CN102321564A - Genetic engineering bacteria for high level expression of cytidine triphosphate synthetase, and construction method thereof - Google Patents
Genetic engineering bacteria for high level expression of cytidine triphosphate synthetase, and construction method thereof Download PDFInfo
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Abstract
The present invention discloses genetic engineering bacteria for high level expression of cytidine triphosphate synthetase. The genetic engineering bacteria is classified and named Escherichia coli. The strain was preserved in the China general microbiological culture collection center on July 8th, 2011. The preservation number of the genetic engineering bacteria is CGMCC No. 5048. The present invention further discloses a construction method for the genetic engineering bacteria and an application of the genetic engineering bacteria. According to the present invention, cytidine triphosphate synthetase gene pyrG in corynebacterium glutmicum ATCC13032 is firstly efficiently expressed in escherichia coli Rosetta; with adopting the recombinant genetic engineering bacteria, the cytidine triphosphate synthetase gene pyrG can be efficiently expressed, the cytidine triphosphate synthetase is secreted and expressed; IPTG is adopted for inducing the expression of the recombinant escherichia coli, the enzyme activity is 10 U/mg; lactose is adopted for self-induction of the expression of the recombinant escherichia coli, the enzyme activity is 7 U/mg.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of genetic engineering bacterium and construction process thereof that efficiently expresses the cytidine synthetic enzyme.
Background technology
Cytidine triphosphate (being called for short CTP) is participated in the biosynthetic process of phospholipids (Yelkin TTS, kephalin, serine phosphatide, sphingophospholipid etc.) in vivo.Phospholipids is the important component of nervous tissue, also is a kind of natural fat emulsifier.Have the brain of perfecting and nervous tissue, promote body inner cholesterol, fatty transportation to prevent the effect of piling up thereby increase intelligence with memory and have.Clinically, CTP is used to treat cerebral trauma as biochemical drug, the disturbance of consciousness due to the nerve injury.The disturbance of consciousness due to cerebral concussion, neurosis, the nerve injury, fatty liver etc. there is significant curative effect, in addition in treatment cerebrovascular accident and sequela thereof; Dysfunction after cerebral concussion, traumatic stupor, the craniocerebral operations; Cerebrovascular sclerosis, senile dementia; Peripheral nerve injury; Also there is good effect aspects such as children's cerebral dysgenesis.
CTP is synthetic to mainly contain three kinds of chemical synthesiss, photosynthetic phosphoric acid method, biological synthesis process.Since shortcomings such as the synthetic agents useful for same of chemical method is many, complex process, conditional request height, long reaction time, the little large-scale industrial production of realization easily.Along with the fast development of biotechnology, the synthetic of CTP changed enzymatic conversion research over to from chemical process.
Enzyme process is enzyme source catalytic production CTP with the cytidine synthetic enzyme promptly.Cytidine synthetic enzyme (E.C.6.3.4.2) is at Stimulina, ATP, GTP, Mg
2+Participate in down, but catalysis UTP generates CTP, ADP and Pi, is the biosynthetic key enzyme of CTP.Therefore, utilizing genetic engineering means, clone highly active cytidine synthase gene, make up engineering strain great expression cytidine synthetic enzyme, is one step of raw material to produce CTP with UTP, is feasible way of the efficient synthetic of CTP.This approach has advantages such as technology is simple, mild condition, the cycle is short, by product is few, and cleanliness without any pollution, can realize the CTP production process energy-conservation, consumption reduction, reduce discharging.
Summary of the invention
Technical problem to be solved by this invention provides a kind of genetic engineering bacterium that efficiently expresses the cytidine synthetic enzyme.
The technical problem that the present invention also will solve provides the construction process of said gene engineering bacteria.
The technical problem that the present invention will solve at last provides the application of said gene engineering bacteria in efficiently expressing the cytidine synthetic enzyme.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is following:
A kind of genetic engineering bacterium that efficiently expresses the cytidine synthetic enzyme; Its called after ETEC (Escherichia coli) of classifying; This bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, institute of microbiology of the Chinese Academy of Sciences on July 8th, 2011; Postcode: 100101, deposit number is CGMCC No.5048.
