Low pyrogen staphylokinase and its preparation method

A technology for staphylokinase and raw materials, applied in the field of preparation of staphylokinase, can solve the problems of unreported purity and endotoxin content, low pyrogen, unreported endotoxin content, etc., and achieves good thrombolytic effect, low pyrogen, and overall quality excellent effect

Active Publication Date: 2006-07-26
CHENGDU DIAO JIUHONG PHARMA FACTORY
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AI Technical Summary

Problems solved by technology

[0006] 1. One-step purification using CM-cellulose column chromatography with continuous gradient elution of sodium chloride (Sako TO Overproduction of staphylokinase in Escherichia coli and its characterization. FEBS. 1985, 149 (3): 557-63), SDS-PAGE purity in About 90%, endotoxin content not reported
[0007] 2. Purification by ion exchange chromatography and molecular sieve method (Gerlach D, Kraft R, Behnke DPurification and characterization of the bacterial plasminogen activatorstaphylokinase secreted by a recombinant Bacillus subtilis. Zentralb Bakteriol Mikrobiol Hyg [A]. 1988, 269 (3): 314- 22.), the SDS-PAGE purity is about 90%, and the endotoxin content has not been reported
[0012] 2. CN1307129A, mainly used two-step column chromatography (SP-Sepharose F.F, and Phenyl-Sepharose F.F) to purify, and the purity and endotoxin content were not reported
[0016] The above purification method is only to improve the purity of staphylokinase. For those skilled in the art, for genetically engineered drugs, microorganism-derived drugs and other biochemical drugs, especially for staphylokinase, at the same time, there is no suitable method to achieve high purity and low heat source. method report

Method used

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  • Low pyrogen staphylokinase and its preparation method
  • Low pyrogen staphylokinase and its preparation method
  • Low pyrogen staphylokinase and its preparation method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] The preparation of embodiment one engineering bacteria

[0054] Using the complete DNA of Staphylococcus aureus capable of secreting staphylokinase as a template, primers 1 and 2 were used for PCR amplification: Primer 1 (SEQ ID NO.1): 5'GGT GAA TTC ATG TCA AGT TCA TTC GAC AA3';

[0055] Primer 2 (SEQ ID NO. 2): 5'GGA GGA TCC TTA TTT CTT TTC TAT AAC AA 3'.

[0056] Primer 1 comprises EcoR I site, and primer 2 comprises BamH I site, obtains staphylokinase gene, and the DNA sequence (5' to 3 ') of this staphylokinase gene is shown in Table 1:

[0057] Table 1 DNA sequence of staphylokinase gene (SEQ ID NO.3)

[0058] Tcaagttcat tcgacaaagb aaaatataaa 30

[0059] aaaggcgatg acgcgagtta ttttgaacca 60

[0060] acaggcccgt atttgatggt aaatgtgact 90

[0061] ggagttgatg gtaaaggaaa tgaattgcta 120

[0062] tcccctcatt atgtcgagtt tccttattaaa 150

[0063] cctgggacta cacttacaaa agaaaaaatt 180

[0064] gaatactatg tcgaatggga cttagatgcg 210

[0065] acagcatata aagagtttad agtagttgaa ...

Embodiment 2

[0095] The preparation of embodiment two staphylokinase

[0096] 1. Materials and equipment

[0097] Bacteria-breaking tools: homogenizer;

[0098] Ultrafiltration system: Millipore Pellicon 0.5m 2 ×3, the membrane molecular weight cut off is 5000;

[0099] SDS-PAGE electrophoresis scanner: DS-700Bio-Rad;

[0100] Chromatography column: 50×400mm, 100×400mm Shanghai Yarong Biochemical Instrument Factory;

[0101] Chromatography medium: DEAE-Sepharose F.F, CM-Sepharose F.F, and Q-Sepharose F.F are all products of Pharmacia Biotech, and the particle diameters are all 90 μm.

[0102] Chromatographic detection system: UV detector (HD-93-1 type) Shanghai Jinda Biochemical Instrument Factory.

[0103] 2. Preparation process

[0104] The wet weight obtained from fermentation was 1610g engineering bacteria, and 10000ml pH8.010mM phosphate buffer was used to suspend the bacteria, and 9000ml supernatant was obtained after breaking the bacteria. The r-SAK content of the supernatant ...

Embodiment 3

[0114] The preparation of embodiment three staphylokinase

[0115] 1, material and equipment (with embodiment two)

[0116] 2. Preparation process

[0117] Take engineering bacteria with a wet weight of 800g, suspend the bacteria with 5500ml pH8.010mM phosphate buffer, and obtain 5000ml supernatant after breaking the bacteria. The supernatant is scanned by SDS-PAGE electrophoresis. The r-SAK content is 38.9%, and the protein concentration is 10.40 mg / ml, the total protein content is 52000mg, and the r-SAK content is 20228mg.

[0118] The above supernatant was washed with 15% (NH 4 ) 2 SO 4 After precipitation treatment, ultrafiltration (5KD) was used to desalt, and the medium was converted to pH 8.010mM phosphate buffer. Concentrate to 1000ml, protein concentration is 43.4mg / ml (total protein amount 43400mg).

[0119] DEAE-Sepharose F.F permeation chromatography column: 100×400mm (Shanghai Yarong Biochemical Instrument Factory), DEAE-Sepharose F.F column height 300mm, mo...

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Abstract

This invention belongs to the biology project pharmacy filed, which provides a Staphylokinase (SAK) with low pyrogen and it's preparing method. The Staphylokinase has low pyrogen, high purify and strong activity, which is completely fit for the medicinal demand. This invention includes the following steps: a breaking up the material containing with the Staphylokinase and distilling the floating liquid; b. sieving the product from step a with the DEAD gelatin rob, then removing salt and condensing; c. sieving the product form step b with CM gelatin rob, collecting the sample apex, condensing the washing liquid and transforming the medium; d. sieving the product from step c with the Q gelatin rob to obtain the Staphylokinase. The Staphylokinase of this invention has good bolt dissolving effect, and it is safe and efficient for users..

Description

technical field [0001] The invention relates to the field of bioengineering and pharmacy, in particular to a method for preparing staphylokinase (SAK). Background technique [0002] As we all know, the content of pyrogens in drugs directly affects the safety of drug use, and due to the particularity of their sources, the removal and control of pyrogens has always been a key point in their production and use The key issue. The main pyrogen substance endotoxin is quite stable under high temperature, strong acid and strong alkali. Traditional heating, distillation, filtration, reverse osmosis, activated carbon powder, and various column chromatography methods can only remove or inactivate part of endotoxin . Most methods are difficult to completely remove endotoxin at one time, and even after multiple steps, it still fails to meet clinical requirements. Moreover, due to the extremely heterogeneous properties of endotoxin, it is difficult to find a targeted method for removin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N9/50A61K38/43
Inventor 吴洽庆杨卫华梁波黄坤石金发刘忠荣及元乔王若竹李伯刚
Owner CHENGDU DIAO JIUHONG PHARMA FACTORY
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