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Method and uses for expressing polypeptide in endosperm using cereal non-storage protein as fusion vector

A non-storage, carrier technology, applied in animal/human proteins, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of low expression, no biological activity, poor solubility, etc., to solve the problem of low expression , the effect of preventing pollution

Active Publication Date: 2006-12-27
WUHAN HEALTHGEN BIOTECHNOLOGY CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The invention not only effectively solves the problems of low expression, poor solubility, and no biological activity of other expression systems, but also can completely eliminate the pollution of animal pathogens

Method used

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  • Method and uses for expressing polypeptide in endosperm using cereal non-storage protein as fusion vector
  • Method and uses for expressing polypeptide in endosperm using cereal non-storage protein as fusion vector
  • Method and uses for expressing polypeptide in endosperm using cereal non-storage protein as fusion vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] [Example 1] Cloning of Gt13a promoter and signal peptide

[0057] In order to clone the prolamin Gt13a gene promoter and signal peptide from the rice genome sequence, using the primer sequence 1, using the standard polymerase chain (PCR) reaction, amplified 1284 bases from the genomic DNA of the Taipei 309 variety The amplified DNA fragment was digested with restriction endonucleases NaeI and XhoI, and cloned into the vector plasmid pBI221 to generate the expression vector pOsPMP2 specifically expressed in rice endosperm cells, see figure 1 , through DNA sequence analysis, this DNA fragment has obvious promoter characteristics and sequence of signal peptide (SEQ ID NO.1).

Embodiment 2

[0058] [Example 2] artificially synthesized a fusion protein containing optimized rice genetic codes and a gene of interest.

[0059] The amino acid sequences of the rice Bip gene (registration number: AAB63469), wheat PDI gene (registration number: CAI30637) and the target gene human insulin-like growth factor-1 (registration number: CAA01955) were obtained from the NIBC database , using the DNA analysis software Mark Vector Mac Vector to convert the fusion protein gene into the nucleotide sequence of the rice genetic code, the optimized fusion protein and the deoxynucleotide sequence and genetic code change results of the target gene are shown in Table 1, but Their amino acid sequences are identical. These recombinant fusion proteins and target genes were artificially synthesized by Blue Heron Biotechnology Company in the United States. In the process of gene synthesis, Myyl and XhoI restriction sites were added to both ends of the gene, and cloned into pUC119 (Blue Heron B...

Embodiment 3

[0060] [Example 3] Construction of a rice-specific expression vector for the fusion protein.

[0061] 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. pOsPMP3 plasmid DNA is digested with restriction endonuclease NaeI and NcoI, pOsPMP2 (see SEQ ID NO.2), pOsBipC and pOsPDIC plasmid DNA are digested with suitable endonuclease simultaneously, fusion protein gene is connected with expression vector plasmid pOsPMP2, Escherichia coli strain DH10B was then transformed to produce expression vector plasmids pOsPMP25 and pOsPMP26. For the restriction enzyme map of its plasmid, see figure 2 and image 3 .

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Abstract

The invention discloses a method of using corn non-storage protein as molten carrier in endosperm expression polypeptide and its application, which includes: first obtaining rice astopic promoter and signal peptide; secondarily constructing rice albuminous cell specific expression carrier; thirdly using rice preference codon to prepare rice non-storage protein Bip gene C-end which is used as molten carrier, wheat PD1 gene C end and para-insulin growth factor-1 gene; fourthly constructing expression fusion protein carrier; fifthly obtaining genetically modified rice and barley plant with more than 0.3 % seed dry weight of their fusion protein expression. The invention is of simple process, convenient operation and low cost, and can be used in corn endosperm to express various polypeptides.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and more specifically relates to a method and application for expressing polypeptides in endosperm by using grain non-storage proteins as fusion proteins, that is, using endosperm cells of grain crops such as rice and barley as bioreactors, and using fusion protein technology strategy, the endosperm cell-specific fusion protein expression vector is introduced into rice and barley cells through transgenic methods, and the method of specifically expressing and accumulating fusion proteins in the endosperm cells of transgenic rice / barley and the plant-derived protein produced by this method peptide. Background technique [0002] Small molecular polypeptides usually refer to polypeptides with an amino acid sequence of less than 100. In recent years, they have been widely used in medicine, disease treatment, and molecular vaccines. These include antibody su...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N15/82C12N15/62C12N15/66C12N15/12
CPCC12N15/8257C07K14/65
Inventor 杨代常谢婷婷
Owner WUHAN HEALTHGEN BIOTECHNOLOGY CORP
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