The above-mentioned genetic engineering bacterium that efficiently expresses the cytidine synthetic enzyme; It is the intestinal bacteria that comprise cytidine synthetic enzyme pyrG gene, and said cytidine synthetic enzyme pyrG gene source is in Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032.
Wherein, the nucleotide sequence of described cytidine synthetic enzyme pyrG gene is shown in SEQ ID NO:1, and mrna length is 1665bp, its 554 amino acid of coding and a terminator codon, and coded aminoacid sequence is shown in SEQ ID NO:2.
The above-mentioned construction process that efficiently expresses the genetic engineering bacterium of cytidine synthetic enzyme; From Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032, extract cytidine synthetic enzyme pyrG gene through the polymerase chain reaction; Make up recombinant expression plasmid carrier pET28a-pyrG; With recombinant plasmid thermal shock transformed into escherichia coli Rosetta, carry out abduction delivering then, screening obtains the highest bacterial strain CGMCC No.5048 of a strain yield of enzyme.
The above-mentioned application of genetic engineering bacterium in efficiently expressing the cytidine synthetic enzyme that efficiently expresses the cytidine synthetic enzyme.Through cultivating the said gene engineering bacteria, utilize the expression of cytidine synthase gene pyrG in IPTG or the lactose-induced genetic engineering bacterium, collect thalline, the ultrasonication cell, the supernatant that obtains is as crude enzyme liquid.
Beneficial effect: the present invention efficiently expresses cytidine synthase gene pyrG among Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 first in intestinal bacteria Rosetta.The engineering strain of reorganization can efficiently express cytidine synthase gene pyrG, and secreting, expressing cytidine synthetic enzyme, IPTG abduction delivering recombination bacillus coli, enzyme are lived and be 10U/mg; Lactose self-induction express recombinant intestinal bacteria, enzyme live and are 7U/mg.
The description of drawings book
Fig. 1 is plasmid construction figure of the present invention.
Fig. 2 is for the Corynebacterium glutamicum gene group being the electrophorogram behind the template PCR.Wherein, the 1st road: maker2000, the 10th, 11,12,13 road: ATCC13032pyrG.
Embodiment
According to following embodiment, can understand the present invention better.Yet, those skilled in the art will readily understand that the described content of embodiment only is used to explain the present invention, and the present invention that should also can not limit in claims to be described in detail.
Embodiment 1: the clone of cytidine synthase gene pyrG.
Corynebacterium glutamicum (Corynebacterinm glutamicum) ATCC13032 is by this laboratory preservation, and substratum is formed (g.L-1) as follows: yeast soaks powder 5g, Tryptones 10g, and sodium-chlor 10g mends zero(ppm) water to 1L.ATCC13032 is inoculated in the 30mL substratum with Corynebacterium glutamicum (Corynebacterium glutamicum), and 37 ℃ are cultured to logarithmic phase, uses genome DNA extracting reagent kit to extract genome.
The used primer of construction of expression vector adds restriction enzyme site, and primer sequence is following:
Upstream primer:
5’CGGGATCCATGACCTCTAGTCGAAAAGTCCG3’
5’GGAATTCCATATGATGACCTCTAGTCGAAAAGTCC3’
Downstream primer (CRII-anti contains Hind III) is:
5’GGAATTCCTAAGGGTGGACACGCAGCTCC3’
5’CCCAAGCTTCTAAGGGTGGACACGCAG3’
All primers are synthetic by Nanjing Genscript Biotechnology Co., Ltd..
The PCR condition of gene:
94 ℃ of sex change 5min, circulate 30 times by following parameter: 94 ℃ of sex change 1min, 60 ℃ of annealing 50s, 72 ℃ are extended 1.5min.Last 72 ℃ are extended 10min
Gel electrophoresis separation and purification PCR product (Fig. 1) also carries out glue and reclaims.Glue is reclaimed product be connected to PMD
18-simpleT-vector connects product transformed into escherichia coli DH5a competent cell, coats ammonia benzyl resistant panel.
