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Production of recombinant human serum albumin with rice-embryo milk cell as biological reactor

A technology of human serum albumin and endosperm cells, which is applied in the field of genetic engineering, can solve the problems of no biological activity, poor solubility, and low expression level, and achieve the effects of safe and reliable products, low production costs, and easy storage and processing

Active Publication Date: 2009-09-16
WUHAN HEALTHGEN BIOTECHNOLOGY CORP
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0004] Aiming at the shortcomings of existing prokaryotic and eukaryotic host bioreactors, such as low expression, poor solubility, no biological activity and unsafety, the inventors used the protein body of rice endosperm cells, one of the main foods for human beings, as a recombinant The storage site of the protein uses the rice endosperm-specific promoter and signal peptide to mediate recombinant human serum albumin into the endomembrane system of rice endosperm cells and store it in the protein body of rice endosperm, so that recombinant human serum albumin Proteins can accumulate in large quantities in rice seeds and eventually reach a higher level. The present invention can not only overcome the problems of low expression, poor solubility, and no biological activity in other expression systems, but also completely eliminate the pollution of animal pathogens

Method used

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  • Production of recombinant human serum albumin with rice-embryo milk cell as biological reactor
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  • Production of recombinant human serum albumin with rice-embryo milk cell as biological reactor

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Embodiment 1

[0032] [Example 1] Cloning of Gt13a promoter and signal peptide

[0033] 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 base DNA fragment ( figure 1 ), the amplified fragment was digested with restriction endonucleases NaeI and XhoI, cloned into the vector plasmid pBI221, and the expression vector pOsPMP1 specifically expressed in rice endosperm cells was produced. Through DNA sequence analysis, this DNA fragment has an obvious promoter Sequence of signature and signal peptide (SEQ ID NO: 3).

Embodiment 2

[0034] [Example 2] Synthesis of human serum albumin gene containing rice genetic code.

[0035] The amino acid sequence of the human serum albumin gene sequence (gene bank accession number is CAA01491), using the DNA analysis software Mark vector MacVector to convert the human serum albumin gene into the nucleotide sequence of rice genetic codes, the modified human serum albumin The deoxynucleotide sequence of the protein gene has changed by 25.5%, and the genetic code has changed by 71.1%, but its amino acid sequence is completely the same. Then, according to the codon-optimized deoxynucleotide sequence, the recombinant human serum albumin gene was artificially synthesized by PCR extension method. During gene synthesis, Myyl and XhoI restriction sites were added to both ends of the gene, and cloned into the pUC19 plasmid vector to generate the rice codon-optimized human serum albumin gene (pOsHSA).

Embodiment 3

[0036] [Example 3] A rice-specific vector for expressing human serum albumin was constructed.

[0037] The pOsHSA plasmid DNA was digested with restriction endonucleases Myyl and XhoI, and the pOsPMP1 plasmid DNA was digested with NaeI and XhoI at the same time, the human serum albumin gene was connected to the vector plasmid pOsPMP1, and then transformed into E. coli strain DH10B to generate the expression vector plasmid pOsPMP2. For the restriction enzyme map of its plasmid, see figure 2 .

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Abstract

Production of recombinant human serum albumin with rice albuminous cell as biological reactor is carried out by taking rice albuminous cell protein as storage site for recombinant protein, taking rice albuminous specific expression starter and signal peptide, entering inductive recombinant human serum albumin into inner-film system of rice serum albumin cell and storing it in rice albuminous protein. It is cheap, has more yield and higher expression.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a method for producing medical recombinant human serum albumin by using corn seed rice endosperm cells as a bioreactor. That is, rice is used as the host and endosperm cells are used as the bioreactor. Through DNA recombination technology, the vector specifically expressing human serum albumin in rice endosperm cells is introduced into rice cells, and human serum albumin is specifically expressed and accumulated in large quantities in the endosperm cells of transgenic rice. A method for serum albumin and recombinant human serum albumin of plant origin produced by the method. Background technique [0002] In the 1950s, the production of major pharmaceutical products used bacteria as bioreactors. However, because bacteria belong to prokaryotes, they do not have the protein synthesis and processing system of eukaryotes. However, the biological activity of some proteins i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/10C12N15/14C12N15/82C12N15/65A01H4/00C07K14/765
Inventor 杨代常
Owner WUHAN HEALTHGEN BIOTECHNOLOGY CORP
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