CN102212538A - Adenylate cyclase, and coding gene, vector, strain and application thereof - Google Patents
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Abstract
The invention provides adenylate cyclase, and a coding gene, a vector, a strain and application thereof. The DNA sequence provided by the invention for coding adenylate cyclase has the sequence shown in SEQ ID No.: 1, or a nucleotide sequence represented by the formula . In addition, the invention also provides adenylate cyclase coded by the DNA sequence, a recombinant vector containing the DNA sequence, a host cell containing the recombinant vector and application of the adenylate cyclase in production. The escherichia coli genetic engineering strain can efficiently express adenylate cyclase, and the induced enzyme activity can reach 7U/mg or 10U/mg. The strain is applied to the production of cyclic adenosine monophosphate, and has the advantages of simple process, mild condition, short period, few byproducts and the like.
Description
Technical field
The invention belongs to bioengineering field, be specifically related to a kind of adenylate cyclase, its encoding gene, carrier, bacterial strain and application.
Background technology
Cyclic monophosphate (cyclic adenosine monophosphate, be called for short cAMP) be a kind of material that extensively exists in the human body with physiologically active, it is as intracellular second messenger, and, protein synthesis etc. synthetic to carbohydrate metabolism, metabolism of fat, nucleic acid brought into play important regulatory role.CAMP is used for the treatment of chronic heart failure, pulmonary heart disease, myocardial infarction, myocarditis and cardiogenic shock clinically; The palpitaition of improving rheumatic heart disease, symptom such as out of breath, uncomfortable in chest are had certain effect; Can improve the curative effect of acute leukemia, also can be used for the inducer remission of acute leukemia in conjunction with chemotherapy; In addition, senile chronic bronchitis, various hepatitis and psoriatic also there is certain curative effect.CAMP also can be used as pharmaceutical intermediate and prepares dibutyryl cyclic adenosine monophosphate and Meglumine Cyclic Adenylate, improve fat-soluble, thereby bring into play more effective physiology and pharmacological action.CAMP also can be used for additive for farm animal feed, and the effect of simulation tethelin promotes growth of animals or poultry under isolated condition, increases high-quality poultry product output.
The production method of cAMP has three kinds of chemical synthesis, fermentation method and enzyme process, and industrialization is both at home and abroad at present produced and all adopted with AMP is the chemical synthesis of raw material.The reagent costliness that chemical synthesis is related, and adopt a large amount of pyridines as solvent has pungency to the skin and the respiratory tract of human body, and is very big to human body harm, and causes environmental pollution, and synthetic very high to processing requirement.Raw materials for production and the reagent price is higher, production cost is higher.Utilize brevibacterium liquefaciens, dialister bacterium, coryneform bacteria, Arthrobacter etc. to prepare cAMP and also have restriction with fermentation method, lower as productive rate, efficiently expressing of key enzyme adenylate cyclase is machine-processed unclear, and the relation of ionic environment and cytoactive and regulation mechanism remain further to be studied.
Enzyme process promptly is enzyme source catalytic production cAMP with the adenylate cyclase.Adenylate cyclase (E.C.4.6.1.1) is at Mg
2+Participate in down, but catalysis ATP generates cAMP and PPi, is the biosynthetic key enzyme of cAMP.Therefore, exploitation is means with the genetically engineered, clones highly active adenylate cyclase gene, makes up engineering strain great expression adenylate cyclase, with ATP be one step of raw material to produce cAMP, and be feasible way of cAMP synthetic.
Summary of the invention
Therefore, the purpose of this invention is to provide a kind of gene that efficiently expresses adenylate cyclase.
Another object of the present invention provides the recombinant vectors that comprises said gene.
Another purpose of the present invention provides host cell that comprises above-mentioned recombinant vectors and preparation method thereof.
In addition, also purpose of the present invention provides the application of said gene, recombinant vectors and host cell.
The objective of the invention is to be achieved through the following technical solutions.On the one hand, the invention provides a kind of dna sequence dna of the adenylate cyclase that is used to encode, it has the base sequence shown in SEQ ID NO.:1.
The present invention also provides a kind of adenylate cyclase, has the aminoacid sequence shown in SEQ ID NO.:2.
