Nucleic acid structure and expression carrier for enhancing recombinant protein production and mass-production of recombinant protein

A technology of nucleic acid constructs and recombinant vectors, applied in the direction of hemoglobin/myoglobin, the use of vectors to introduce foreign genetic material, biochemical equipment and methods, etc., can solve problems such as difficulties, achieve the promotion of efficiency, facilitate the cultivation operation, shorten the the effect of time

Inactive Publication Date: 2005-12-28
FENG CHIA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0016] However, to the applicant's knowledge, there are still difficulties in achieving the goal of high yields of recombinant proteins with the techniques currently available in the field of biotechnology

Method used

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  • Nucleic acid structure and expression carrier for enhancing recombinant protein production and mass-production of recombinant protein
  • Nucleic acid structure and expression carrier for enhancing recombinant protein production and mass-production of recombinant protein
  • Nucleic acid structure and expression carrier for enhancing recombinant protein production and mass-production of recombinant protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0110] Example 1. Construction of an expression plasmid comprising the Vitella hyaline hemoglobin structural gene (vgb) and thioredoxin structural gene (trxA)

[0111] Experimental Materials:

[0112] The intermediate cells (intermediate cells) used in the DNA cloning process were Escherichia coli (Escherichia coli) XL1-Blue (Stratagene Co.), and were cultured at 37°C in Luria-Bertani (LB) medium (Miller, J.H. (1972 ), Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), then the transformed Escherichia coli was cultured in the medium supplemented with antibiotics (the dosage of antibiotics was 15 μg / mL for streptomycin, and 15 μg / mL for ampicillin Penicillin was used at 50 μg / mL).

[0113] Vitreoscilla sp. (Murray strain no.389) was donated by Dr. Webster (Department of Biology, Illinois Institute of Technology, Chicago, USA), and cultured at 30°C in a medium containing 1.5% yeast Extract (yeast extract), 1.5% peptone (peptone) and 0.02...

Embodiment 2

[0149] Example 2. Production of thioredoxin, Vitreola hemoglobin, and fusion protein of thioredoxin and Vitiligo hemoglobin with Escherichia coli

[0150] According to the plasmid transformation method described in the above "general experimental method", the recombinant plasmids pGB-VHb, pGB-Trx, pGB-TV1 and pGB-TV2 obtained in Example 1 were respectively transformed into E. coli strain BL21 (DE3) (Novagen Co.), and obtained recombinant strains BL21(DE3) / pGB-VHb, BL21(DE3) / pGB-Trx, BL21(DE3) / pGB-TV1 and BL21(DE3) / pGB-TV2.

[0151] Colonies of each recombinant strain were selected from the solid-state culture dish, inoculated into 5 mL of LB medium containing 15 μg / mL streptomycin, and cultured overnight at 37°C. Afterwards, the overnight cultured cells were inoculated into a 250 mL flask containing 20 mL of LB medium (containing 15 μg / mL streptomycin), and when the initial cell density reached 0.05 (OD 550 ), culture the newly inoculated bacteria solution in a constant tempe...

Embodiment 3

[0160] Example 3. Production of heterologous protein -- human interferon alpha 2 with transformed Escherichia coli

[0161] The plasmid pET-IFN contains a T7 promoter to control the expression of the human interferon α2 structural gene, and its construction method is described below.

[0162] The following two primers were synthesized according to the nucleotide sequence of the human interferon α2 structural gene (E.Austruy et al. (1998), Cancer Gene Ther., 5:247-256):

[0163] forward primer

[0164] 5'-tgctctcatatgttg atctgcctcaaac-3' (SEQ ID NO: 5)

[0165] reverse primer

[0166] 5'-cgtgctcgag ttaattattccttacttcttaaac-3' (SEQ ID NO: 6)

[0167] The above two primers were designed to have cleavage sites for restriction enzymes NdeI and XhoI (marked underlined), respectively.

[0168] Plasmid pIFN2A purified using QIAprep® Spin Miniprep kit (obtained from Professor Xu Zuan of the National Institutes of Health, which contains the human interferon α2 gene introduced into t...

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Abstract

Nucleic acid construction and expression carrier for improving recombinant protein production and production of recombinant protein are disclosed. The process is carried out by cloning first nucleic acid sequence of coded thioredoxin and second nucleic acid sequence of coded ferrohemoglobin into recombinant host cell, reinforcing the recombinant host cell production and helping to release intracellular stress.

Description

technical field [0001] The present invention discloses nucleic acid constructs and expression vectors for improving the production of recombinant polypeptide / protein (recombinantpolypeptide / protein), as well as methods for mass production of recombinant polypeptide / protein, wherein one of the first encoding thioredoxin (thioredoxin) The nucleic acid sequence and a second nucleic acid sequence encoding hemoglobin are cloned into a recombinant host cell, thereby enhancing the productivity of the recombinant host cell to produce a selected gene product and helping the recombinant host cell to alleviate the excess of the gene product Intracellular stress due to production. Background technique [0002] "Production of recombinant polypeptide / protein" can be regarded as a very important genetic engineering technology in the field of biotechnology. Its basic principle is to express a desired gene product (for example, industrial and agricultural enzymes, therapeutic proteins, The ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/805C12N5/10C12N9/02C12N15/63C12N15/67C12N15/70
CPCC12N9/0036C07K14/805C07K2319/00C12N15/67C12N15/70
Inventor 赵云鹏王祉雯陈柏庭陈裕星
Owner FENG CHIA UNIVERSITY
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