence in vivo, recover lowered immunologic competence resulted by reduction of leucopenia induced by chemotherapy and radiotherapy of cancer.

Description

technical field [0001] The invention belongs to genetic engineering products in the field of biomedicine, and more particularly relates to a method for preparing transgenic rice seeds, which utilizes a rice endosperm-specific expression expression platform to efficiently express human macrophage colony-stimulating factor (rhGM) in the rice seeds. -CSF), and then the transgenic rice seeds are directly used as oral agents for the treatment of leucopenia (Leucopenia) caused by chemotherapy and radiotherapy for cancer, autologous and allogeneic bone marrow transplantation, and aplastic anemia and bone marrow failure caused by scald and burns. Thrombocytopenia, leukemia relief in the treatment of leukemia, anemia and cancer, among other drugs. Background technique [0002] Human granulocyte macrophage colony stimulating factor (Granulocyte Macrophage Colony Stimulating Factor, GM-CSF) gene consists of 127 amino acids, is a small amount of secreted protein in the human body, granu...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/27C12N15/82A01H1/00A01H5/12A61K36/899A61K9/20A61K9/48A61K9/14A61P7/00
Inventor 杨代常
Owner WUHAN HEALTHGEN BIOTECHNOLOGY CORP
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