On the other hand, the invention provides a kind of recombinant vectors that is used to express adenylate cyclase, it contains above-mentioned dna sequence dna.
Preferably, described recombinant vectors is the pattern of fusion expression vector, is preferably pET28a.
Another aspect the invention provides a kind of host cell, and it contains above-mentioned recombinant vectors.
Preferably, described host cell is intestinal bacteria.
More preferably, described colibacillary deposit number is CGMCC No.4706, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on March 24th, 2011, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, institute of microbiology of the Chinese Academy of Sciences (postcode: 100101).
The present invention also provides the method for preparing above-mentioned host cell, and it comprises the recombinant vectors that structure is above-mentioned, and utilizes this recombinant vectors transformed host cell.
Preferably, described method comprises that structure comprises the recombinant vectors pET28a of above-mentioned dna sequence dna, utilizes this recombinant vectors by the thermal shock transformed into escherichia coli, and according to product expression amount screening bacterial strain.
Also on the one hand, the invention provides above-mentioned dna sequence dna, above-mentioned recombinant vectors or the above-mentioned application of host cell in producing adenylate cyclase.Preferably, described adenylate cyclase has the aminoacid sequence shown in SEQ ID NO.:2.
In addition, the invention provides a kind of production method of adenylate cyclase, it comprises and adopts above-mentioned dna sequence dna, above-mentioned recombinant vectors or above-mentioned host cell to produce adenylate cyclase.Preferably, described adenylate cyclase has the aminoacid sequence shown in SEQ ID NO.:2.
The present invention also provides a kind of production method of cyclic monophosphate, and it comprises and adopts the adenylate cyclase enzyme catalysis adenosine triphosphate of above-mentioned dna sequence dna, above-mentioned recombinant vectors or above-mentioned host cell production to produce cyclic monophosphate.Preferably, described adenylate cyclase has the aminoacid sequence shown in SEQ IDNO.:2.
In sum, the invention provides a kind of gene that efficiently expresses adenylate cyclase, its nucleotide sequence is (the following cya gene that is called again) shown in SEQ IDNO:1, and this adenylate cyclase cya mrna length is 1125bp, encode 374 amino acid and a terminator codon.The present invention also provides a kind of expression vector, and it contains the gene of above-mentioned nucleotide sequence shown in SEQ IDNO:1.This expression vector preferably contains the secreted expression carrier pET28a-cya just like gene shown in the SEQ IDNO:1.The present invention provides a kind of host cell again, contains above-mentioned expression vector.This host cell preferably contains the intestinal bacteria of pET28a-cya expression vector.Above-mentioned host cell is more preferably expressed the colibacillus engineering strain of adenylate cyclase, its called after colon bacillus (Escherichia coli) of classifying, deposit number CGMCC No.4706.The present invention also provides the method that makes up host cell, move from Arthrobacter (Arthrobacter A302) by gene step and to extract the cya gene, make up recombinant expression plasmid carrier pET28a-cya, with recombinant plasmid thermal shock transformed into escherichia coli Rosetta, carry out abduction delivering then, screen the highest bacterial strain CGMCCNo.4706 of a strain yield of enzyme.In addition, the present invention also provides the above-mentioned application of gene in producing cAMP that efficiently expresses adenylate cyclase.Above-mentioned application is embodied in and utilizes recombinant bacterial strain CGMCC No.4706 to produce cAMP, its method is as follows: cultivate the reorganization bacterium, utilize the expression of adenylate cyclase gene cya in IPTG or the lactose-induced recombinant plasmid, collect thalline, the ultrasonication cell, the supernatant that obtains is as crude enzyme liquid.At Mg
2+Existing down, is substrate with ATP, and as catalyzer, catalysis generates cAMP with crude enzyme liquid.
