CN101121932A - Method for preparing phycocyanin fluorescence protein - Google Patents
Method for preparing phycocyanin fluorescence protein Download PDFInfo
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- CN101121932A CN101121932A CNA2007100291238A CN200710029123A CN101121932A CN 101121932 A CN101121932 A CN 101121932A CN A2007100291238 A CNA2007100291238 A CN A2007100291238A CN 200710029123 A CN200710029123 A CN 200710029123A CN 101121932 A CN101121932 A CN 101121932A
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Abstract
The invention discloses a method for preparing phycocyanin fluorescent protein. The phycocyanin fluorescent protein is prepared by using phycobiliprotein beta lyase to catalyze phycocyanobilin and phycocyanin apoprotein for covalent combination. The invention is environment-friendly because bioprocess is adopted to produce phycocyanin fluorescent protein. Phycocyanin fluorescent protein is applicable to the fields of foodstuff, health-care and medical functional materials, particularly applicable to biological and medical molecule monitoring and as well as fluorescent probe.
Description
Technical field
The invention belongs to chromoprotein material field in the biotechnology, be specifically related to a kind of preparation method of phycocyanin fluorescence protein.
Technical background
Phycobiliprotein (phycobiliprotein) is the function ingredients that blue-green algae and red algae photosynthesis are caught the recovery compound.According to its absorption spectrum and fluorescence spectral characteristic, phycobiliprotein can be divided into phycoerythrin (phycoerythrin, abbreviation CPE), phycoerythrocyanin (pec) (phycoerythrocyanin, abbreviation PEC), Phycocyanins, C-(phycocyanin, be called for short CPC) and become Phycocyanins, C-(allophycocyanin is called for short APC).Phycoerythrin, phycoerythrocyanin (pec), Phycocyanins, C-and change Phycocyanins, C-contain alpha (α) and beta (β) subunit, phycobilin in each subunit (phycobilin) is by the sulfydryl covalent attachment of thioether bond and phycobiliprotein apoprotein (apo-phycobiliprotein) cysteine residues, its kind and with the spectral quality of the interaction decision phycobiliprotein of apoprotein.Phycobilin and apoprotein covalent attachment form specific conformation, make phycoerythrin mainly absorb the visible light of about 560nm, the fluorescence of the about 580nm of emission; Phycoerythrocyanin (pec) absorbs the visible light of about 570nm, the fluorescence of the about 630nm of emission; Phycocyanins, C-absorbs the visible light of about 620nm, the fluorescence of the about 640nm of emission; Become the visible light that Phycocyanins, C-absorbs about 650～660nm, launch the fluorescence of about 660～670nm.Phycoerythrin bonded prothetic group pigment is phycoerythrobilin (phycoerythrobilin is called for short PEB); The alpha subunit bonded prothetic group pigment of phycoerythrocyanin (pec) is the purple courage element of algae (phycoviolobilin is called for short PVB); The beta subunit bonded prothetic group pigment of phycoerythrocyanin (pec) is phycocyanobilin (phycocyanobilin is called for short PCB); Phycocyanins, C-and change Phycocyanins, C-bonded prothetic group pigment all are phycocyanobilin PCB.
The alpha subunit of Phycocyanins, C-can be produced (Tooley A J by the bacillus coli gene engineering bacteria, Cai Y A, Glazer A N.Biosynthesis of a fluorescent cyanobacterial C-phycocyanin holo-alphasubunit in a heterologous host[J] .Proc Natl Acad Sci USA, 2001,98 (19): 10560～10565).Phycocyanins, C-has the raising body immunity, promotes functions such as animal cell substitution, can grow by anticancer, and cancer cells is had very strong lethality, is a kind of desirable photosensitizers of no side effects, but the side effect of ameliorate tumor chemotherapy.Phycocyanins, C-is as a kind of natural pigment, and tone is extremely beautiful sky blue, also can be used as senior natural pigment and is applied in the foods and cosmetics.Phycocyanins, C-can be used for preparing fluorescent probe owing to also have the intensive fluorescence on molecular biology, be applicable to the research of aspects such as immunology, cytology, physiology and molecular biology.
The phycocyanobilin biosynthesis gene is made up of ho1 and pcyA, can produce phycocyanobilin (F.T.Landgraf by the bacillus coli gene engineering bacteria, C.Forreiter, et al.Recombinant holophytochrome in Echerichiacoli[J] .FEBS Letters, 2001,508:459-462).Different with forefathers' method, also there is homologous beta lyase in the slr2049 gene beta lyase of encoding respectively among alr0617 gene and the Synechocystis sp.PCC 6803 in other blue-green algae in Anabaena sp.PCC7120.The beta subunit and the homologous protein covalent attachment thereof of these beta lyases energy catalysis phycocyanobilins and Phycocyanins, C-, thus phycocyanin fluorescence protein generated with good photoluminescent property.In the blue-green algae and red algae of the overwhelming majority, all have coding phycoerythrin apoprotein, Phycocyanins, C-apoprotein, become the gene of the required enzyme of Phycocyanins, C-apoprotein, beta lyase and phycocyanobilin biosynthesizing, wherein some lacks PE and has and have phycoerythrocyanin (pec) in the blue-green algae of heterocyst; Do not have phycobilisome in the latent algae, do not have the gene of the change Phycocyanins, C-in the said gene.
The phycobiliprotein type fluorescence protein can be applied to food, makeup, medicine and bioengineering field.The phycobiliprotein type fluorescence protein that uses mainly extracts (separation method of high-purity biliprotein, CN1344723,2002.04.17 from blue-green algae and red algae at present.A kind of bloom blue algae prepares the method for Phycocyanins, C-, CN1563083,2005.01.12).Present method does not utilize algae as raw material, but utilizes gene engineering method to produce Phycocyanins, C-.Phycocyanin fluorescence protein has good photoluminescent property, in order to develop Phycocyanins, C-class novel fluorescence probe, and can the molecular designing phycocyanin fluorescence protein.Present method is that Phycocyanins, C-class chromoprotein matter functional materials is applied to food, makeup, medicine and bioengineering field and lays a good foundation.
Summary of the invention
The object of the present invention is to provide a kind of method for preparing phycocyanin fluorescence protein, it is to use beta lyase catalysis phycocyanobilin and Phycocyanins, C-class apoprotein covalent attachment, thus the preparation phycocyanin fluorescence protein; The expression plasmid that will contain beta lyase genes, Phycocyanins, C-class apoprotein gene and phycocyanobilin biosynthetic enzyme genes changes the host bacterium successively over to, obtain the corresponding engineering bacterium, the genetic engineering bacterium that obtains is by this method produced phycocyanin fluorescence protein.
The purpose of this invention is to provide a kind of method for preparing phycocyanin fluorescence protein, comprise following:
(1) adopts gene engineering method, beta lyase genes or its homologous gene are cloned in expression vector, obtain beta lyase expression plasmid; The using gene engineering method is cloned Phycocyanins, C-class apoprotein gene or its homologous gene in second expression vector similarly, obtains the apoprotein expression plasmid.Similarly the using gene engineering method with phycocyanobilin biosynthetic enzyme genes (ho1 and pcyA) or its homologous gene clone in the 3rd and (or) in the 4th expression vector, obtain phycocyanobilin synthetic enzyme expression plasmid.