Single bacterium colony on the picking LB flat board is inoculated in the 50mL centrifuge tube that the 5mLLB liquid nutrient medium is housed, and 37 ℃, 220rpm cultivated 8-12 hour down.Utilize the plasmid extraction kit of Shen, Shanghai ability lottery industry ltd to extract plasmid, and send Nanjing Genscript Biotechnology Co., Ltd.'s order-checking bacterium liquid.Sequence length is 1665bp, shown in SEQ ID No:1.
Embodiment 2: the structure of gene pyrG expression vector.
According to nucleotide sequence (shown in SEQ ID No:1) the Design Expression primer of the pyrG gene that obtains, introduce NdeI and HindIII restriction enzyme site respectively at the 5 ' end and the 3 ' end of primer, pcr amplification connects PMD
18-simple T-vector makes up PMD 18T-pyrG recombinant plasmid.Adopt NdeI and HindIII double digestion PMD 18T-pyrG recombinant plasmid and pET28a plasmid, ligase enzyme is cut product, obtains recombinant plasmid pET28a-pyrG, transformed into escherichia coli DH5a.Picking list bacterium colony is inoculated in the 50mL centrifuge tube that the 5mLLB liquid nutrient medium is housed, and 37 ℃, 220rpm cultivated 8-12 hour down.Extract plasmid, obtain to contain the recombinant expression plasmid of pyrG gene, and the reading frame of the affirmation recombinant plasmid that checks order is correct through PCR and double digestion screening.
Embodiment 3: express the structure and the screening of the colibacillus engineering strain of Corynebacterium glutamicum ATCC13032pyrG gene.
With the recombinant plasmid transformed intestinal bacteria Rosetta that obtains, picking list bacterium colony, inoculation 5mL contains in the LB liquid nutrient medium of paraxin and kalamycin, and 37 ℃, 220rpm cultivated 12 hours down.Extracting plasmid, is template with the plasmid, the PCR checking.
Embodiment 4:IPTG abduction delivering recombination bacillus coli.
Picking reorganization bacterium intestinal bacteria Rosetta (pET28a-pyrG) and the contrast bacterium intestinal bacteria Rosetta (DE3) that contain the pET28a empty plasmid to the LB liquid nutrient medium that contains 50mg/L kalamycin and 20mg/L paraxin, 37 ℃, the 220rpm overnight cultures.Be inoculated into respectively in the fresh medium by 2% inoculum size then, 37 ℃, 220rpm is cultured to OD600 and is about at 0.6 o'clock, adds IPTG to final concentration 0.8mM, 30 ℃; 220rpm, behind the abduction delivering 6h, 8000rpm, 4 ℃ of centrifugal 5min; Bacterium mud is resuspended with 200mM Tris-HCl (PH8.0), ultrasonication cell (power 200W, ultrasonic 3s, intermittently 4s; Be total to 3min), 6500rpm, 4 ℃ of centrifugal 5min, the enzyme of measuring in the supernatant is lived.
The enzyme reaction system comprises 3.0mmol/l uridylic trinucleotide (UTP), 3.0mmol/l adenine trinucleotide (ATP), 3.0mmol/l guanine trinucleotide (GTP), 35mmol/l tris-Hcl, 10mmol/l Mgcl2,2mmol/l Stimulina; Under pH6.0～7.5,20～30 ℃, 150～300rpm condition, react 15～30h, obtain 5 '-cytidine.30 ℃ of reaction 60min boil the 5min enzyme that goes out in the boiling water, centrifugal, supernatant is crossed film.HPLC detects the content of CTP.
Enzyme biopsy survey method (HPLC): the Lichrospher C18 of Huaiyin, Jiangsu Han Bang Science and Technology Ltd. [4.6 * 250mm, 5um] chromatographic column; Moving phase: the phosphoric acid triethylamine solution of 100% methyl alcohol pH6.6; Column temperature: room temperature; Detect wavelength: 260nm; Flow velocity: 0.8mLmin
-1Sample size: 20 μ L.Below identical.
Enzyme work is defined as and generates the needed enzyme amount of 1 micromole CTP at 30 ℃ of following PMs and be defined as enzyme U of unit that lives.Below identical.