As seen, the present invention has found the gene cya of adenylate cyclase in the Arthrobacter (this laboratory called after Arthrobacter A302) first, and has realized adenylate cyclase gene efficiently expressing in intestinal bacteria Rosetta.Recombinant strain can efficiently express adenylate cyclase gene cya, expresses the adenylate cyclase of solubility, is applied to produce cAMP, has obtained good effect.IPTG abduction delivering recombination bacillus coli, enzyme are lived and are 10U/mg; Lactose self-induction express recombinant intestinal bacteria, enzyme live and are 7U/mg.This recombination bacillus coli is applied to produce adenylate cyclase, can be one step of raw material to generate cAMP with ATP, have advantages such as technology is simple, mild condition, the cycle is short, by product is few, and a cleanliness without any pollution, can realize the cAMP production process energy-conservation, consumption reduction, reduce discharging.
The preservation of biomaterial
Arthrobacter (Arthrobacter sp.) A302, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on January 18th, 2010, depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, (postcode: 100101), deposit number is CGMCC No.3584 to institute of microbiology of the Chinese Academy of Sciences.
Recombinant bacterial strain colon bacillus (Escherichia coli) Rosetta (pET28a-cya), be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on March 24th, 2011, depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, institute of microbiology of the Chinese Academy of Sciences (postcode: 100101), deposit number CGMCC No.4706.
Description of drawings
Fig. 1 is the electrophoresis result of cya full-length gene PCR, and wherein swimming lane 1 is 2000bp Marker, and swimming lane 2 is the PCR product.
Fig. 2 is the electrophoresis result of construction of recombinant plasmid and checking, and wherein Fig. 2 A is the electrophoresis result of plasmid double digestion checking, and swimming lane 1 is a pET28a-cya recombinant plasmid double digestion product; Swimming lane 2 is 2000bp Marker; Fig. 2 B is the electrophoresis result of plasmid PCR checking, and swimming lane 1 is for being the PCR product that template obtains with the pET28a-cya recombinant plasmid; Swimming lane 2 is for being the PCR product that template obtains with water; Swimming lane 3 is 2000bp Marker.
Embodiment
Followingly the present invention is described with reference to specific embodiment.It will be appreciated by those skilled in the art that these embodiment only are used to illustrate the present invention, the scope that it does not limit the present invention in any way.
Except as otherwise noted, below among each embodiment employed biomaterial, carrier, bacterial strain, reagent, test kit etc. all can be purchased approach and obtain by routine, the biological gene engineering operative technique that wherein relates to, as plasmid extract, enzyme cuts digestion, fragment recovery, nucleic acid fragment and plasmid vector ligation and clone and screening etc., be the routine operation in this area or operate with reference to the specification sheets of corresponding product.
Embodiment 1The screening of Arthrobacter Arthrobacter A302
With the bacterial strain that separates the product cyclic monophosphate obtain from soil is starting strain, will be through N
+(vacuum tightness is 2 * 10 to ion beam mutagenesis in the low energy ion implanter target chamber
-4, the injection energy is 20kev, dosage is 5 * 10
13Ionscm
-2.s
-1) after spore suspension after 100 times of sterilized water dilutions, get 0.1ml and coat on the extractum carnis plate culture medium 30 ℃ and cultivated 3 days down, therefrom select single bacterium colony extractum carnis slant culture 2 days.Switching one encircles in the 500mL that 30mL liquid seed culture medium (glucose 10g/L, peptone 10g/L, yeast extract paste 5g/L, extractum carnis 10g/L, NaCl 3g/L) is housed shakes in the bottle, cultivates 20 hours in 30 ℃ of following 250rpm shaking tables.4mL transfer again in 40mL liquid fermentation medium (glucose 50g/L, K are housed
2HPO
410g/L, KH
2PO
410g/L, MgSO
41g/L, urea 5g/L, peptone 5g/L) 500mL shake in the bottle, cultivated 72 hours in 30 ℃ of following 280rpm shaker fermentations.Through primary dcreening operation, obtain the 88 strains mutant strain higher than starting strain output.With carrying out multiple sieve under the same condition, finally obtain the highest strains A of output 302, cyclic phosphoric acid glycosides output production peak reaches 2.4g/L, improves 2 times than the 1.2g/L of original strain.
This strains A 302 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) at present, depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.3584, and preservation date is on January 18th, 2010.About the details of Arthrobacter A302 (Arthrobacter sp.), the Chinese invention patent application 201010191515.6 that can submit on June 4th, 2010 referring to the applicant.