(2) change beta lyase expression plasmid or apoprotein expression plasmid or phycocyanobilin synthetic enzyme expression plasmid over to supporting host bacterium successively; After these expression plasmids all change the host bacterium over to, obtain the corresponding engineering bacterium, use this engineering bacteria and can produce phycocyanin fluorescence protein by fermentation engineering; After the fermentation, pass through Ni
2+Affinity chromatography, can purify obtains corresponding phycocyanin fluorescence protein.
The present invention compared with prior art has the following advantages:
1, do not use algae as raw material but utilize intestinal bacteria to produce phycocyanin fluorescence protein, the intestinal bacteria breeding is fast, can shorten the cycle greatly;
2, compare with algae, the Bacillus coli cells wall is broken easily, can save the energy in the purge process;
3, produce by intestinal bacteria, target protein content height has the His-tag mark, and does not almost have kin albumen in the system, and it is convenient to extract;
4, conveniently carry out the phycobiliprotein molecular designing, help developing fluorescence phycobiliprotein class chromoprotein matter functional materials.
Description of drawings
Fig. 1 prepares the absorption and the fluorescence spectrum of phycocyanin fluorescence protein for the present invention; Wherein solid line is an absorption spectrum, and dotted line is a fluorescence spectrum.
Embodiment
The present invention is described in further detail below in conjunction with drawings and Examples:
Embodiment 1:
(1) adopts gene engineering method, can from GeneBank, find.The complete sequence of algae kind Anabaena sp.PCC7120 and Synechocystis sp.PCC 6803 is finished after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, and Synechocystis sp.PCC 6803 is numbered in GeneBank: BA000022, can find required gene from complete sequence; Alr0617 gene clone among the Anabaena sp.PCC7120 is in the pCDFDuet-1 of Novagen company, and the gained plasmid can be expressed the beta lyase pCDFDuet-alr0617 in intestinal bacteria.
Gene order according to finding among the GeneBank has designed primer, utilizes corresponding algae kind to extract total DNA as masterplate, obtains required fragment by pcr amplification.By the restriction enzyme site that designs in the upstream and downstream primer, target fragment is changed on the restriction enzyme site same in the corresponding plasmid.
(2) the using gene engineering method is with Phycocyanins, C-class apoprotein gene; Phycocyanins, C-B clones in the expression vector pET30 of Novagen company, and the gained plasmid can be expressed apoprotein Phycocyanins, C-B pET30-Phycocyanins, C-B in intestinal bacteria; Be lyase alr0617 or slr2049 in pCDFDuet, be in second multiple clone site, restriction enzyme site is Nde I and Xho I; When lyase and apoprotein during, the invariant position of lyase in carrier simultaneously at pCDFDuet, just the another one multiple clone site many an apoprotein gene.
Apoprotein cpcB is in pET30 between EcoRV and Xho I; When apoprotein was followed lyase simultaneously at pCDFDuet, apoprotein was in first multiple clone site, and used restriction enzyme site is EcoR I and Pst I.
The PCB synthetic enzyme is 2 genes (HO1 and PcyA), when they at same carrier the time, ho1 is in first multiple clone site, between Nco I and Pst I, pcyA is second multiple clone site, between Nde I and Xho I; As HO1 and PcyA not at same carrier the time, their position in corresponding carrier also is same restriction enzyme site.
(3) the using gene engineering method is cloned phycocyanobilin biosynthetic enzyme genes (ho1 and pcyA) in the pACYCDuet-1 of Novagen company, and the gained plasmid can be expressed HO1 and PcyA pACYCDuet-ho1-pcyA simultaneously in intestinal bacteria.The step of gene insertion vector is: according to gene order design primer, divide upstream and downstream, and a pair of.The downstream, upstream all has restriction enzyme site respectively; Utilize corresponding algae kind to extract total DNA as masterplate.Obtain required gene fragment through pcr amplification, the gained gene fragment is carried out electrophoresis, reclaim.The gained fragment is carried out enzyme and is cut with the enzyme of design, also carrier is cut with same enzyme simultaneously, reclaims gene fragment and carrier respectively; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4-6 hour) under the effect of T4 ligase enzyme; Connect the product conversion and enter intestinal bacteria, utilize to contain antibiotic plate screening, the picking clone, checking correctly gets final product.
(4) combination with used plasmid changes in the intestinal bacteria, and transform mode is as follows:
The single bacterium colony of picking coli strain BL21 (DE3) from the flat board of fresh coating is inoculated in 5mL LB substratum, and 37 ℃ of shaking culture are spent the night.Get 100 μ L saturated culture, the aseptic 5mL LB substratum that is forwarded to, 37 ℃ of shaking culture are transferred to bacterium liquid in the aseptic centrifuge tube of 5mL to OD600=0.3～0.4 o'clock, place 10min on ice.Centrifugal 1min (10000g, 4 ℃) supernatant discarded, the collecting precipitation thalline.The resuspended thalline of 0.1mol/L CaCl2 sterile solution with the 1mL precooling, centrifugal 30s (10000g, 4 ℃) remove supernatant, bacterial sediment is resuspended with the 0.1mol/L CaCl2 of 100 μ L precoolings, be positioned on ice, the plasmid that adds a certain amount of connection product or build, the mixing ice bath is placed 30min, 42 ℃ of heat-shocked 90s, add 300 μ L LB substratum behind the ice bath 5min, 37 ℃ of low-speed oscillations are cultivated 45min, and aseptic being coated on contained on the corresponding antibiotic LB culture medium flat plate, is inverted in 37 ℃ of incubators to forming visible mono-clonal bacterial plaque.
Change pCDFDuet-alr0617, pET30-Phycocyanins, C-B and pACYCDuet-ho1-pcyA over to intestinal bacteria, after these plasmids all change intestinal bacteria over to, obtain colibacillus engineering.This engineering bacteria is inoculated in the LB substratum of pH7.0,37 ℃ of shaking culture are to OD
600Be 0.5 to 0.8, add isopropylthio-(isoprophylthio-β-D-galactoside, be called for short IPTG) to final concentration be 1mmol/L, 20 ℃ to 37 ℃ vibrations were expressed 12 to 16 hours, produced phycocyanin fluorescence protein phycocyanobilin-Phycocyanins, C-B.Centrifugal collection somatic cells, behind adding damping fluid, the smudge cells, centrifugal collection supernatant liquor passes through Ni
2+Affinity chromatography, can purify obtains corresponding phycocyanin fluorescence protein phycocyanobilin-Phycocyanins, C-B.
Embodiment 2:
(1) adopts gene engineering method, can from GeneBank, find.The complete sequence of algae kind Anabaena sp.PCC7120 and Synechocystis sp.PCC 6803 is finished after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, and Synechocystis sp.PCC 6803 is numbered in GeneBank: BA000022, can find required gene from complete sequence; Alr0617 gene clone among the Anabaena sp.PCC7120 is in the pCDFDuet-1 of Novagen company, and the gained plasmid can be expressed the beta lyase pCDFDuet-alr0617 in intestinal bacteria.
Gene order according to finding among the GeneBank has designed primer, utilizes corresponding algae kind to extract total DNA as masterplate, obtains required fragment by pcr amplification.By the restriction enzyme site that designs in the upstream and downstream primer, target fragment is changed on the restriction enzyme site same in the corresponding plasmid.
(2) the using gene engineering method is cloned Phycocyanins, C-class apoprotein gene Phycocyanins, C-B (C155I) in the expression vector pET30 of Novagen company, the gained plasmid can be expressed apoprotein Phycocyanins, C-B (C155I) pET30-Phycocyanins, C-B (C155I) in intestinal bacteria; Be lyase alr0617 or slr2049 in pCDFDuet, be in second multiple clone site, restriction enzyme site is Nde I and Xho I; When lyase and apoprotein during, the invariant position of lyase in carrier simultaneously at pCDFDuet, just the another one multiple clone site many an apoprotein gene.
