CN104388504A - Method for preparing receptor-ligand binding sulfonamide antibiotic protein - Google Patents
Method for preparing receptor-ligand binding sulfonamide antibiotic protein Download PDFInfo
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- CN104388504A CN104388504A CN201410686178.6A CN201410686178A CN104388504A CN 104388504 A CN104388504 A CN 104388504A CN 201410686178 A CN201410686178 A CN 201410686178A CN 104388504 A CN104388504 A CN 104388504A
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- albumen
- prokaryotic expression
- recombinant plasmid
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Abstract
The invention discloses a method for preparing a receptor-ligand binding sulfonamide antibiotic protein. The method comprises the following steps: firstly preparing a prokaryotic expression vector, and then isolating and purifying Escherichia coli strains to obtain dihydrofolate synthase; with a genome of Escherichia coli as a template, taking the obtained dihydrofolate synthetase gene fragment and cloning into the prokaryotic expression vector to obtain a cloning vector, and extracting recombinant plasmids from the cloning vector and the prokaryotic expression vector; culturing the recombinant plasmids and Escherichia coli in a culture medium to obtain a positive clone strain, and then continuously culturing the positive clone strain in the culture medium and centrifuging to obtain a thallus; concentrating the thallus and polyethylene glycol octylphenyl ether, and centrifuging to obtain a protein; and finally purifying the protein. According to the method for preparing the receptor-ligand binding sulfonamide antibiotic protein provided by the invention, a rapid detection product produced under the action of high binding force has sensitivity higher than that of the same product produced by an antibody binding principle.
Description
Technical field
The present invention relates to a kind of preparation method of sulfa antibiotics albumen of analgesics screening platform.
Background technology
In current detection fresh milk, antibiotic method is varied, and traditional method has: microbiological method, chromatography, Immunological Method etc.Although these traditional method sensitivity are higher, detecting step is loaded down with trivial details, plant and instrument and operator require higher, consuming time longer, generally all wants more than two hours.And be primary for production efficiency enterprise, so rapid detection enjoys favor in enterprise.But quick detection kit is import reagent box at present, and cost is higher, a test kit price detecting 96 samples is more than 3000 yuan.This fund making an annual medium and small dairy enterprises be used in microbiotic detection field at least wants hundreds of thousands of.It is huge that the disappearance of domestic effective testing product fast and high import reagent make domestic milk-product microbiotic detect market potential.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of sulfa antibiotics albumen of analgesics screening platform, its associated proteins used is the natural binding proteins of the Sulphadiazine Sodium transformed by gene engineering, and the product that the rapid detection product produced under the effect of high-bond is produced than antibodies principle of the same type has higher sensitivity.
In order to achieve the above object, technical scheme of the present invention is:
A kind of preparation method of sulfa antibiotics albumen of analgesics screening platform, comprise the steps: first to prepare prokaryotic expression carrier (PET-28a), then by colon bacillus (E.Coli) strains separation, purifying obtains dihydrofolate synthetase, then be that template is transferred the dihydrofolate synthetase gene fragment of acquisition and is cloned in prokaryotic expression carrier (PET-28a) and obtains cloning vector with the genome of colon bacillus (E.Coli), then cloning vector and prokaryotic expression carrier (PET-28a) are extracted recombinant plasmid, then recombinant plasmid and intestinal bacteria (BL21) are cultivated in the medium and obtain positive colony bacterial classification, then positive colony bacterial classification is continued in the medium to cultivate, centrifugal acquisition thalline, then thalline and Triton X-100 (TritonX-100) are concentrated, centrifugal acquisition albumen, finally by protein purification.
First fragment is reclaimed after gel electrophoresis after using restriction endonuclease (XhoI and EcoRI) to carry out double digestion respectively to cloning vector and prokaryotic expression carrier (PET-28a) when described cloning vector and prokaryotic expression carrier (PET-28a) extract recombinant plasmid, then with ligase enzyme (T4 DNA), recovery fragment is connected the prokaryotic expression carrier be built into containing goal gene, then the prokaryotic expression carrier transformation of E. coli (DH5 α) containing goal gene is extracted recombinant plasmid afterwards.