Embodiment 2The clone of Arthrobacter adenylate cyclase gene cya
1, the genomic extraction of Arthrobacter
Arthrobacter (Arthrobacter) A302 is inoculated in the 30mL substratum, and (substratum is formed (g.L
-1) as follows: glucose 10g, peptone 10g, acid hydrolyzed casein 10g, yeast extract paste 5g, extractum carnis 10g, sodium-chlor 3g mends distilled water to 1L) in, 30 ℃ are cultured to logarithmic phase, use bacterial genomes DNA extraction test kit (available from Beijing lid Ning Jinnuo Bioisystech Co., Ltd) to extract genome.
2, amplification conserved sequence
According to the aminoacid sequence of existing adenylate cyclase in the ncbi database, the degenerated primer of design amplification conserved sequence is as follows:
S220：5’-GCTCTCCGCCCGGAA(A/G)(A/C/T)T(A/G/C/T)TGG(A/C)G-3’
AS1：5’-CAGCCGGGCGGC(A/G/C/T)A(A/G/T)(A/G)TT(A/G/C/T)AC-3’
The PCR reaction system is as follows:
The PCR condition is as follows:
94 ℃ of sex change 5min; Circulate 30 times by following parameter: 94 ℃ of sex change 1min, 52 ℃ of annealing 30s, 72 ℃ are extended 30s; Last 72 ℃ are extended 10min.
The degenerated primer pcr amplification obtains the fragment of length for about 750bp, and is close with expection length, and this fragment of gel electrophoresis separation and purification is also carried out glue and reclaimed.Glue is reclaimed product be connected to PMD18T-vector (available from TAKARA company), connect product and transform the escherichia coli DH5a competent cell that adopts the Calcium Chloride Method preparation, coat 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 plasmid extraction kit (available from Shanghai Shenergy Biocolor BioScience ﹠ Technology Company) to extract plasmid, and this auspicious science and technology (Nanjing) company limited order-checking of trust money, conserved sequence length is 768bp, its nucleotide sequence is seen SEQ IDNO:3.
3, the gene step moves
1) the C end step moves
The primer that moves three-wheel PCR according to the 768bp conserved sequence design C end step of above-mentioned gained is as follows:
SP1：5’-TGGACGCCCTCGAAGAAGTACTGGT-3’
SP2：5’-CCCGGCGCATGAACGAAAAAACCCT-3’
SP3：5’-TCCTGTACATCGCAGAAACCCCCGC-3’
With the genome is template, and increase other required reagent and degenerated primer adopt Genomewalking kit (available from TAKARA company), and first round PCR system is as follows:
100 times of diluents with first round PCR product are template, and second to take turns the PCR reaction system as follows:
With second 50 times of diluents of taking turns the PCR product is template, and third round PCR reaction system is as follows:
The reaction conditions of three-wheel PCR is as follows respectively:
The fragment of about 900bp of third round PCR product gained is carried out the gel electrophoresis separation and purification and glue reclaims, and this auspicious science and technology (Nanjing) company limited order-checking of trust money.The gained fragment contains the adenylate cyclase gene sequence of 354bp, with the overlapping 180bp of conserved sequence, shows successfully and move 174bp until terminator to the C end step that its sequence is seen SEQ IDNO:4.
2) the N end step moves
Aminoacid sequence design degenerated primer according to existing adenylate cyclase in the ncbi database and embrane-associated protein before thereof is as follows:
J200：5’-GGAGGGCGGCCGG(A/G)A(C/T)GA(A/G)(A/C)G(A/G/C/T)AT-3’
AS941：5’-TAGTCCAGCACAAGCCCCTTGC-3’
With the genome is template, and the PCR reaction system is as follows:
The PCR reaction conditions is as follows:
94 ℃ of sex change 5min; Circulate 30 times by following parameter: 94 ℃ of sex change 1min, 53 ℃ of annealing 30s, 72 ℃ are extended 1min; Last 72 ℃ are extended 10min.
Gel electrophoresis separation and purification PCR product, and the product of 1000-2000bp is carried out glue reclaim, and this auspicious science and technology (Nanjing) company limited order-checking of trust money.The result shows that success moves 183bp until initiator codon to the N end step, and its sequence is seen SEQ IDNO:5.
