Mutant Alfimeprase engineering strain, and preparation method and applications thereof

A technology of engineering strains and plasmin, applied in the biological field, can solve problems such as oxidation, re-embolization, and limited sources of raw materials, and achieve high activity and stable strains

Active Publication Date: 2012-10-10
ANHUI FENGYUAN PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The efficacy of these drugs has been clinically proven to be positive, but there are still many problems, such as bleeding, body oxidation and re-embolization, limited sources of raw materials, etc.

Method used

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  • Mutant Alfimeprase engineering strain, and preparation method and applications thereof
  • Mutant Alfimeprase engineering strain, and preparation method and applications thereof
  • Mutant Alfimeprase engineering strain, and preparation method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Using the amino acid sequence (GenBank No.P28891) of the venom Fibrolase protein published by the National Center for Biotechnology (NCBI) in the United States, the EQR at its N-terminal was mutated into serine S, and its amino acid sequence was shown in SEQ ID No. .1 shown. Combined with the codon bias of eukaryotic organisms, the nucleotide sequence is designed, and the nucleotide sequence is shown in SEQ ID No.2.

[0029] Four rounds of PCR reactions were used to obtain the gene fragment encoding Alfimeprase containing the required restriction enzyme site, His×6 tag, and EK site. The PCR schematic diagram is as follows figure 1 shown. The reaction system used in each round of PCR is as follows:

[0030] The first round of PCR with SEQ ID No.3 and SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8, SEQ ID No.9 and SEQ ID No. .10, SEQ ID No.11 and SEQ ID No.12, SEQ ID No.13 and SEQ ID No.14, SEQ ID No.15 and SEQ ID No.16 are primers for PCR amplif...

Embodiment 2

[0091] according to Figure 4 The method shown was used to construct the expression vector pPic9K-His-Alf. The pBS-Alf constructed in Example 1 and the expression vector pPIC9K were digested with EcoR I and Not I ( Figure 5 ), purify and collect the digested fragments, and connect them to obtain the recombinant expression vector pPIC9K-His6-Alf. The double digestion and connection system are as follows:

[0092] Enzyme digestion reaction system 1: Enzyme digestion reaction system 2:

[0093] pBS-Alf plasmid 10μl pPIC9k plasmid 10μl

[0094] EcoR I (12u / μl) 2.0μl EcoR I (12u / μl) 1.5μl

[0095] Not I (10u / μl) 2.0μl Not I (10u / μl) 1.5μl

[0096] 10x Digestion Buffer 6μl 10x Digestion Buffer 6μl

[0097] 10xBSA 6μl 10xBSA 6μl

[0098] Sterile water 34μl Sterile water 34.5μl

[0099] Total volume 60μl Total volume 60μl

[0100] Connection reaction system:

[0101] Recover 1.5 μl of purified pPIC9K plasmid

[0102] Recover and purify the target fragment His6-Alf 5 μl

[...

Embodiment 3

[0108] Shake flask culture in BMG / MY liquid medium, liquid volume 100ml, shaker speed 250rpm, when the OD600 value reaches 1-1.5, add sterile methanol 1-2ml to induce expression, then add once every 24 hours . Sampling was carried out continuously for electrophoresis determination, and cultured for 5 days.

[0109] Collect the fermentation broth, centrifuge at 10,000 rpm, concentrate in vacuum at low temperature, gradually add ammonium sulfate to the concentrated solution to a final concentration of 50%, and keep stirring gently, centrifuge the precipitate at 3,000 rpm, remove the supernatant, and redissolve the precipitate with physiological saline. The solution was purified according to the operating instructions of the His-Bind protein purification kit from Novagen. The purified protein solution was collected, cut with enterokinase EK, precipitated with ammonium sulfate again, centrifuged, precipitated and dialyzed for redissolution to obtain active Alfimeprase.

[0110] ...

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Abstract

The invention relates to a preparation method of a recombinant viper venom protein. A mutant Alfimeprase gene of artificially synthesized Agkistrodon contortrix is utilized, and the gene has the nucleotide sequence shown as SEQ ID No.2. The gene is linked to yeast expression plasmids pPIC9K to construct a high efficiency expression vector pPIC9K-His-Alf, a Pichia yeast strain GS115 is introduced,and the vector is named as FY-Alf. Induced expression is carried out with methanol, the expressed recombinant protein is purified and then subjected to activity verification by a fibrin plate assay, and the result shows that the recombinant protein Alfimeprase expressed by the yeast strain GS115 containing pPIC9K-His-Alf plasmids has high fibrinolytic activity.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a mutant snake venom fibrinolytic enzyme engineering bacterium, its preparation method and application. The mutant protein has better fibrinolytic activity but no hemorrhagic activity. Background technique [0002] Alfimeprase (ALF) is a zinc metalloprotease developed through gene recombination technology. It is an N-terminal mutant of fibrolytic enzyme Fibrolase (the first three amino acids EQR are replaced by Ser), and it is a protein containing 201 amino acids. The zinc single-chain polypeptide has three pairs of disulfide bonds inside the active molecule, and has high homology with other reported zinc metalloproteases. Alfimeprase and Fibrolase have the same fibrinolytic activity, and can be used to treat Peripheral Arterial Occlusive (PAO), which is common and high incidence in the elderly population. Alfimeprase can directly act on fibrin or fibrinogen α-chain to degrade it in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/81C12N15/57C12N9/68
Inventor 黄飞盛太奎鞠岚岚余宏燕
Owner ANHUI FENGYUAN PHARM CO LTD
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