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Method of expressing human apolipoprotein ApoA I inside Pichia yeast cell

A technology of apolipoprotein and intracellular expression, applied in the field of bioengineering

Inactive Publication Date: 2005-07-13
FUDAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no reports on the expression of rApoAI by Pichia pastoris at home and abroad

Method used

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  • Method of expressing human apolipoprotein ApoA I inside Pichia yeast cell
  • Method of expressing human apolipoprotein ApoA I inside Pichia yeast cell
  • Method of expressing human apolipoprotein ApoA I inside Pichia yeast cell

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Experimental program
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Effect test

Embodiment 1

[0023] Embodiment 1, Construction and Identification of Recombinant Expression Plasmids

[0024] Using the plasmid pPIC9k-apoA I with the artificially synthesized gene apoA I as a template, primers were designed for PCR amplification. Forward primer: 5'-GGGGATCCAAACGATGGATGAACCACCTCAGTCTCCATGG-3', BamH I site was introduced at the 5' end, reverse primer: 5'-GCAAATGGCATTCTGACATCC-3'. The PCR reaction conditions were set according to conventional settings. The PCR product was double-digested with EcoR I and BamH I, and the intracellular expression plasmid pPIC3.5k was also double-digested with EcoR I and BamH I, followed by ligation and transformation. The recombinant expression plasmid pPIC3.5k-apoA I was extracted from the transformant, identified by enzyme digestion and DNA sequence determination, which proved that the construction of the recombinant expression plasmid was completely correct.

Embodiment 2

[0025] Example 2, Electrotransformation of recombinant expression plasmid Pichia GS115

[0026] Inoculate Pichia yeast GS115(his4) in YPD medium, shake culture at 28-30°C until OD600=1.3-1.5, centrifuge, collect the bacteria, wash once with pre-cooled sterile water and 1mol / L sorbitol, Finally, 200 μl of 1mol / L sorbitol was used to suspend to obtain GS115 competent cells. Take 80 μl of GS115 competent cells and mix them with 5 μg of recombinant expression plasmid digested with Bgl II, transform by electric shock under the conditions of 1300V, 25μF, 200Ω, suspend with 1ml of 1mol / L sorbitol, spread the RDB plate, and incubate at 28-30℃ After 3-4 days, more than 1400 transformants were obtained as a result.

Embodiment 3、G418

[0027] Example 3, Screening of G418 highly resistant yeast transformants

[0028] More than 1,400 colonies of yeast transformants grown on RDB plates were planted on YPD plates with G418 concentrations of 1.5, 3.0, and 4.0 mg / ml, cultured at 28-30°C, and G418-resistant strains were screened step by step. Results 27 strains were obtained on the plate containing 4mg / ml G418.

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Abstract

The present invention belongs to the field of biological engineering technology, and is especially method of expressing human apoolipoprotein ApoA I inside Pichia yeast cell. Artificially synthesized ApoA I gene is inserted into cell to express plasmid pPIC3.5k, Pichia yeast strain GS115 is electric shock introduced, and after shake flask fermentation and methanol induction, there is obvious rApoA I protein expression inside yeast cell with the molecular weight identical with that of ApoA I extracted from human blood plasma.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a method for expressing human apolipoprotein ApoA I. Specifically, the rApoA I protein is highly expressed in yeast cells by constructing a recombinant engineered strain of Pichia pastoris through shake flask fermentation and induction. Background technique [0002] Human apolipoprotein ApoA I (apolipoprotein AI) is composed of 243 amino acid residues and has a molecular mass of 28.3KD. It is mainly synthesized in the liver and is an important component of high-density lipoprotein (high-density lipoprotein, HDL) and also the function of HDL. the main executor. ApoA I plays an important role in the transport and metabolism of cholesterol, anti-atherosclerosis, anti-endotoxin, anti-inflammation, anti-virus and inhibition of tissue damage caused by acute phase inflammatory response, so it has become one of the focuses of lipid metabolism research one. [0003] Current stud...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/87C12P21/02
Inventor 宋大新赵志安杨婷婷张淼张彦
Owner FUDAN UNIV
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