Described recombinant plasmid and intestinal bacteria (BL21) cultivate in the medium first take out in-70 DEG C when obtaining positive colony bacterial classification containing competence intestinal bacteria (BL21) 50 μ L put into centrifuge tube inserts wet ice dissolve 15min, then the recombinant plasmid adding 5 μ L in centrifuge tube mixes rear standing 30min on ice, then will in 42 DEG C after heating in water bath 90s ice bath leave standstill 2min, then recombinant plasmid and intestinal bacteria (BL21) are added 800 μ L containing penbritin LB substratum in 37 DEG C time shaking culture 1h, then get after 50 μ L cultures painting LB agar plates cultivate 12h in 37 DEG C and obtain single bacterium colony, then after choosing single bacterium colony LB substratum shaking culture 12h, upgrading grain obtains positive colony bacterial classification.
Described positive colony bacterial classification continues to cultivate in the medium, centrifugal acquisition thalline time first by positive colony strain inoculation in LB substratum, cultivate after 24h in 37 DEG C of vibrations and culture is forwarded to LB substratum, 37 DEG C of shaking culture are 0.8 to OD600; Then in LB substratum, add sec.-propyl-β-D-Thiogalactopyranoside (IPTG) makes its final concentration be lmmol/L, in 4 DEG C, 6000rmp centrifugal 15min results thalline after continuation cultivation 4h.
Thalline and Triton X-100 (TritonX-100) are concentrated, during centrifugal acquisition albumen, first thalline is resuspended in phosphoric acid buffer (PBS) and is placed on ultrasonication in ice bath, then being concentrated into ultimate density after adding Triton X-100 (TritonX-100) is 1%, then mixture is stirred 30min, then in 4 DEG C, collect albumen after centrifugal 15 min of 12000 rmp.
Described albumen imidazoles purifying.
The invention has the beneficial effects as follows: a kind of preparation method of sulfa antibiotics albumen of analgesics screening platform, its associated proteins used is the natural binding proteins of the Sulphadiazine Sodium transformed by gene engineering, and the product that the rapid detection product produced under the effect of high-bond is produced than antibodies principle of the same type has higher sensitivity.
Embodiment
Embodiment 1
A kind of preparation method of sulfa antibiotics albumen of analgesics screening platform, comprise the steps: first to prepare prokaryotic expression carrier (PET-28a), then by colon bacillus (E.Coli) strains separation, purifying obtains dihydrofolate synthetase, then be that template is transferred the dihydrofolate synthetase gene fragment of acquisition and is cloned in prokaryotic expression carrier (PET-28a) and obtains cloning vector with the genome of colon bacillus (E.Coli), then cloning vector and prokaryotic expression carrier (PET-28a) are extracted recombinant plasmid, then recombinant plasmid and intestinal bacteria (BL21) are cultivated in the medium and obtain positive colony bacterial classification, then positive colony bacterial classification is continued in the medium to cultivate, centrifugal acquisition thalline, then thalline and Triton X-100 (TritonX-100) are concentrated, centrifugal acquisition albumen, finally by protein purification.
First fragment is reclaimed after gel electrophoresis after using restriction endonuclease (XhoI and EcoRI) to carry out double digestion respectively to cloning vector and prokaryotic expression carrier (PET-28a) when described cloning vector and prokaryotic expression carrier (PET-28a) extract recombinant plasmid, then with ligase enzyme (T4 DNA), recovery fragment is connected the prokaryotic expression carrier be built into containing goal gene, then the prokaryotic expression carrier transformation of E. coli (DH5 α) containing goal gene is extracted recombinant plasmid afterwards.