3) clone of adenylate cyclase gene cya
Primer according to the full length sequence design amplification cya full length sequence that obtains after the splicing is as follows:
uAC2：5’-CAGTCCGGCGGGGCGTTGGATTAC-3’
dAC：5’-TTAGTCCAGCACAAGCCCCTTGCC-3’
The PCR reaction system is as follows:
The PCR reaction conditions is as follows:
94 ℃ of sex change 5min; Circulate 30 times by following parameter: 94 ℃ of sex change 1min, 53 ℃ of annealing 30s, 72 ℃ are extended 80s; Last 72 ℃ are extended 10min.
The PCR product (electrophoresis result is seen Fig. 1) of the about 1100bp of gel electrophoresis separation and purification carries out after glue reclaims this auspicious science and technology (Nanjing) company limited order-checking of trust money.The result shows, cya mrna length 1125bp, encode 374 amino-acid residues and a terminator, its sequence such as SEQ IDNO:1.
Embodiment 3The structure of cya expression vector
Primer is expressed in nucleotide sequence design according to the cya gene that obtains, introduces NdeI and EcoRI restriction enzyme site (seeing the horizontal line part) respectively at the 5 ' end and the 3 ' end of primer, and primer sequence is as follows:
sAC：5’-TTC
CATATGATGAACGATGAGGACCAGCA-3’
asAC：5’-CCG
GAATTCTTAGTCCAGCACAAGCCCCT-3’
The PCR reaction system is as follows:
The PCR reaction conditions is as follows:
94 ℃ of sex change 5min; Circulate 30 times by following parameter: 94 ℃ of sex change 1min, 53 ℃ of annealing 30s, 72 ℃ are extended 80s; Last 72 ℃ are extended 10min.
The PCR product of the about 1100bp of gel electrophoresis separation and purification carries out glue and reclaims.Glue is reclaimed product be connected to PMD18T-simple, make up the PMD18T-cya recombinant plasmid.
Adopt NdeI and EcoRI double digestion PMD18T-cya recombinant plasmid and several different plasmids (comprising pET28a plasmid, pET15b plasmid, pET22b plasmid etc.), and ligase enzyme cuts product, obtain corresponding recombinant plasmid.The result shows that plasmid pET28a obviously is better than other plasmids.
Adopt this recombinant plasmid pET28a-cya to transform different host'ss (comprising intestinal bacteria, subtilis and pichia spp etc.), the result shows that intestinal bacteria obviously are better than other hosts.
Adopt this recombinant plasmid pET28a-cya transformed into escherichia coli DH5a, picking list bacterium colony is inoculated in the 50mL centrifuge tube that 5mL LB liquid nutrient medium is housed, and 37 ℃, 220rpm cultivated 8-12 hour down.Extract plasmid, screen the recombinant expression plasmid that obtains to contain the cya gene through PCR and double digestion, the electrophoresis checking the results are shown in Figure 2A and 2B, and correct through the reading frame of order-checking affirmation recombinant plasmid.
Embodiment 4Express the structure and the screening of the colibacillus engineering strain of Arthrobacter cya gene
The recombinant plasmid that obtains is passed through thermal shock transformed into escherichia coli Rosetta, concrete operations are as follows: get 1 μ L recombinant plasmid, join in the 100 μ L intestinal bacteria Rosetta competent cell liquid, after placing 30min on ice, 42 ℃ of thermal shock 90s, take out immediately and put 2min on ice, add 900 μ LLB liquid nutrient mediums, 37 ℃, 220rpm cultivates after 1 hour down, get the LB solid medium that the coating of 150 μ L nutrient solutions contains paraxin and kalamycin, 37 ℃ of single bacterium colonies of cultivating gained after 12 hours are the reorganization bacterium.
Extracting plasmid, is template with the plasmid, carries out the PCR checking.
Carry out after the abduction delivering, obtain the highest bacterial strain Rosetta (pET28a-cya) of a strain yield of enzyme through screening, its deposit number is CGMCC No.4706.