Through detecting, the enzyme of pyrG is lived and is 10U/mg among the reorganization bacterium intestinal bacteria Rosetta (pET28a-pyrG), and the enzyme of pyrG is lived and is 0.74U/mg in the contrast bacterium.
Embodiment 5: lactose self-induction express recombinant intestinal bacteria.
Picking reorganization bacterium intestinal bacteria Rosetta (pET28a-pyrG) and the contrast bacterium intestinal bacteria Rosetta (DE3) that contain the pET28a empty plasmid to the LB liquid nutrient medium that contains 50mg/L kalamycin and 20mg/L paraxin, 37 ℃, the 220rpm overnight cultures.Be inoculated into respectively to lactinated LB liquid nutrient medium by 2% inoculum size then, form (g.L-1) as follows: 2g glucose, 3g lactose, 10gNaCl, 15g Tryptones.37 ℃, 220rpm is cultured to 3h.30 ℃ then, 220rpm, abduction delivering 12h.8000rpm, 4 ℃ of centrifugal 5min collect bacterium mud, and are resuspended with 200mMTris-HCl (PH8.0), ultrasonication cell (power 200W, ultrasonic 3s, intermittently 4s, 3min altogether), 6500rpm, 4 ℃ of centrifugal 5min, the enzyme of measuring in the supernatant is lived.Through detecting, the enzyme of pyrG is lived and is 7U/mg among the reorganization bacterium intestinal bacteria Rosetta (pET28a-pyrG), and the enzyme of pyrG is lived and is 0.89U/mg in the contrast bacterium.
Claims (5)
1. genetic engineering bacterium that efficiently expresses the cytidine synthetic enzyme; Its called after ETEC (Escherichia coli) of classifying; This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 8th, 2011, and deposit number is CGMCC No.5048.
2. genetic engineering bacterium according to claim 1; It is characterized in that; It is the intestinal bacteria that comprise cytidine synthetic enzyme pyrG gene, and said cytidine synthetic enzyme pyrG gene source is in Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032.
3. genetic engineering bacterium according to claim 2 is characterized in that, the nucleotide sequence of described cytidine synthetic enzyme pyrG gene is shown in SEQ ID NO:1.
4. the described construction process that efficiently expresses the genetic engineering bacterium of cytidine synthetic enzyme of claim 1; It is characterized in that; From Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032, extract cytidine synthetic enzyme pyrG gene through the polymerase chain reaction; Make up recombinant expression plasmid carrier pET28a-pyrG; With recombinant plasmid thermal shock transformed into escherichia coli Rosetta, carry out abduction delivering then, screening obtains the highest bacterial strain CGMCC No.5048 of a strain yield of enzyme.
5. the described application of genetic engineering bacterium in efficiently expressing the cytidine synthetic enzyme that efficiently expresses the cytidine synthetic enzyme of claim 1.
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| CN103540570A (en) * | 2013-10-30 | 2014-01-29 | 南京工业大学 | Site-specific mutagenesis modified cytidine triphosphate (CTP) synthetase |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1420172A (en) * | 2001-11-19 | 2003-05-28 | 杭州华大基因研发中心 | High-temp resistant CTP synthetase gene, polypeptide coded therewith and preparing method thereof |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1420172A (en) * | 2001-11-19 | 2003-05-28 | 杭州华大基因研发中心 | High-temp resistant CTP synthetase gene, polypeptide coded therewith and preparing method thereof |
Non-Patent Citations (2)
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| KALINOWSKI J.: "Corynebacterium glutamicum ATCC 13032,IS fingerprint type 4-5, complete genome; segment 5/10", 《GENBANK 登录号：BX927152》 * |
| 刘仲奇，等: "三磷酸胞苷合成酶基因在鼻咽癌纯瘤组织中的表达", 《中华耳鼻喉头颈外科杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103540570A (en) * | 2013-10-30 | 2014-01-29 | 南京工业大学 | Site-specific mutagenesis modified cytidine triphosphate (CTP) synthetase |
| CN103540570B (en) * | 2013-10-30 | 2015-09-23 | 南京工业大学 | A kind of Fixedpoint mutation modified cytidine synthetic enzyme |
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| CN102321564B (en) | 2014-04-23 |
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