Apoprotein cpcB is in pET30 between EcoRV and Xho I; When apoprotein was followed lyase simultaneously at pCDFDuet, apoprotein was in first multiple clone site, and used restriction enzyme site is EcoR I and Pst I.
The PCB synthetic enzyme is 2 genes (H01 and PcyA), when they at same carrier the time, ho1 is in first multiple clone site, between Nco I and Pst I, pcyA is second multiple clone site, between Nde I and Xho I; As HO1 and PcyA not at same carrier the time, their position in corresponding carrier also is same restriction enzyme site.
(3) the using gene engineering method is cloned phycocyanobilin biosynthetic enzyme genes (ho1 and pcyA) in the pACYCDuet-1 of Novagen company, and the gained plasmid can be expressed HO1 and PcyA pACYCDuet-ho1-pcyA simultaneously in intestinal bacteria.The step of gene insertion vector is: according to gene order design primer, divide upstream and downstream, and a pair of.The downstream, upstream all has restriction enzyme site respectively; Utilize corresponding algae kind to extract total DNA as masterplate.Obtain required gene fragment through pcr amplification, the gained gene fragment is carried out electrophoresis, reclaim.The gained fragment is carried out enzyme and is cut with the enzyme of design, also carrier is cut with same enzyme simultaneously, reclaims gene fragment and carrier respectively; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4-6 hour) under the effect of T4 ligase enzyme; Connect the product conversion and enter intestinal bacteria, utilize to contain antibiotic plate screening, the picking clone, checking correctly gets final product.
(4) combination with used plasmid changes in the intestinal bacteria, and transform mode is as follows:
The single bacterium colony of picking coli strain BL21 (DE3) from the flat board of fresh coating is inoculated in 5mL LB substratum, and 37 ℃ of shaking culture are spent the night.Get 100 μ L saturated culture, the aseptic 5mL LB substratum that is forwarded to, 37 ℃ of shaking culture are transferred to bacterium liquid in the aseptic centrifuge tube of 5mL to OD600=0.3～0.4 o'clock, place 10min on ice.Centrifugal 1min (10000g, 4 ℃) supernatant discarded, the collecting precipitation thalline.The resuspended thalline of 0.1mol/L CaCl2 sterile solution with the 1mL precooling, centrifugal 30s (10000g, 4 ℃) remove supernatant, bacterial sediment is resuspended with the 0.1mol/L CaCl2 of 100 μ L precoolings, be positioned on ice, the plasmid that adds a certain amount of connection product or build, the mixing ice bath is placed 30min, 42 ℃ of heat-shocked 90s, add 300 μ L LB substratum behind the ice bath 5min, 37 ℃ of low-speed oscillations are cultivated 45min, and aseptic being coated on contained on the corresponding antibiotic LB culture medium flat plate, is inverted in 37 ℃ of incubators to forming visible mono-clonal bacterial plaque.
Change pCDFDuet-alr0617, pET30-Phycocyanins, C-B (C155I) and pACYCDuet-ho1-pcyA over to intestinal bacteria, obtain colibacillus engineering after these plasmids all change intestinal bacteria over to, this engineering bacteria is produced phycocyanin fluorescence protein phycocyanobilin-Phycocyanins, C-B (C155I) by fermentation energy.Protein purification techniques routinely, can purify obtains corresponding phycocyanin fluorescence protein phycocyanobilin-Phycocyanins, C-B (C155I).
Embodiment 3:
(1) adopts gene engineering method, can from GeneBank, find.The complete sequence of algae kind Anabaena sp.PCC7120 and Synechocystis sp.PCC 6803 is finished after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, and Synechocystis sp.PCC 6803 is numbered in GeneBank: BA000022, can find required gene from complete sequence; The slr2049 gene clone is in the pCDFDuet-1 of Novagen company among the Synechocystis sp.PCC 6803, and the gained plasmid can be expressed the beta lyase pCDFDuet-slr2049 in intestinal bacteria.
Gene order according to finding among the GeneBank has designed primer, utilizes corresponding algae kind to extract total DNA as masterplate, obtains required fragment by pcr amplification.By the restriction enzyme site that designs in the upstream and downstream primer, target fragment is changed on the restriction enzyme site same in the corresponding plasmid.
(2) the using gene engineering method is cloned Phycocyanins, C-class apoprotein gene Phycocyanins, C-B in the expression vector pET30 of Novagen company, and the gained plasmid can be expressed apoprotein Phycocyanins, C-B pET30-Phycocyanins, C-B in intestinal bacteria; Be lyase alr0617 or slr2049 in pCDFDuet, be in second multiple clone site, restriction enzyme site is Nde I and Xho I; When lyase and apoprotein during, the invariant position of lyase in carrier simultaneously at pCDFDuet, just the another one multiple clone site many an apoprotein gene.
Apoprotein cpcB is in pET30 between EcoRV and Xho I; When apoprotein was followed lyase simultaneously at pCDFDuet, apoprotein was in first multiple clone site, and used restriction enzyme site is EcoR I and Pst I.
The PCB synthetic enzyme is 2 genes (HO1 and PcyA), when they at same carrier the time, ho1 is in first multiple clone site, between Nco I and Pst I, pcyA is second multiple clone site, between Nde I and Xho I; As HO1 and PcyA not at same carrier the time, their position in corresponding carrier also is same restriction enzyme site.
(3) the using gene engineering method is cloned phycocyanobilin biosynthetic enzyme genes (ho1 and pcyA) in the pACYCDuet-1 of Novagen company, and the gained plasmid can be expressed HO1 and PcyA pACYCDuet-ho1-pcyA simultaneously in intestinal bacteria.The step of gene insertion vector is: according to gene order design primer, divide upstream and downstream, and a pair of.The downstream, upstream all has restriction enzyme site respectively; Utilize corresponding algae kind to extract total DNA as masterplate.Obtain required gene fragment through pcr amplification, the gained gene fragment is carried out electrophoresis, reclaim.The gained fragment is carried out enzyme and is cut with the enzyme of design, also carrier is cut with same enzyme simultaneously, reclaims gene fragment and carrier respectively; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4-6 hour) under the effect of T4 ligase enzyme; Connect the product conversion and enter intestinal bacteria, utilize to contain antibiotic plate screening, the picking clone, checking correctly gets final product.
(4) combination with used plasmid changes in the intestinal bacteria, and transform mode is as follows:
The single bacterium colony of picking coli strain BL21 (DE3) from the flat board of fresh coating is inoculated in 5mL LB substratum, and 37 ℃ of shaking culture are spent the night.Get 100 μ L saturated culture, the aseptic 5mL LB substratum that is forwarded to, 37 ℃ of shaking culture are transferred to bacterium liquid in the aseptic centrifuge tube of 5mL to OD600=0.3～0.4 o'clock, place 10min on ice.Centrifugal 1min (10000g, 4 ℃) supernatant discarded, the collecting precipitation thalline.The resuspended thalline of 0.1mol/L CaCl2 sterile solution with the 1mL precooling, centrifugal 30s (10000g, 4 ℃) remove supernatant, bacterial sediment is resuspended with the 0.1mol/L CaCl2 of 100 μ L precoolings, be positioned on ice, the plasmid that adds a certain amount of connection product or build, the mixing ice bath is placed 30min, 42 ℃ of heat-shocked 90s, add 300 μ L LB substratum behind the ice bath 5min, 37 ℃ of low-speed oscillations are cultivated 45min, and aseptic being coated on contained on the corresponding antibiotic LB culture medium flat plate, is inverted in 37 ℃ of incubators to forming visible mono-clonal bacterial plaque.