Described recombinant plasmid and intestinal bacteria (BL21) cultivate in the medium first take out in-70 DEG C when obtaining positive colony bacterial classification containing competence intestinal bacteria (BL21) 50 μ L put into centrifuge tube inserts wet ice dissolve 15min, then the recombinant plasmid adding 5 μ L in centrifuge tube mixes rear standing 30min on ice, then will in 42 DEG C after heating in water bath 90s ice bath leave standstill 2min, then recombinant plasmid and intestinal bacteria (BL21) are added 800 μ L containing penbritin LB substratum in 37 DEG C time shaking culture 1h, then get after 50 μ L cultures painting LB agar plates cultivate 12h in 37 DEG C and obtain single bacterium colony, then after choosing single bacterium colony LB substratum shaking culture 12h, upgrading grain obtains positive colony bacterial classification.
Described positive colony bacterial classification continues to cultivate in the medium, centrifugal acquisition thalline time first by positive colony strain inoculation in LB substratum, cultivate after 24h in 37 DEG C of vibrations and culture is forwarded to LB substratum, 37 DEG C of shaking culture are 0.8 to OD600; Then in LB substratum, add sec.-propyl-β-D-Thiogalactopyranoside (IPTG) makes its final concentration be lmmol/L, in 4 DEG C, 6000rmp centrifugal 15min results thalline after continuation cultivation 4h.
Thalline and Triton X-100 (TritonX-100) are concentrated, during centrifugal acquisition albumen, first thalline is resuspended in phosphoric acid buffer (PBS) and is placed on ultrasonication in ice bath, then being concentrated into ultimate density after adding Triton X-100 (TritonX-100) is 1%, then mixture is stirred 30min, then in 4 DEG C, collect albumen after centrifugal 15 min of 12000 rmp.
Described albumen imidazoles purifying.
A kind of diaphragm type one-way valve of the present embodiment, its structure is simple, easy to use, improves the work-ing life of check valve while improving one-way sealing.
Claims (6)
1. the preparation method of the sulfa antibiotics albumen of an analgesics screening platform, it is characterized in that comprising the steps: first to prepare prokaryotic expression carrier (PET-28a), then by colon bacillus (E.Coli) strains separation, purifying obtains dihydrofolate synthetase, then be that template is transferred the dihydrofolate synthetase gene fragment of acquisition and is cloned in prokaryotic expression carrier (PET-28a) and obtains cloning vector with the genome of colon bacillus (E.Coli), then cloning vector and prokaryotic expression carrier (PET-28a) are extracted recombinant plasmid, then recombinant plasmid and intestinal bacteria (BL21) are cultivated in the medium and obtain positive colony bacterial classification, then positive colony bacterial classification is continued in the medium to cultivate, centrifugal acquisition thalline, then thalline and Triton X-100 (TritonX-100) are concentrated, centrifugal acquisition albumen, finally by protein purification.
2. the preparation method of the sulfa antibiotics albumen of a kind of analgesics screening platform according to claim 1, it is characterized in that: after first using restriction endonuclease (XhoI and EcoRI) to carry out double digestion respectively to cloning vector and prokaryotic expression carrier (PET-28a) when described cloning vector and prokaryotic expression carrier (PET-28a) extract recombinant plasmid, after gel electrophoresis, reclaim fragment, then with ligase enzyme (T4 DNA), recovery fragment is connected the prokaryotic expression carrier be built into containing goal gene, then the prokaryotic expression carrier transformation of E. coli (DH5 α) containing goal gene is extracted recombinant plasmid afterwards.
3. the preparation method of the sulfa antibiotics albumen of a kind of analgesics screening platform according to claim 1, it is characterized in that: described recombinant plasmid and intestinal bacteria (BL21) cultivate in the medium first take out in-70 DEG C when obtaining positive colony bacterial classification containing competence intestinal bacteria (BL21) 50 μ L put into centrifuge tube inserts wet ice dissolve 15min, then the recombinant plasmid adding 5 μ L in centrifuge tube mixes rear standing 30min on ice, then will in 42 DEG C after heating in water bath 90s ice bath leave standstill 2min, then recombinant plasmid and intestinal bacteria (BL21) are added 800 μ L containing penbritin LB substratum in 37 DEG C time shaking culture 1h, then get after 50 μ L cultures painting LB agar plates cultivate 12h in 37 DEG C and obtain single bacterium colony, then after choosing single bacterium colony LB substratum shaking culture 12h, upgrading grain obtains positive colony bacterial classification.