Embodiment 5IPTG abduction delivering recombination bacillus coli
Reorganization bacterium intestinal bacteria Rosetta (pET28a-cya) that filter out among the picking embodiment 4 and the contrast bacterium intestinal bacteria Rosetta (DE3) that contain the pET28a empty plasmid are to the LB liquid nutrient medium that contains 50mg/L kalamycin and 20mg/L paraxin, 37 ℃, the 220rpm overnight incubation.
Be inoculated into respectively in the fresh medium by 2% inoculum size, 37 ℃, 220rpm is cultured to OD600 and is about at 0.6 o'clock, add IPTG to final concentration 0.8mM, 30 ℃, 220rpm, behind the abduction delivering 7h, 8000rpm, 4 ℃ of centrifugal 5min, bacterium mud is resuspended with 200mM Tris-HCl (pH8.0), ultrasonication cell (power 200W, ultrasonic 3s, intermittently 4s is total to 3min), 6500rpm, 4 ℃ of centrifugal 5min measure the enzyme of supernatant liquor and live.
The enzyme reaction system comprises 1mM ATP and 5mM MgSO
4, 30 ℃ of reaction 15min boil the 5min enzyme that goes out in the boiling water, and centrifugal, supernatant liquor is crossed film.HPLC detects the content of cAMP, is specially the Lichrospher C18[4.6x250mm of Huaiyin, Jiangsu Han Bang Science and Technology Ltd., 5um] chromatographic column; Moving phase: V (methyl alcohol): V (the phosphoric acid triethylamine solution of pH6.6)=15: 85; Column temperature: room temperature; Detect wavelength: 254nm; Flow velocity: 0.8mLmin
-1Sample size: 20 μ L.
Enzyme work is defined as and generates the needed enzyme amount of 1 micromole cAMP at 30 ℃ of following per minutes and be defined as enzyme U of unit that lives.
After testing, the enzyme of cya is lived and is 10U/mg among the reorganization bacterium intestinal bacteria Rosetta (pET28a-cya), and the enzyme work of cya is 7.4 * 10 in the contrast bacterium
-4U/mg.
Embodiment 6Lactose self-induction express recombinant intestinal bacteria
Reorganization bacterium intestinal bacteria Rosetta (pET28a-cya) that filter out among the picking embodiment 4 and the contrast bacterium intestinal bacteria Rosetta (DE3) that contain the pET28a empty plasmid are to the LB liquid nutrient medium that contains 50mg/L kalamycin and 20mg/L paraxin, 37 ℃, the 220rpm overnight incubation.
Be inoculated into respectively to lactinated LB liquid nutrient medium by 2% inoculum size, form (g.L
-1) as follows: 2g glucose, 3g lactose, 10gNaCl, 15g peptone, 25g yeast extract paste.37 ℃, 220rpm is cultured to 3h.30 ℃ then, 220rpm, abduction delivering 12h.8000rpm, 4 ℃ of centrifugal 5min collect bacterium mud, and are resuspended with 200mM Tris-HCl (pH8.0), ultrasonication cell (power 200W, ultrasonic 3s, intermittently 4s, 3min altogether), 6500rpm, 4 ℃ of centrifugal 5min measure the enzyme of supernatant liquor and live, specifically with reference to definition among the embodiment 5 and method.
After testing, the enzyme of cya is lived and is 7U/mg among the reorganization bacterium intestinal bacteria Rosetta (pET28a-cya), and the enzyme work of cya is 8.9 * 10 in the contrast bacterium
-4U/mg.
Claims (11)
1. the dna sequence dna of the adenylate cyclase that is used to encode, it has the base sequence shown in SEQ ID NO.:1.
2. adenylate cyclase, it has the aminoacid sequence shown in SEQ ID NO.:2.
3. recombinant vectors that is used to express adenylate cyclase, it contains the described dna sequence dna of claim 1.
4. recombinant vectors according to claim 3 is characterized in that described recombinant vectors is the pattern of fusion expression vector, is preferably pET28a.
5. host cell, it contains claim 3 or 4 described recombinant vectorss.
6. host cell according to claim 5 is characterized in that, described host cell is intestinal bacteria; Preferably, described colibacillary deposit number is CGMCC No.4706.
7. the method for preparing claim 5 or 6 described host cells, it comprises structure claim 3 or 4 described recombinant vectorss, and utilizes this recombinant vectors transformed host cell.
