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Mutant venin fibrinolytic enzyme gene and preparation method thereof

A technology of fibrinolytic enzyme and gene, applied in the field of mutant snake venom fibrinolytic enzyme gene and its preparation, can solve the problems of re-embolization, oxidation, and limited sources of raw materials, and achieve the effect of eliminating cumbersome steps and being easy to obtain

Active Publication Date: 2012-09-05
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 venin fibrinolytic enzyme gene and preparation method thereof
  • Mutant venin fibrinolytic enzyme gene and preparation method thereof
  • Mutant venin fibrinolytic enzyme gene and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Utilizing the amino acid sequence of the fibrolase protein (GenBank No.P28891) published by the American National Center for Biotechnology (NCBI) database, the QQR at its N-terminus was mutated into serine S, and its amino acid sequence is 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.

[0025] 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:

[0026] 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 amplification ...

Embodiment 2

[0086] 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:

[0087] Enzyme digestion reaction system 1: Enzyme digestion reaction system 2:

[0088] pBS-Alf plasmid 10μl pPic9k plasmid 10μl

[0089] EcoR I (12u / μl) 2.0μl EcoR I (12u / μl) 1.5μl

[0090] Not I (10u / μl) 2.0μl Not I (10u / μl) 1.5μl

[0091] 10x Digestion Buffer 6μl 10x Digestion Buffer 6μl

[0092] 10xBSA 6μl 10xBSA 6μl

[0093] Sterile water 34μl Sterile water 34.5μl

[0094] Total volume 60μl Total volume 60μl

[0095] Connection reaction system:

[0096] Recover 1.5 μl of purified pPIC9K plasmid

[0097] Recover and purify the target fragment His6-Alf 5 μl

[...

Embodiment 3

[0103] Shake flask culture in BMG / MY liquid medium (recipe: 1% yeast extract, 2% peptone, 100mM potassium phosphate pH6.0, 0.00004% biotin, 1% glycerol, 0.5% methanol.), liquid volume 100ml, shaker speed 250rpm, when the OD600 value reaches 1-1.5, add sterile methanol 1-2ml to induce expression, and then add once every 24 hours. Sampling was carried out continuously for electrophoresis determination, and cultured for 5 days.

[0104] 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 ...

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Abstract

The invention relates to a synthesis method of a mutant venin fibrinolytic enzyme gene, in particular to a full-length target gene segment prepared by the segmented synthesis of target genes and the artificial synthesis of the overlapping polymerase chain reaction (PCR) technology. The amino acid sequence (GenBank No.P28891) which is published by the national center for biotechnology information (NCBI), of the venin Fibrolase protein of the Agkistrodon contortrix is utilized to design 14 segments of amino acid sequences of which average length is 60bp, wherein the sequences are shown in SEQ ID No.3-16; and the extension and amplification of the synthetized segments are performed to obtain the complete target gene. By adopting the preparation method in the invention, the coding genes of Alfimeprase is easier to obtain, the complicated step that mRNA is converted to cDNA through reverse transcription is eliminated and the corresponding mutant sites can be designed according to needs.

Description

technical field [0001] The invention relates to biotechnology, in particular to a mutant snake venom fibrinolytic enzyme gene and a preparation method thereof. Background technique [0002] Peripheral Arterial Occlusive (PAO) is a common disease in the elderly in which peripheral atherosclerosis leads to arterial stenosis or even occlusion, resulting in corresponding ischemic spasm or necrosis of distal tissues. The prevalence of PAO in the elderly population at home and abroad is basically 12% to 25%, and it has a tendency to increase year by year. [0003] Alfimeprase (ALF) is a zinc metalloprotease developed through gene recombination technology, and it is an N-terminal mutant of fibrolytic enzyme Fibrolase (the first three amino acids QQR are replaced by S), a 201-amino acid protein containing 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 has ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/57C12N15/63C12N9/68
Inventor 黄飞盛太奎鞠岚岚黄熠
Owner ANHUI FENGYUAN PHARM CO LTD
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