Change pCDFDuet-slr2049, pET30-Phycocyanins, C-B and pACYCDuet-ho1-pcyA over to intestinal bacteria, obtain colibacillus engineering after these plasmids all change intestinal bacteria over to, this engineering bacteria is produced phycocyanin fluorescence protein phycocyanobilin-Phycocyanins, C-B by fermentation energy.Protein purification techniques routinely, can purify obtains corresponding phycocyanin fluorescence protein phycocyanobilin-Phycocyanins, C-B.
Embodiment 4:
(1) adopts gene engineering method, can from GeneBank, find.The complete sequence of algae kind Anabaena sp.PCC7120 and Synechocystis sp.PCC 6803 is finished after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, and Synechocystis sp.PCC 6803 is numbered in GeneBank: BA000022, can find required gene from complete sequence; The slr2049 gene clone is in the pCDFDuet-1 of Novagen company among the Synechocystis sp.PCC 6803, and the gained plasmid can be expressed the beta lyase pCDFDuet-slr2049 in intestinal bacteria.
Gene order according to finding among the GeneBank has designed primer, utilizes corresponding algae kind to extract total DNA as masterplate, obtains required fragment by pcr amplification.By the restriction enzyme site that designs in the upstream and downstream primer, target fragment is changed on the restriction enzyme site same in the corresponding plasmid.
(2) the using gene engineering method is cloned Phycocyanins, C-class apoprotein gene Phycocyanins, C-B (C155I) in the expression vector pET30 of Novagen company, the gained plasmid can be expressed apoprotein Phycocyanins, C-B (C155I) pET30-Phycocyanins, C-B (C155I) in intestinal bacteria; Be lyase alr0617 or slr2049 in pCDFDuet, be in second multiple clone site, restriction enzyme site is Nde I and Xho I; When lyase and apoprotein during, the invariant position of lyase in carrier simultaneously at pCDFDuet, just the another one multiple clone site many an apoprotein gene.
Apoprotein cpcB is in pET30 between EcoRV and Xho I; When apoprotein was followed lyase simultaneously at pCDFDuet, apoprotein was in first multiple clone site, and used restriction enzyme site is EcoR I and Pst I.
The PCB synthetic enzyme is 2 genes (HO1 and PcyA), when they at same carrier the time, ho1 is in first multiple clone site, between Nco I and Pst I, pcyA is second multiple clone site, between Nde I and Xho I; As HO1 and PcyA not at same carrier the time, their position in corresponding carrier also is same restriction enzyme site.
(3) the using gene engineering method is cloned phycocyanobilin biosynthetic enzyme genes (ho1 and pcyA) in the pACYCDuet-1 of Novagen company, and the gained plasmid can be expressed HO1 and PcyA pACYCDuet-ho1-pcyA simultaneously in intestinal bacteria.The step of gene insertion vector is: according to gene order design primer, divide upstream and downstream, and a pair of.The downstream, upstream all has restriction enzyme site respectively; Utilize corresponding algae kind to extract total DNA as masterplate.Obtain required gene fragment through pcr amplification, the gained gene fragment is carried out electrophoresis, reclaim.The gained fragment is carried out enzyme and is cut with the enzyme of design, also carrier is cut with same enzyme simultaneously, reclaims gene fragment and carrier respectively; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4-6 hour) under the effect of T4 ligase enzyme; Connect the product conversion and enter intestinal bacteria, utilize to contain antibiotic plate screening, the picking clone, checking correctly gets final product.
(4) combination with used plasmid changes in the intestinal bacteria, and transform mode is as follows:
The single bacterium colony of picking coli strain BL21 (DE3) from the flat board of fresh coating is inoculated in 5mL LB substratum, and 37 ℃ of shaking culture are spent the night.Get 100 μ L saturated culture, the aseptic 5mL LB substratum that is forwarded to, 37 ℃ of shaking culture are transferred to bacterium liquid in the aseptic centrifuge tube of 5mL to OD600=0.3～0.4 o'clock, place 10min on ice.Centrifugal 1min (10000g, 4 ℃) supernatant discarded, the collecting precipitation thalline.The resuspended thalline of 0.1mol/L CaCl2 sterile solution with the 1mL precooling, centrifugal 30s (10000g, 4 ℃) remove supernatant, bacterial sediment is resuspended with the 0.1mol/L CaCl2 of 100 μ L precoolings, be positioned on ice, the plasmid that adds a certain amount of connection product or build, the mixing ice bath is placed 30min, 42 ℃ of heat-shocked 90s, add 300 μ L LB substratum behind the ice bath 5min, 37 ℃ of low-speed oscillations are cultivated 45min, and aseptic being coated on contained on the corresponding antibiotic LB culture medium flat plate, is inverted in 37 ℃ of incubators to forming visible mono-clonal bacterial plaque.
Change pCDFDuet-slr2049, pET30-Phycocyanins, C-B (C155I) and pACYCDuet-ho1-pcyA over to intestinal bacteria, obtain colibacillus engineering after these plasmids all change intestinal bacteria over to, this engineering bacteria is produced phycocyanin fluorescence protein phycocyanobilin-Phycocyanins, C-B (C155I) by fermentation energy.Protein purification techniques routinely, can purify obtains corresponding phycocyanin fluorescence protein phycocyanobilin-Phycocyanins, C-B (C155I).
Embodiment 5:
(1) adopts gene engineering method, can from GeneBank, find.The complete sequence of algae kind Anabaena sp.PCC7120 and Synechocystis sp.PCC 6803 is finished after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, and Synechocystis sp.PCC 6803 is numbered in GeneBank: BA000022, can find required gene from complete sequence; Alr0617 among the Anabaena sp.PCC7120 and Phycocyanins, C-B gene clone are in the pCDFDuet-1 of Novagen company, and the gained plasmid can be expressed beta lyase and Phycocyanins, C-B pCDFDuet-alr0617-Phycocyanins, C-B simultaneously in intestinal bacteria.
Gene order according to finding among the GeneBank has designed primer, utilizes corresponding algae kind to extract total DNA as masterplate, obtains required fragment by pcr amplification.By the restriction enzyme site that designs in the upstream and downstream primer, target fragment is changed on the restriction enzyme site same in the corresponding plasmid.
(2) the using gene engineering method is cloned phycocyanobilin biosynthetic enzyme genes (ho1 and pcyA) in the pACYCDuet-1 of Novagen company, and the gained plasmid can be expressed HO1 and PcyA pACYCDuet-ho1-pcyA simultaneously in intestinal bacteria; Be lyase alr0617 or slr2049 in pCDFDuet, be in second multiple clone site, restriction enzyme site is Nde I and Xho I; When lyase and apoprotein during, the invariant position of lyase in carrier simultaneously at pCDFDuet, just the another one multiple clone site many an apoprotein gene.
Apoprotein cpcB is in pET30 between EcoRV and Xho I; When apoprotein was followed lyase simultaneously at pCDFDuet, apoprotein was in first multiple clone site, and used restriction enzyme site is EcoR I and Pst I.