4. the preparation method of the sulfa antibiotics albumen of a kind of analgesics screening platform according to claim 1, it is characterized in that: described positive colony bacterial classification continue in the medium cultivate, centrifugal acquisition thalline time first by positive colony strain inoculation in LB substratum, after 24h is cultivated in 37 DEG C of vibrations, culture is forwarded to LB substratum, 37 DEG C of shaking culture are 0.8 to OD600; Then in LB substratum, add sec.-propyl-β-D-Thiogalactopyranoside (IPTG) makes its final concentration be lmmol/L, in 4 DEG C, 6000rmp centrifugal 15min results thalline after continuation cultivation 4h.
5. the preparation method of the sulfa antibiotics albumen of a kind of analgesics screening platform according to claim 1, it is characterized in that: thalline and Triton X-100 (TritonX-100) are concentrated, during centrifugal acquisition albumen, first thalline is resuspended in phosphoric acid buffer (PBS) and is placed on ultrasonication in ice bath, then being concentrated into ultimate density after adding Triton X-100 (TritonX-100) is 1%, then mixture is stirred 30min, then in 4 DEG C, collect albumen after centrifugal 15 min of 12000 rmp.
6. the preparation method of the sulfa antibiotics albumen of a kind of analgesics screening platform according to claim 1, is characterized in that: described albumen imidazoles purifying.
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| CN201410686178.6A CN104388504A (en) | 2014-11-26 | 2014-11-26 | Method for preparing receptor-ligand binding sulfonamide antibiotic protein |
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| CN201410686178.6A CN104388504A (en) | 2014-11-26 | 2014-11-26 | Method for preparing receptor-ligand binding sulfonamide antibiotic protein |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105018406A (en) * | 2015-07-29 | 2015-11-04 | 中国农业大学 | Recombinant Escherichia coli for producing protein capable of detecting sulfonamide as well as construction method and application of recombinant Escherichia coli |
| CN105137086A (en) * | 2015-07-29 | 2015-12-09 | 中国农业大学 | Kit and test strip for detecting sulfonamides and use thereof |
| CN118028426A (en) * | 2024-04-12 | 2024-05-14 | 哈尔滨工业大学(深圳)(哈尔滨工业大学深圳科技创新研究院) | Method for detecting sulfonamide pollutants of escherichia coli in-vitro expression vector based on DHPS protein |
-
2014
- 2014-11-26 CN CN201410686178.6A patent/CN104388504A/en active Pending
Non-Patent Citations (4)
| Title |
|---|
| ANIRUDDHA ACHARI ET AL.: "Crystal structure of the anti-bacterial sulfonamide drug target dihydropteroate synthase", 《NATURE STRUCTURAL BIOLOGY》 * |
| JEFFREY TOY ET AL.: "Cloning and Expression of the Gene Encoding Lactobacillus casei Folylpoly-y-glutamate Synthetase in Escherichia coli and Determination of Its Primary Structure", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| PHILIPPE M. HAUSER ET AL. : "Isolation of the Pneumocystis carinii dihydrofolate synthase gene and functional complementation in Saccharomyces cerevisiae", 《FEMS MICROBIOL LETT》 * |
| 梁晓: "基于二氢叶酸合成酶的磺胺类药物多残留快速检测分析研究", 《中国博士学位论文全文数据库 农业科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105018406A (en) * | 2015-07-29 | 2015-11-04 | 中国农业大学 | Recombinant Escherichia coli for producing protein capable of detecting sulfonamide as well as construction method and application of recombinant Escherichia coli |
| CN105137086A (en) * | 2015-07-29 | 2015-12-09 | 中国农业大学 | Kit and test strip for detecting sulfonamides and use thereof |
| CN118028426A (en) * | 2024-04-12 | 2024-05-14 | 哈尔滨工业大学(深圳)(哈尔滨工业大学深圳科技创新研究院) | Method for detecting sulfonamide pollutants of escherichia coli in-vitro expression vector based on DHPS protein |
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Application publication date: 20150304 |