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Method for improving secernment efficiency of recombined protein

A recombinant protein and efficiency technology, applied in the biological field to achieve the effect of improving the efficiency of recombinant protein secretion

Inactive Publication Date: 2008-05-07
WUXI KINGENEW BIOTECHNOLOGY LIMITED COMPANY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the recombinant protein does not enter the lumen of the endoplasmic reticulum containing complex protein complexes during non-canonical secretion, the decrease in secretion efficiency due to transport block caused by steric conformation does not occur

Method used

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  • Method for improving secernment efficiency of recombined protein
  • Method for improving secernment efficiency of recombined protein
  • Method for improving secernment efficiency of recombined protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Construction of Cell Secreted Expression Vector

[0028] 1.1 Construction of classic secretion expression vector of human catalase

[0029] The whole construction process of the classic secretion vector is shown in Figure 1, and the details are as follows:

[0030] Using the EST cDNA clone of Invitrogen Company IMAGE number: 4515735 as a template, and based on the full-length cDNA sequence of the human catalase gene (accession number NM 001752) included in NCBI, primers were designed to amplify the sequence of the gene coding region. NheI and PstI restriction sites were introduced into the two ends respectively, and the stop codon of the gene was deleted. The upstream primer is NheI F: 5'-TTGCTAGCAGATGAAGGTCCTCATCC-3', and the downstream primer is PstI R: 5'-TTCTGCAGCAGATTTGCCTTCTCCCCT-3'. The PCR product was recovered and connected to the pMD19-T vector for sequencing. After the sequence was confirmed to be correct, EcoRI and PstI were used for double diges...

Embodiment 2

[0044] Example 2 CHO cell transfection and expression detection

[0045] 2.1 Cell culture and transient transfection

[0046] CHO cells will be planted in 6-well cell culture plates, and when they are cultured in DMEM medium containing 10% fetal bovine serum to 70-80% confluence, according to the instructions, use Lipofectamine 2000 liposomes to secrete the expression vector psighCAT- EGFP and pSShCAT-EGFP and control empty vector pEGFP-N1 were transferred into the cells.

[0047] 2.2 RT-PCR and Western blot detection

[0048] The transfected CHO cells were cultured in a 37°C incubator for 24 hours, the total RNA was extracted with TRIzol, and the residual DNA was removed with DNase, as a template, after reverse transcription with Oligo dT primers to synthesize cDNA strands, the following pair of primers were used to amplify A 184bp fragment in the human catalase gene was added. Upstream primer: hCAT F 5'-TGCTGAATGAGGAACAGAGG-3'; downstream primer: hCAT R5'-GTGTGAATCGCATTCT...

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Abstract

The invention provides a method for improving the secretion efficiency of recombinant protein, which is to fuse the secretory sequence SS in the homeotic domain of mouse transcription factor Engrailed 2 to the N-terminal of the recombinant protein, the amino acid sequence of the secretory sequence SS For: Ala Gln Glu Leu Ser Leu Asn Glu Ser Gln. The secretion efficiency of the human catalase obtained by the method of the invention is 2.3 times higher than that of the classic secretion guided by the signal peptide of bovine beta casein, which provides an effective solution for improving the secretion efficiency of the recombinant protein in industrial production.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for improving the secretion efficiency of recombinant proteins. Background technique [0002] Humans have used recombinant DNA technology to produce therapeutic pharmaceutical proteins for decades. At present, people mainly use prokaryotic bacterial expression system and eukaryotic yeast and mammalian cell expression system to produce recombinant medicinal protein. Bacteria grow fast, and it is relatively easy to genetically modify them, especially in industrial production, where the cost is much lower than the large-scale culture system of mammalian cells. However, due to some inherent defects of the bacterial expression system, it is difficult to express some complex proteins derived from higher eukaryotes. One of the main reasons is that bacteria lack a perfect post-translational modification system, which easily causes the expressed recombinant protein to form an incor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N15/62C12N15/53A61K38/16A61K38/44
Inventor 何祖勇汤波李宁
Owner WUXI KINGENEW BIOTECHNOLOGY LIMITED COMPANY
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