The PCB synthetic enzyme is 2 genes (HO1 and PcyA), when they at same carrier the time, ho1 is in first multiple clone site, between Nco I and Pst I, pcyA is second multiple clone site, between Nde I and Xho I; As HO1 and PcyA not at same carrier the time, their position in corresponding carrier also is same restriction enzyme site.
(3) change pCDFDuet-alr0617-Phycocyanins, C-B and pACYCDuet-ho1-pcyA over to intestinal bacteria successively, obtain colibacillus engineering after these plasmids all change intestinal bacteria over to, this engineering bacteria is produced phycocyanin fluorescence protein phycocyanobilin-Phycocyanins, C-B by fermentation energy.Protein purification techniques routinely, can purify obtains corresponding phycocyanin fluorescence protein phycocyanobilin-Phycocyanins, C-B.The step of gene insertion vector is: according to gene order design primer, divide upstream and downstream, and a pair of.The downstream, upstream all has restriction enzyme site respectively; Utilize corresponding algae kind to extract total DNA as masterplate.Obtain required gene fragment through pcr amplification, the gained gene fragment is carried out electrophoresis, reclaim.The gained fragment is carried out enzyme and is cut with the enzyme of design, also carrier is cut with same enzyme simultaneously, reclaims gene fragment and carrier respectively; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4-6 hour) under the effect of T4 ligase enzyme; Connect the product conversion and enter intestinal bacteria, utilize to contain antibiotic plate screening, the picking clone, checking correctly gets final product.
Embodiment 6:
1) adopts gene engineering method, can from GeneBank, find.The complete sequence of algae kind Anabaena sp.PCC7120 and Synechocystis sp.PCC 6803 is finished after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, and Synechocystis sp.PCC 6803 is numbered in GeneBank: BA000022, can find required gene from complete sequence; Alr0617 among the Anabaena sp.PCC7120 and Phycocyanins, C-B (C155I) gene clone are in the pCDFDuet-1 of Novagen company, the gained plasmid can be expressed beta lyase and Phycocyanins, C-B (C155I) simultaneously pCDFDuet-alr0617-Phycocyanins, C-B (C155I) in intestinal bacteria.
Gene order according to finding among the GeneBank has designed primer, utilizes corresponding algae kind to extract total DNA as masterplate, obtains required fragment by pcr amplification.By the restriction enzyme site that designs in the upstream and downstream primer, target fragment is changed on the restriction enzyme site same in the corresponding plasmid.
(2) the using gene engineering method is cloned phycocyanobilin biosynthetic enzyme genes (ho1 and pcyA) in the pACYCDuet-1 of Novagen company, and the gained plasmid can be expressed HO1 and PcyA pACYCDuet-ho1-pcyA simultaneously in intestinal bacteria; Be lyase alr0617 or slr2049 in pCDFDuet, be in second multiple clone site, restriction enzyme site is Nde I and Xho I; When lyase and apoprotein during, the invariant position of lyase in carrier simultaneously at pCDFDuet, just the another one multiple clone site many an apoprotein gene.
Apoprotein cpcB is in pET30 between EcoRV and Xho I; When apoprotein was followed lyase simultaneously at pCDFDuet, apoprotein was in first multiple clone site, and used restriction enzyme site is EcoR I and Pst I.
The PCB synthetic enzyme is 2 genes (HO1 and PcyA), when they at same carrier the time, ho1 is in first multiple clone site, between Nco I and Pst I, pcyA is second multiple clone site, between Nde I and Xho I; As HO1 and PcyA not at same carrier the time, their position in corresponding carrier also is same restriction enzyme site.
(3) step of gene insertion vector is: according to gene order design primer, divide upstream and downstream, and a pair of.The downstream, upstream all has restriction enzyme site respectively; Utilize corresponding algae kind to extract total DNA as masterplate.Obtain required gene fragment through pcr amplification, the gained gene fragment is carried out electrophoresis, reclaim.The gained fragment is carried out enzyme and is cut with the enzyme of design, also carrier is cut with same enzyme simultaneously, reclaims gene fragment and carrier respectively; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4-6 hour) under the effect of T4 ligase enzyme; Connect the product conversion and enter intestinal bacteria, utilize to contain antibiotic plate screening, the picking clone, checking correctly gets final product.
Change pCDFDuet-alr0617-Phycocyanins, C-B (C155I) and pACYCDuet-ho1-pcyA over to intestinal bacteria, obtain colibacillus engineering after these plasmids all change intestinal bacteria over to, this engineering bacteria is produced phycocyanin fluorescence protein phycocyanobilin-Phycocyanins, C-B (C155I) by fermentation energy.Protein purification techniques routinely, can purify obtains corresponding phycocyanin fluorescence protein phycocyanobilin-Phycocyanins, C-B (C155I).
Embodiment 7:
(1) adopts gene engineering method, can from GeneBank, find.The complete sequence of algae kind Anabaena sp.PCC7120 and Synechocystis sp.PCC 6803 is finished after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, and Synechocystis sp.PCC 6803 is numbered in GeneBank: BA000022, can find required gene from complete sequence; Slr2049 among the Synechocystis sp.PCC 6803 and Phycocyanins, C-B gene clone are in the pCDFDuet-1 of Novagen company, the gained plasmid can be expressed beta lyase and Phycocyanins, C-B pCDFDuet-slr2049-Phycocyanins, C-B simultaneously in intestinal bacteria.
Gene order according to finding among the GeneBank has designed primer, utilizes corresponding algae kind to extract total DNA as masterplate, obtains required fragment by pcr amplification.By the restriction enzyme site that designs in the upstream and downstream primer, target fragment is changed on the restriction enzyme site same in the corresponding plasmid.
(2) the using gene engineering method is cloned phycocyanobilin biosynthetic enzyme genes (ho1 and pcyA) in the pACYCDuet-1 of Novagen company, and the gained plasmid can be expressed HO1 and PcyA pACYCDuet-ho1-pcyA simultaneously in intestinal bacteria; Be lyase alr0617 or slr2049 in pCDFDuet, be in second multiple clone site, restriction enzyme site is Nde I and Xho I; When lyase and apoprotein during, the invariant position of lyase in carrier simultaneously at pCDFDuet, just the another one multiple clone site many an apoprotein gene.
Apoprotein cpcB is in pET30 between EcoRV and Xho I; When apoprotein was followed lyase simultaneously at pCDFDuet, apoprotein was in first multiple clone site, and used restriction enzyme site is EcoR I and Pst I.
The PCB synthetic enzyme is 2 genes (HO1 and PcyA), when they at same carrier the time, ho1 is in first multiple clone site, between Nco I and Pst I, pcyA is second multiple clone site, between Nde I and Xho I; As HO1 and PcyA not at same carrier the time, their position in corresponding carrier also is same restriction enzyme site.
(3) step of gene insertion vector is: according to gene order design primer, divide upstream and downstream, and a pair of.The downstream, upstream all has restriction enzyme site respectively; Utilize corresponding algae kind to extract total DNA as masterplate.Obtain required gene fragment through pcr amplification, the gained gene fragment is carried out electrophoresis, reclaim.The gained fragment is carried out enzyme and is cut with the enzyme of design, also carrier is cut with same enzyme simultaneously, reclaims gene fragment and carrier respectively; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4-6 hour) under the effect of T4 ligase enzyme; Connect the product conversion and enter intestinal bacteria, utilize to contain antibiotic plate screening, the picking clone, checking correctly gets final product.
