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Process for preparing human apolipoprotein A-I

An apolipoprotein and protein technology, applied in the field of bioengineering, can solve problems such as difficulty in separation and purification, and achieve the effect of high-efficiency secretion and expression

Inactive Publication Date: 2005-03-16
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Difficulty in separation and purification due to intracellular expression

Method used

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  • Process for preparing human apolipoprotein A-I
  • Process for preparing human apolipoprotein A-I
  • Process for preparing human apolipoprotein A-I

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Obtaining recombinant apoA-I expression plasmid

[0031] According to the apoA-I gene sequence, the expression plasmid DNA of the insect expression system was used as a template to amplify the natural fragment by PCR, and Xhol I and EcoR I restriction sites were set at the N-terminus and C-terminus respectively, and the above-mentioned apoA-I gene fragment The expression plasmid was obtained by inserting into the P. pastoris secreted expression vector through Xhol I and EcoRI double digestion.

Embodiment 2

[0032] Example 2 Expression plasmid electrotransformation Pichia pastoris GS115

[0033]The constructed expression plasmid pPIC9k-apoA-I was digested with BglII and linearized. The host bacteria P.pastorisGS115 (his mut+) was cultured to an OD of 1.6-1.8 to prepare electrocompetent cells, mixed with the above plasmids, and then electrotransformed with GIBCOL BRL electrotransformer CELL-PORATOR, where the voltage was 1500V, the capacitance was 50μF, and the resistance was 4kΩ , add 0.5ml 1mol / L cold sorbitol to the transformation cup, take 200μl and smear it on the MD plate, culture at 30℃ until a single colony appears, and more than 1000 transformants are obtained as a result.

Embodiment 3

[0034] Example 3 Screening of highly resistant Pichia recombinant strains

[0035] Spread more than 1000 transformants on YPD plates containing G418 at 1mg / ml, 2mg / ml, 3mg / ml, and 4mg / ml in turn, and culture them at 28-30°C. As a result, 22 transformants were obtained on plates containing 4mg / mlG418 Turn.

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Abstract

The invention concerns bioengineering technology field. The invention relates to a method for preparing human apoprotein A-I(ApoA-I), especially a highly effective method of obtaining human apoprotein A-I(ApoA-I) through using Pichia pastoris system, constructing recombinant engineered bacterium, expressing ,separating and purifying. The invention recombines natural apoA-I gene to P.pastoris host bacterium by gene engineering approach. Expressed product amount is highly to 200mg / L. The expressed products of electrophoresis grade purity ApoA-I protein is obtained through cold acetone fractional precipitation and sepharose separation and purification. The expressed protein are identical with human blood derived ApoA-I protein after western-blot identification, protein spectrum detection, N-terminal amino acid sequencing and cell associativity determination.

Description

technical field [0001] The invention belongs to the technical field of bioengineering and relates to a method for preparing human apolipoprotein A-I. Specifically, it relates to a method for efficiently producing human apolipoprotein A-I by constructing recombinant engineering strains, expressing, separating and purifying using the Pichia pastoris system. Background technique [0002] High-density lipoprotein (high density lipoprotein, HDL) is an important lipoprotein in plasma. HDL participates in the reverse transport of cholesterol, transports cholesterol from extrahepatic cells to the liver, transforms and clears it, and thus has anti-atherosclerotic effects . In addition, HDL also has the functions of anti-inflammation, anti-endotoxin, promoting cell division, linking lipopolysaccharide, improving abnormal contraction of arteries, reducing the secretion of epidermal factor by vascular endothelial cells, stimulating endothelial cells to synthesize prostacyclin and prolo...

Claims

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Application Information

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IPC IPC(8): C07K1/14C12N1/19C12N15/12C12N15/63C12N15/81C12P21/02
Inventor 吴满平宋大新周佩钟江赵志安
Owner FUDAN UNIV
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