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Antibiotic peptide buforin II and porcine INF-alpha fusion expression pichia pastoris, and preparation method and applications thereof

A technology of -6his-bufforinii-zeocin and Pichia pastoris, applied in the field of biotechnology, can solve the problems of inability to directly apply, troublesome purification and the like, and achieve the effects of easy purification, high expression amount and simple purification process

Active Publication Date: 2010-06-02
GUANGDONG HINAPHARM PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the use of Escherichia coli to express buforin II has the advantages of short cycle time and low cost of cultivation, there are also disadvantages such as subsequent purification is troublesome and cannot be directly applied.

Method used

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  • Antibiotic peptide buforin II and porcine INF-alpha fusion expression pichia pastoris, and preparation method and applications thereof
  • Antibiotic peptide buforin II and porcine INF-alpha fusion expression pichia pastoris, and preparation method and applications thereof
  • Antibiotic peptide buforin II and porcine INF-alpha fusion expression pichia pastoris, and preparation method and applications thereof

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Experimental program
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Effect test

Embodiment 1

[0042] The construction of Pichia pastoris engineered bacteria expressing the fusion expression of antibacterial peptide buforin II and porcine INF-α and the preparation method of antibacterial peptide buforin II.

[0043] 1 material

[0044] 1.1 Strain and carrier

[0045] See Table 1 for strains and vector genotypes and sources.

[0046] Table 1 Strain and carrier genotype and source

[0047]

[0048] 1.2 Tool enzyme

[0049] RNaseA and Proteinase K were purchased from Shanghai Shenggong Company; restriction endonucleases were purchased from promega; 2 * PCR master Mix was purchased from Beijing Tiangen Biotechnology Company.

[0050] 1.3 Kit

[0051] Small, medium and large extraction plasmid kits, and yeast genome kits are products of QIAGEN.

[0052] 1.4 Medium

[0053] LB liquid medium: 5g of yeast extract; 10g of peptone; 5g of NaCl; add water to make up to 1L.

[0054] LB solid medium: add agar powder at a final concentration of 1.5% (m / v) to the LB liquid m...

Embodiment 2

[0063] An engineering bacterium Pichia pastoris FZM 2009 The method for preparing fusion protein buforin II and INF-α, the steps are as follows:

[0064] A. Material and solution preparation

[0065] The engineering bacterium used is Pichia pastoris FZM provided by the present invention 2009 .

[0066] The engineering bacteria medium is an optimized medium for experimental fermentation, and the mass fraction% of each component is: glucose 4%, ammonia water 150μL, KH 2 PO 4 0.7%, MgSO 4 .7H 2 O, 0.03% FeSO 4 .7H 2 O, 0.05% MnSO 4 .H 2 O0.05%, 0.1% Peptone, pH 5.5, the culture condition is 30°C, add ammonia every 8h, add methanol once every 24h, 0.4% PTM1 (v / v).

[0067] The formula of PTM1 is: copper sulfate (CuSO 4 ) 6g / L, potassium iodate (KI) 0.08g / L, magnesium sulfate monohydrate (MnSO 4 ·H 2 O) 3g / L, sodium molybdate dihydrate 0.2g / L, boric acid (H 3 BO 3 ) 0.02g / L, zinc chloride (ZnCl 2 ) 20g / L, cobalt chloride 0.5g / L, iron sulfate heptahydrate (FeSO 4 ....

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Abstract

The invention discloses an antibiotic peptide buforin II and porcine INF-alpha fusion expression pichia pastoris (Pichia pastoris FZM2009, CCTCC NO:M209259), and a preparation method and applications thereof. The method comprises the steps: A, the construction of expression cassette PAOX1-alpha Factor-IFN alpha-6xHis-bufforin II-Zeocinr; B the construction of expression engineering bacteria Pichia pastoris FZM 2009, wherein the engineering bacteria is resulted from the screening of antibiotics Zeocin and PCR identification; C, the preparation of fusion protein, which comprises the steps: 1) the recovery is implemented on a YPD plate containing 100mug / ml of Zeocin; 2) single clone is inoculated to a YPD-containing culture medium for vibration and culture; 3) 1 volume of culture is inoculated into 10 volumes of a fermentation culture medium for culture; 4) supernatant is obtained by centrifugation; 5) loading buffer with double volume is added into the supernatant, and electrophoresis detection is performed; 6) the supernatant is subject to a molecular sieve gel column to collect components; 7) the collected components are lyophilized to result in fusion protein; 8) buforin II and IFN alpha are obtained by the enzyme digestion of enterokinase on the fusion protein. The fusion protein buforin II and IFN alpha in fusion expression are applied to pig feeds. The method has great feasibility, simple and convenient operation, high expression, low cost and strong bioactivity.

Description

technical field [0001] The present invention relates to the field of biotechnology, and more specifically relates to a Pichia pastoris engineered strain fused to express antimicrobial peptide buforin II and porcine INF-α, and also relates to a preparation method of antimicrobial peptide buforin II and INF-α, antimicrobial peptide buforin II and porcine INF-α INF-α can be widely used in animal feed, and it is a new type of antibacterial drug after further purification. Background technique [0002] In 1996, Park first discovered buforin I in the gastric tissue of the Asian toad, Bufo bufo gargarizans. BuforinI and histone H 2 The N-terminus of A has sequence homology, which is cleaved by pepsin C isozymes Cb and Ca. 2 A's Tyr 39 -Ala 40 from the peptide bond. Buforin II is cleaved from buforin I under the action of intracellular protease Lys-C, it is buforin I from Thr 16 to Lys 36 A potent antimicrobial peptide with a total of 21 amino acid residues, buforin II has st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/62C12N15/81C12P21/02A23K1/16A23K1/18C12R1/84
Inventor 唐志如冯泽猛印遇龙张友明
Owner GUANGDONG HINAPHARM PHARMA CO LTD
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