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Expression human seralbumin carrier and engineering bacterium

A technology for human serum albumin and yeast expression vectors, applied in the direction of using vectors to introduce foreign genetic material, fungi, and microbial-based methods, can solve the problem of low HSA production and achieve broad application prospects

Active Publication Date: 2006-11-01
NCPC NEW DRUG RES & DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, US5707828 introduced the HSA expression plasmid pHSA413 into Pichia pastoris GS115 to obtain engineering bacteria GS115: pHSA 413-6. The disadvantage is that the engineering bacteria is used to ferment and produce HSA. The HSA yield is low, and the HSA expression level is 3.39. g / L medium supernatant (dry cell weight 101g DCW / L, induced by methanol for 237 hours)

Method used

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  • Expression human seralbumin carrier and engineering bacterium
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  • Expression human seralbumin carrier and engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1, the construction of the vector pAZP-HSA expressing HSA and engineering bacteria Pichia pastoris (Pichia pastoris) HSA75-10 CGMCC No.1360

[0024] 1. Isolation of HSA mRNA from human liver tissue

[0025] Isolation of messenger RNA (mRNA) from livers of accident-dead children. The specific method is as follows:

[0026] Add 210 ml of lysate (4 mol / L thiocyanidine, 0.1 mol / L Tris-HCl, 0.1 mol / L 2-mercaptoethanol, pH 7.5) to 10.5 g of frozen human liver tissue to homogenate. Centrifuge at 10,000 rpm at 4°C for 10 minutes to pellet cell debris. Transfer the supernatant to a new centrifuge tube, add 1 mol / L phosphoric acid 0.04 times the volume of the supernatant and 95% acetic acid 0.5 times the volume of the supernatant, and place at -20°C for 2 hours. Then centrifuge at 7500 rpm at 4°C for 10 minutes. The precipitate was resuspended in 50ml washing solution (6mol / L guanidine hydrochloride, 10mmol / L Na 2 EDTA, 10mmol / L DTT, pH7.0), centrifuged at 5500rpm...

Embodiment 2

[0099] Embodiment 2, the molecular weight determination of the HSA expressed by Pichia pastoris (Pichia pastoris) HSA75-10 CGMCC No.1360

[0100] 1. Purification of HSA expressed by Pichia pastoris HSA75-10 CGMCC No.1360

[0101] Various buffers used in the purification process are shown in Table 2.

[0102] name

Buffer formulation

Solution A

25mmol / L sodium acetate at pH4.5

Solution B

50mmol / L sodium phosphate at pH7.0, 0.1mol / L sodium chloride, 10mmol / L sodium octanoate

Solution C

50mmol / L sodium phosphate at pH6.0, 0.1mol / L sodium chloride

Solution D

50mmol / L sodium phosphate at pH6.0, 0.2mol / L sodium chloride

[0103] (1) Heat treatment of fermentation broth

[0104] 7L of the fermentation supernatant of Pichia pastoris (Pichia pastoris) HSA75-10CGMCC No.1360 prepared according to the method of Example 1 step 7 was filtered with a 0.22um membrane (manufactured by MILLIPORE), and then 2% activated carb...

Embodiment 3

[0119] Example 3, C-terminal and N-terminal sequence determination of HSA expressed by Pichia pastoris (Pichia pastoris) HSA75-10 CGMCC No.1360

[0120] The HSA purified in step 1 in step 2 was submitted to the Shanghai Institute of Biochemistry and Cells, Chinese Academy of Sciences and the State Key Laboratory of Protein Engineering and Plant Genetic Engineering, Peking University for C-terminal and N-terminal sequencing. The C-terminal sequencing results showed that Pichia pastoris ( The C-terminal sequence of HSA expressed by Pichia pastoris) HSA75-10 CGMCC No.1360 is AALGL, and the N-terminal sequencing results show that the N-terminal sequence of HSA expressed by Pichia pastoris (Pichia pastoris) HSA75-10 CGMCC No.1360 is DAHKSEVAHRFKDLG, identical to the C-terminal and N-terminal sequences of HSA purified from human blood.

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Abstract

The invention discloses a carrier and engineering bacteria to express the human serum albumin. The carrier is the plasmid which inserts the encoding human serum albumin sequence into the multi clone locus of the Pichia pastoris expression carrier. The 5'end of the encoding sequence of the serum albumin is connected to the encoding sequence of the secretory signal peptide and the leading peptide which the 5' end is connected to the sequence GAAACG of the AOX1 gene for the Pichia pastoris. The bacteria including the expression carrier especially the Pichia pastoris HSA75-10 CGMCCNo.1360 can be cultured to get the high yield of the human serum albumin (10g / L of the supernate) which is 80% of the secretory protein.

Description

technical field [0001] The invention relates to a carrier and engineering bacteria expressing human serum albumin in the field of biotechnology. Background technique [0002] Human serum albumin (hereinafter referred to as HSA) is the main protein component in plasma, consisting of 585 amino acid residues. Human serum albumin is synthesized in the liver and then secreted into the blood. Its main function is to maintain normal osmotic pressure in the blood, and it also has the function of binding and transporting small molecular substances in the blood. The current HSA preparation is a kind of blood preparation extracted from human plasma. It is mainly used for clinical correction of acute blood volume reduction caused by major surgery, trauma, organ transplantation, etc., and the imbalance of water, electrolyte and colloid caused by various reasons. Prevention and control of shock; also used for acute gastrointestinal bleeding, renal dialysis, and serious chronic diseases, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C12N5/14C12R1/84
Inventor 高健贾茜李梅彦邓建慧
Owner NCPC NEW DRUG RES & DEV
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