Change pCDFDuet-slr2049-Phycocyanins, C-B and pACYCDuet-ho1-pcyA over to intestinal bacteria, obtain colibacillus engineering after these plasmids all change intestinal bacteria over to, this engineering bacteria is produced phycocyanin fluorescence protein phycocyanobilin-Phycocyanins, C-B by fermentation energy.Protein purification techniques routinely, can purify obtains corresponding phycocyanin fluorescence protein phycocyanobilin-Phycocyanins, C-B.
Embodiment 8:
1) adopts gene engineering method, can from GeneBank, find.The complete sequence of algae kind Anabaena sp.PCC7120 and Synechocystis sp.PCC 6803 is finished after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, and Synechocystis sp.PCC 6803 is numbered in GeneBank: BA000022, can find required gene from complete sequence; Slr2049 among the Synechocystis sp.PCC 6803 and Phycocyanins, C-B (C155I) gene clone are in the pCDFDuet-1 of Novagen company, the gained plasmid can be expressed beta lyase and Phycocyanins, C-B (C155I) simultaneously pCDFDuet-slr2049-Phycocyanins, C-B (C155I) in intestinal bacteria.
Gene order according to finding among the GeneBank has designed primer, utilizes corresponding algae kind to extract total DNA as masterplate, obtains required fragment by pcr amplification.By the restriction enzyme site that designs in the upstream and downstream primer, target fragment is changed on the restriction enzyme site same in the corresponding plasmid.
(2) the using gene engineering method is cloned phycocyanobilin biosynthetic enzyme genes (ho1 and pcyA) in the pACYCDuet-1 of Novagen company, and the gained plasmid can be expressed HO1 and PcyA pACYCDuet-ho1-pcyA simultaneously in intestinal bacteria; Be lyase alr0617 or slr2049 in pCDFDuet, be in second multiple clone site, restriction enzyme site is Nde I and Xho I; When lyase and apoprotein during, the invariant position of lyase in carrier simultaneously at pCDFDuet, just the another one multiple clone site many an apoprotein gene.
Apoprotein cpcB is in pET30 between EcoRV and Xho I; When apoprotein was followed lyase simultaneously at pCDFDuet, apoprotein was in first multiple clone site, and used restriction enzyme site is EcoR I and Pst I.
The PCB synthetic enzyme is 2 genes (HO1 and PcyA), when they at same carrier the time, ho1 is in first multiple clone site, between Nco I and Pst I, pcyA is second multiple clone site, between Nde I and Xho I; As HO1 and PcyA not at same carrier the time, their position in corresponding carrier also is same restriction enzyme site.
(3) step of gene insertion vector is: according to gene order design primer, divide upstream and downstream, and a pair of.The downstream, upstream all has restriction enzyme site respectively; Utilize corresponding algae kind to extract total DNA as masterplate.Obtain required gene fragment through pcr amplification, the gained gene fragment is carried out electrophoresis, reclaim.The gained fragment is carried out enzyme and is cut with the enzyme of design, also carrier is cut with same enzyme simultaneously, reclaims gene fragment and carrier respectively; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4-6 hour) under the effect of T4 ligase enzyme; Connect the product conversion and enter intestinal bacteria, utilize to contain antibiotic plate screening, the picking clone, checking correctly gets final product.
Change pCDFDuet-slr2049-Phycocyanins, C-B (C155I) and pACYCDuet-ho1-pcyA over to intestinal bacteria, obtain colibacillus engineering after these plasmids all change intestinal bacteria over to, this engineering bacteria is produced phycocyanin fluorescence protein phycocyanobilin-Phycocyanins, C-B (C155I) by fermentation energy.Protein purification techniques routinely, can purify obtains corresponding phycocyanin fluorescence protein phycocyanobilin-Phycocyanins, C-B (C155I).
Embodiment 9:
(1) adopts gene engineering method, can from GeneBank, find.The complete sequence of algae kind Anabaena sp.PCC7120 and Synechocystis sp.PCC 6803 is finished after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, and Synechocystis sp.PCC 6803 is numbered in GeneBank: BA000022, can find required gene from complete sequence; Alr0617 gene clone among the Anabaena sp.PCC7120 is in the pCDFDuet-1 of Novagen company, and the gained plasmid can be expressed the beta lyase pCDFDuet-alr0617 in intestinal bacteria.
Gene order according to finding among the GeneBank has designed primer, utilizes corresponding algae kind to extract total DNA as masterplate, obtains required fragment by pcr amplification.By the restriction enzyme site that designs in the upstream and downstream primer, target fragment is changed on the restriction enzyme site same in the corresponding plasmid.
(2) the using gene engineering method is cloned Phycocyanins, C-class apoprotein gene Phycocyanins, C-B in the expression vector pCOLADuet-1 of Novagen company, the gained plasmid can be expressed apoprotein Phycocyanins, C-B pCOLADuet-Phycocyanins, C-B in intestinal bacteria; Be lyase alr0617 or slr2049 in pCDFDuet, be in second multiple clone site, restriction enzyme site is Nde I and Xho I; When lyase and apoprotein during, the invariant position of lyase in carrier simultaneously at pCDFDuet, just the another one multiple clone site many an apoprotein gene.
Apoprotein cpcB is in pET30 between EcoRV and Xho I; When apoprotein was followed lyase simultaneously at pCDFDuet, apoprotein was in first multiple clone site, and used restriction enzyme site is EcoR I and Pst I.
The PCB synthetic enzyme is 2 genes (HO1 and PcyA), when they at same carrier the time, ho1 is in first multiple clone site, between Nco I and Pst I, pcyA is second multiple clone site, between Nde I and Xho I; As HO1 and PcyA not at same carrier the time, their position in corresponding carrier also is same restriction enzyme site.
(3) the using gene engineering method is cloned the ho1 of one of phycocyanobilin biosynthesis gene in the pACYCDuet-1 of Novagen company, and the gained plasmid can be expressed HO1 pACYCDuet-ho1 in intestinal bacteria.
(4) the using gene engineering method is cloned the pcyA of one of phycocyanobilin biosynthesis gene in the pETDuet-1 of Novagen company, and the gained plasmid can be expressed PcyA pETDuet-pcyA in intestinal bacteria.
(5) step of gene insertion vector is: according to gene order design primer, divide upstream and downstream, and a pair of.The downstream, upstream all has restriction enzyme site respectively; Utilize corresponding algae kind to extract total DNA as masterplate.Obtain required gene fragment through pcr amplification, the gained gene fragment is carried out electrophoresis, reclaim.The gained fragment is carried out enzyme and is cut with the enzyme of design, also carrier is cut with same enzyme simultaneously, reclaims gene fragment and carrier respectively; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4-6 hour) under the effect of T4 ligase enzyme; Connect the product conversion and enter intestinal bacteria, utilize to contain antibiotic plate screening, the picking clone, checking correctly gets final product.
Change pCDFDuet-alr0617, pCOLADuet-Phycocyanins, C-B, pACYCDuet-ho1 and pETDuet-pcyA over to intestinal bacteria, obtain colibacillus engineering after these plasmids all change intestinal bacteria over to, this engineering bacteria is produced phycocyanin fluorescence protein phycocyanobilin-Phycocyanins, C-B by fermentation energy.Protein purification techniques routinely, can purify obtains corresponding phycocyanin fluorescence protein phycocyanobilin-Phycocyanins, C-B.
Embodiment 10:
(1) adopts gene engineering method, can from GeneBank, find.The complete sequence of algae kind Anabaena sp.PCC7120 and Synechocystis sp.PCC 6803 is finished after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, and Synechocystis sp.PCC 6803 is numbered in GeneBank: BA000022, can find required gene from complete sequence; Alr0617 gene clone among the Anabaena sp.PCC7120 is in the pCDFDuet-1 of Novagen company, and the gained plasmid can be expressed the beta lyase pCDFDuet-alr0617 in intestinal bacteria.