8. method according to claim 7 is characterized in that, described method comprises that structure comprises the recombinant vectors pET28a of the described dna sequence dna of claim 1, and utilizes this recombinant vectors by the thermal shock transformed into escherichia coli, and according to product expression amount screening bacterial strain.
9. the described dna sequence dna of claim 1, claim 3 or 4 described recombinant vectorss or claim 5 or the 6 described host cells application in producing adenylate cyclase.
10. the production method of an adenylate cyclase, it comprises and adopts the described dna sequence dna of claim 1, claim 3 or 4 described recombinant vectorss or claim 5 or 6 described host cells to produce adenylate cyclase.
11. the production method of a cyclic monophosphate, it adenylate cyclase enzyme catalysis adenosine triphosphate that comprises the described dna sequence dna of employing claim 1, claim 3 or 4 described recombinant vectorss or claim 5 or 6 described host cell productions is produced cyclic monophosphate.
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Cited By (8)
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| CN102433292A (en) * | 2011-12-19 | 2012-05-02 | 南京工业大学 | Recombinant escherichia coli for high yield of cyclic adenosine monophosphate and application thereof |
| CN103320373A (en) * | 2013-06-20 | 2013-09-25 | 南京工业大学 | Arthrobacter for overexpression of hypoxanthine phosphoribosyltransferase gene and construction method and application thereof |
| CN103898160A (en) * | 2012-12-25 | 2014-07-02 | 中国科学院深圳先进技术研究院 | Recombinant vector for expressing light-sensitive type adenylate cyclase, applications and construction method of the recombinant vector and treating system for demyelination |
| CN105002234A (en) * | 2015-08-05 | 2015-10-28 | 北京和源泰康生物技术有限公司 | Method for enzymic method catalyzed synthesis cyclic adenosine monophosphate |
| CN110157653A (en) * | 2019-05-09 | 2019-08-23 | 南京工业大学 | Recombinant escherichia coli for high-yield cyclic adenosine monophosphate and application of recombinant escherichia coli in cyclic adenosine monophosphate synthesis |
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| CN102433292A (en) * | 2011-12-19 | 2012-05-02 | 南京工业大学 | Recombinant escherichia coli for high yield of cyclic adenosine monophosphate and application thereof |
| CN102433292B (en) * | 2011-12-19 | 2014-01-22 | 南京工业大学 | Recombinant escherichia coli for high yield of cyclic adenosine monophosphate and application thereof |
| CN103898160A (en) * | 2012-12-25 | 2014-07-02 | 中国科学院深圳先进技术研究院 | Recombinant vector for expressing light-sensitive type adenylate cyclase, applications and construction method of the recombinant vector and treating system for demyelination |
| CN103320373A (en) * | 2013-06-20 | 2013-09-25 | 南京工业大学 | Arthrobacter for overexpression of hypoxanthine phosphoribosyltransferase gene and construction method and application thereof |
| CN103320373B (en) * | 2013-06-20 | 2015-09-23 | 南京工业大学 | Arthrobacter for overexpression of hypoxanthine phosphoribosyltransferase gene and construction method and application thereof |
| CN105002234A (en) * | 2015-08-05 | 2015-10-28 | 北京和源泰康生物技术有限公司 | Method for enzymic method catalyzed synthesis cyclic adenosine monophosphate |
| CN110157653A (en) * | 2019-05-09 | 2019-08-23 | 南京工业大学 | Recombinant escherichia coli for high-yield cyclic adenosine monophosphate and application of recombinant escherichia coli in cyclic adenosine monophosphate synthesis |
| CN112063670A (en) * | 2020-09-24 | 2020-12-11 | 杭州美亚药业股份有限公司 | Method for preparing cyclic adenosine monophosphate by adenylate cyclase |
| CN112442511A (en) * | 2021-01-04 | 2021-03-05 | 郑州大学 | Method for increasing adenylate cyclase expression activity and cAMP content in plants |
| CN112725323A (en) * | 2021-02-05 | 2021-04-30 | 南京工业大学 | Recombinant salt-tolerant adenylate cyclase and coding gene and application thereof |
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