Preparation of new apolipoprotein A-II

A technology of apolipoprotein and A-II, which is applied in the preparation method of apolipoprotein and peptide, animal/human protein, etc., to achieve the effects of low production cost, stable expression system and stable strain

Inactive Publication Date: 2010-12-29
吉林圣元科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there has been no report on the production of human apolipoprotein A-II using Pichia pastoris

Method used

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  • Preparation of new apolipoprotein A-II
  • Preparation of new apolipoprotein A-II
  • Preparation of new apolipoprotein A-II

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Cloning and amplification of human apolipoprotein A-II gene (hApoA-II)

[0043] (1) Trizol method to extract total RNA from human liver tissue

[0044] The freshly isolated human liver tissue was cut into 100 mg size and immediately frozen in liquid nitrogen. Total RNA was extracted from tissues as follows. Take out 300 mg of frozen tissue and put it into a mortar filled with liquid nitrogen, and crush the tissue. Transfer the crushed tissue into a 50ml centrifuge tube, add about 5ml Trizol, and homogenize with a homogenizer at high speed for 15-30s at room temperature. Add 1.0ml of chloroform (200μl / ml Trizol), vortex fully, and place at room temperature for 5min. After centrifugation (12000r / min) at 4°C for 15min, the upper aqueous phase was transferred to another centrifuge tube. Add an equal volume of isopropanol, shake well, precipitate at room temperature for 10 minutes, centrifuge at 4°C (12000r / min) for 15 minutes, and discard the supernatant. Ad...

Embodiment 2

[0080] Example 2: Construction of hApoA-II Pichia secretory expression vector pPICZa / hApoA-II and host cell transformation

[0081] Take 50ng of the plasmid carrying the hApoA-II gene correctly identified by the above sequencing, and set up a PCR reaction system in a 0.2ml EP tube according to the above ratio. Using the above PCR reaction system and conditions, with the help of the upstream primer 5'-AACCTCGAGAAGAGACAGGCAAAGGAGCCATGT-3' (SEQ ID NO: 3) to introduce the partial sequence of the yeast a factor signal peptide and the XhoI site, and using the downstream primer: 5'-GCGTCTAGATCACTGGGTGTTGAGCTTCTTAG-3 '(SEQ ID NO: 4) introduces an EcoR I site. After introducing the above sequences and restriction sites by PCR, recover the target fragment. After cutting with the corresponding enzymes, the plasmid pPICZa cut with the same enzymes was connected to construct the Pichia pastoris expression vector pPICZa / hApoA-II, and then transformed into Escherichia coli, the plasmid was ...

Embodiment 3

[0086] Embodiment 3: Expression and purification of ApoA-II polypeptide

[0087] (1) Expression of recombinant human apolipoprotein A-II (rhApoA-II)

[0088] Inoculate 10 ml of BMGY (pH 6.0) medium with positive clones from the above identification results, and incubate with shaking at 30°C for 24 hours until OD 600 Cells were collected when reaching 2.0-6.0. Resuspend the cell pellet with an equal volume (10ml) of BMMY (pH6.0), culture with shaking at 30°C, and induce expression. During the induction process, methanol was supplemented every 24 hours to a final concentration of 0.5%, and sterilized ultrapure water was supplemented at the same time to keep the total volume of the fermentation broth constant. At the 0, 24, 48, 72, 96, 120, 144, and 168 hours of culture, take 0.5ml of fermentation broth, centrifuge and take the supernatant for protein analysis (SDS-PAGE, N-terminal amino acid sequence analysis, etc.) ).

[0089] (2) Purification of rhApoA-II

[0090] The fer...

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Abstract

The invention relates to a recombinant protein and a production, purification and identification method thereof, in particular to a recombinant human apolipoprotein A-II (rhApoA-II) expressed in saccharomyces pastorianus, and the optimized method for high-density fermentation, production and purification of the recombinant human apolipoprotein A-II.

Description

field of invention [0001] The present invention relates to recombinant protein and its preparation, in particular to the expression of recombinant human apolipoprotein A-II (rhApoA-II) in Pasteur yeast, and the optimized high-density fermentation method for producing recombinant human apolipoprotein A-II . Background of the invention [0002] Apolipoprotein A-II (apolipoprotein A-II, ApoA-II) is synthesized by the liver and small intestine, and the half-life of ApoA-II in human plasma is 4.4 days, which is the second main carrier in serum high-density lipoprotein (HDL). Lipoproteins make up about 20% of the total protein in HDL. Its content in milk microparticles (CM) can reach 7% to 10% of the total apolipoprotein, and there is also a small amount of ApoA-II in very low-density lipoprotein (VLDL), and the concentration of ApoA-II in plasma is 0.35% ~0.5g / L (Luo Jingcong, Liu Bingwen, Lan Tianhe. Separation, purification and identification of human plasma apolipoprotein A-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12P21/02C07K14/775C07K1/18C07K1/20C12R1/84
Inventor 颜炜群苏曼曼
Owner 吉林圣元科技有限责任公司
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