Gene order according to finding among the GeneBank has designed primer, utilizes corresponding algae kind to extract total DNA as masterplate, obtains required fragment by pcr amplification.By the restriction enzyme site that designs in the upstream and downstream primer, target fragment is changed on the restriction enzyme site same in the corresponding plasmid.
(2) the using gene engineering method is cloned Phycocyanins, C-class apoprotein gene Phycocyanins, C-B (C155I) in the expression vector pCOLADuet-1 of Novagen company, the gained plasmid can be expressed apoprotein Phycocyanins, C-B (C155I) pCOLADuet-Phycocyanins, C-B (C155I) in intestinal bacteria; Be lyase alr0617 or slr2049 in pCDFDuet, be in second multiple clone site, restriction enzyme site is Nde I and Xho I; When lyase and apoprotein during, the invariant position of lyase in carrier simultaneously at pCDFDuet, just the another one multiple clone site many an apoprotein gene.
Apoprotein cpcB is in pET30 between EcoRV and Xho I; When apoprotein was followed lyase simultaneously at pCDFDuet, apoprotein was in first multiple clone site, and used restriction enzyme site is EcoR I and Pst I.
The PCB synthetic enzyme is 2 genes (HO1 and PcyA), when they at same carrier the time, ho1 is in first multiple clone site, between Nco I and Pst I, pcyA is second multiple clone site, between Nde I and Xho I; As HO1 and PcyA not at same carrier the time, their position in corresponding carrier also is same restriction enzyme site.
(3) the using gene engineering method is cloned the ho1 of one of phycocyanobilin biosynthesis gene in the pACYCDuet-1 of Novagen company, and the gained plasmid can be expressed HO1 pACYCDuet-ho1 in intestinal bacteria.
(4) the using gene engineering method is cloned the pcyA of one of phycocyanobilin biosynthesis gene in the pETDuet-1 of Novagen company, and the gained plasmid can be expressed PcyA pETDuet-pcyA in intestinal bacteria.
(5) step of gene insertion vector is: according to gene order design primer, divide upstream and downstream, and a pair of.The downstream, upstream all has restriction enzyme site respectively; Utilize corresponding algae kind to extract total DNA as masterplate.Obtain required gene fragment through pcr amplification, the gained gene fragment is carried out electrophoresis, reclaim.The gained fragment is carried out enzyme and is cut with the enzyme of design, also carrier is cut with same enzyme simultaneously, reclaims gene fragment and carrier respectively; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4-6 hour) under the effect of T4 ligase enzyme; Connect the product conversion and enter intestinal bacteria, utilize to contain antibiotic plate screening, the picking clone, checking correctly gets final product.
Change pCDFDuet-alr0617, pCOLADuet-Phycocyanins, C-B (C155I), pACYCDuet-ho1 and pETDuet-pcyA over to intestinal bacteria, obtain colibacillus engineering after these plasmids all change intestinal bacteria over to, this engineering bacteria is produced phycocyanin fluorescence protein phycocyanobilin-Phycocyanins, C-B (C155I) by fermentation energy.Protein purification techniques routinely, can purify obtains corresponding phycocyanin fluorescence protein phycocyanobilin-Phycocyanins, C-B (C155I).
Embodiment 11:
(1) adopts gene engineering method, can from GeneBank, find.The complete sequence of algae kind Anabaena sp.PCC7120 and Synechocystis sp.PCC 6803 is finished after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, and Synechocystis sp.PCC 6803 is numbered in GeneBank: BA000022, can find required gene from complete sequence; The slr2049 gene clone is in the pCDFDuet-1 of Novagen company among the Synechocystissp.PCC 6803, and the gained plasmid can be expressed the beta lyase pCDFDuet-slr2049 in intestinal bacteria.
Gene order according to finding among the GeneBank has designed primer, utilizes corresponding algae kind to extract total DNA as masterplate, obtains required fragment by pcr amplification.By the restriction enzyme site that designs in the upstream and downstream primer, target fragment is changed on the restriction enzyme site same in the corresponding plasmid.
(2) the using gene engineering method is cloned Phycocyanins, C-class apoprotein gene Phycocyanins, C-B in the expression vector pCOLADuet-1 of Novagen company, the gained plasmid can be expressed apoprotein Phycocyanins, C-B pCOLADuet-Phycocyanins, C-B in intestinal bacteria; Be lyase alr0617 or slr2049 in pCDFDuet, be in second multiple clone site, restriction enzyme site is Nde I and Xho I; When lyase and apoprotein during, the invariant position of lyase in carrier simultaneously at pCDFDuet, just the another one multiple clone site many an apoprotein gene.
Apoprotein cpcB is in pET30 between EcoRV and Xho I; When apoprotein was followed lyase simultaneously at pCDFDuet, apoprotein was in first multiple clone site, and used restriction enzyme site is EcoR I and Pst I.
The PCB synthetic enzyme is 2 genes (HO1 and PcyA), when they at same carrier the time, ho1 is in first multiple clone site, between Nco I and Pst I, pcyA is second multiple clone site, between Nde I and Xho I; As HO1 and PcyA not at same carrier the time, their position in corresponding carrier also is same restriction enzyme site.
(3) the using gene engineering method is cloned the ho1 of one of phycocyanobilin biosynthesis gene in the pACYCDuet-1 of Novagen company, and the gained plasmid can be expressed H01 pACYCDuet-ho1 in intestinal bacteria.
(4) the using gene engineering method is cloned the pcyA of one of phycocyanobilin biosynthesis gene in the pETDuet-1 of Novagen company, and the gained plasmid can be expressed PcyA pETDuet-pcyA in intestinal bacteria.
(5) step of gene insertion vector is: according to gene order design primer, divide upstream and downstream, and a pair of.The downstream, upstream all has restriction enzyme site respectively; Utilize corresponding algae kind to extract total DNA as masterplate.Obtain required gene fragment through pcr amplification, the gained gene fragment is carried out electrophoresis, reclaim.The gained fragment is carried out enzyme and is cut with the enzyme of design, also carrier is cut with same enzyme simultaneously, reclaims gene fragment and carrier respectively; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4-6 hour) under the effect of T4 ligase enzyme; Connect the product conversion and enter intestinal bacteria, utilize to contain antibiotic plate screening, the picking clone, checking correctly gets final product.
Change pCDFDuet-slr2049, pCOLADuet-Phycocyanins, C-B, pACYCDuet-ho1 and pETDuet-pcyA over to intestinal bacteria, obtain colibacillus engineering after these plasmids all change intestinal bacteria over to, this engineering bacteria is produced phycocyanin fluorescence protein phycocyanobilin-Phycocyanins, C-B by fermentation energy.Protein purification techniques routinely, can purify obtains corresponding phycocyanin fluorescence protein phycocyanobilin-Phycocyanins, C-B.
Embodiment 12:
(1) adopts gene engineering method, can from GeneBank, find.The complete sequence of algae kind Anabaena sp.PCC7120 and Synechocystis sp.PCC 6803 is finished after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, and Synechocystis sp.PCC 6803 is numbered in GeneBank: BA000022, can find required gene from complete sequence; The slr2049 gene clone is in the pCDFDuet-1 of Novagen company among the Synechocystis sp.PCC 6803, and the gained plasmid can be expressed the beta lyase pCDFDuet-slr2049 in intestinal bacteria.
Gene order according to finding among the GeneBank has designed primer, utilizes corresponding algae kind to extract total DNA as masterplate, obtains required fragment by pcr amplification.By the restriction enzyme site that designs in the upstream and downstream primer, target fragment is changed on the restriction enzyme site same in the corresponding plasmid.
(2) the using gene engineering method is cloned Phycocyanins, C-class apoprotein gene Phycocyanins, C-B (C155I) in the expression vector pCOLADuet-1 of Novagen company, the gained plasmid can be expressed apoprotein Phycocyanins, C-B (C155I) pCOLADuet-Phycocyanins, C-B (C155I) in intestinal bacteria; Be lyase alr0617 or slr2049 in pCDFDuet, be in second multiple clone site, restriction enzyme site is Nde I and Xho I; When lyase and apoprotein during, the invariant position of lyase in carrier simultaneously at pCDFDuet, just the another one multiple clone site many an apoprotein gene.
Apoprotein cpcB is in pET30 between EcoRV and Xho I; When apoprotein was followed lyase simultaneously at pCDFDuet, apoprotein was in first multiple clone site, and used restriction enzyme site is EcoR I and Pst I.
The PCB synthetic enzyme is 2 genes (H01 and PcyA), when they at same carrier the time, ho1 is in first multiple clone site, between Nco I and Pst I, pcyA is second multiple clone site, between Nde I and Xho I; As HO1 and PcyA not at same carrier the time, their position in corresponding carrier also is same restriction enzyme site.
(3) the using gene engineering method is cloned the ho1 of one of phycocyanobilin biosynthesis gene in the pACYCDuet-1 of Novagen company, and the gained plasmid can be expressed HO1 pACYCDuet-ho1 in intestinal bacteria.
(4) the using gene engineering method is cloned the pcyA of one of phycocyanobilin biosynthesis gene in the pETDuet-1 of Novagen company, and the gained plasmid can be expressed PcyA pETDuet-pcyA in intestinal bacteria.
(5) step of gene insertion vector is: according to gene order design primer, divide upstream and downstream, and a pair of.The downstream, upstream all has restriction enzyme site respectively; Utilize corresponding algae kind to extract total DNA as masterplate.Obtain required gene fragment through pcr amplification, the gained gene fragment is carried out electrophoresis, reclaim.The gained fragment is carried out enzyme and is cut with the enzyme of design, also carrier is cut with same enzyme simultaneously, reclaims gene fragment and carrier respectively; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4-6 hour) under the effect of T4 ligase enzyme; Connect the product conversion and enter intestinal bacteria, utilize to contain antibiotic plate screening, the picking clone, checking correctly gets final product.
Change pCDFDuet-slr2049, pCOLADuet-Phycocyanins, C-B (C155I), pACYCDuet-ho1 and pETDuet-pcyA over to intestinal bacteria, obtain colibacillus engineering after these plasmids all change intestinal bacteria over to, this engineering bacteria is produced phycocyanin fluorescence protein phycocyanobilin-Phycocyanins, C-B (C155I) by fermentation energy.Protein purification techniques routinely, can purify obtains corresponding phycocyanin fluorescence protein phycocyanobilin-Phycocyanins, C-B (C155I).
Above-mentioned preparation method is applicable to that beta lyase, Phycocyanins, C-class apoprotein and the phycocyanobilin biosynthetic enzyme genes or its homologous gene that adopt in the various blue-green algaes prepare phycocyanin fluorescence protein, but relate to the disclosed problem of microbial strains, the present invention has only selected for use among the GenBank disclosed blue-green algae Anabaena sp.PCC7120 and Synechocystis sp.PCC 6803 for example the inventive method to be illustrated, and those skilled in the art can adopt other raw material to implement the present invention according to above-mentioned disclosed content.
Claims (5)
1. method for preparing phycocyanin fluorescence protein is characterized in that it may further comprise the steps:
(1) adopts gene engineering method, beta lyase genes or its homologous gene are cloned in expression vector, obtain beta lyase expression plasmid; The using gene engineering method is cloned Phycocyanins, C-class apoprotein gene or its homologous gene in second expression vector, obtains the apoprotein expression plasmid; The using gene engineering method with phycocyanobilin biosynthetic enzyme genes (hol and pcyA) or its homologous gene clone in the 3rd and (or) in the 4th expression vector, obtain the phycocyanobilin synthetic plasmid;
(2) change beta lyase expression plasmid or apoprotein expression plasmid or phycocyanobilin biosynthetic enzyme plasmid over to supporting host bacterium successively; After these expression plasmids all change the host bacterium over to, obtain the corresponding engineering bacterium, use this engineering bacteria and can produce phycocyanin fluorescence protein by fermentation engineering; After the fermentation, protein purification techniques routinely, can purify obtains corresponding phycocyanin fluorescence protein.
2. method according to claim 1 is characterized in that: use beta lyase catalysis phycobilin and Phycocyanins, C-class apoprotein and carry out covalent attachment and prepare phycocyanin fluorescence protein.
3. according to claim 1 and 2 described methods, it is characterized in that: change beta lyase expression plasmid, apoprotein expression plasmid, phycocyanobilin biosynthetic enzyme plasmid over to supporting host bacterium successively; After these expression plasmids all change the host bacterium over to, obtain the corresponding engineering bacterium, use this engineering bacteria and can produce phycocyanin fluorescence protein by fermentation engineering.
4. method according to claim 1 and 2 is characterized in that: the beta lyase genes be meant with Anabaenasp.PCC7120 in the gene of slr2049 dna homolog among alr0617 gene or the Synechocystis sp.PCC 6803; Phycocyanins, C-class apoprotein gene be meant with Anabaena sp.PCC7120 or Synechocystis sp.PCC6803 in Phycocyanins, C-B homologous gene.
5. method according to claim 3 is characterized in that: the beta lyase genes be meant with Anabaena sp.PCC7120 in the gene of slr2049 dna homolog among alr0617 gene or the Synechocystis sp.PCC 6803; Phycocyanins, C-class apoprotein gene be meant with Anabaena sp.PCC7120 or Synechocystis sp.PCC 6803 in Phycocyanins, C-B homologous gene.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101838661B (en) * | 2009-03-14 | 2012-03-28 | 中国科学院海洋研究所 | Preparation method of high stability phycocyanin fuse fluorescent protein |
| CN111323606A (en) * | 2020-03-16 | 2020-06-23 | 中南大学 | Method for detecting antioxidant effect of antioxidant by using oxidative damage of chromoprotein |
| CN114958892A (en) * | 2022-05-31 | 2022-08-30 | 南京林业大学 | Method for producing phycocyanin by expression in bacteria |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101838661B (en) * | 2009-03-14 | 2012-03-28 | 中国科学院海洋研究所 | Preparation method of high stability phycocyanin fuse fluorescent protein |
| CN111323606A (en) * | 2020-03-16 | 2020-06-23 | 中南大学 | Method for detecting antioxidant effect of antioxidant by using oxidative damage of chromoprotein |
| CN111323606B (en) * | 2020-03-16 | 2021-08-10 | 中南大学 | Method for detecting antioxidant effect of antioxidant by using oxidative damage of chromoprotein |
| CN114958892A (en) * | 2022-05-31 | 2022-08-30 | 南京林业大学 | Method for producing phycocyanin by expression in bacteria |
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| CN100537760C (en) | 2009-09-09 |
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