CN101481700B - Preparation of phycoerythrocyanin fluorescent protein combined with phycoerythrobilin - Google Patents

Abstract

The invention discloses a method for preparing phycoerythrocyanin fluorescent protein in combination with phycoerythrobilin (PEB). In the preparation method, phycobiliprotein beta lyase is adopted to catalyze phycoerythrobilin to carry out covalent binding with phycoerythrocyanin apoprotein, so as to prepare the phycoerythrocyanin fluorescent protein in combination with PEB. The method of the invention adopting a bioprocess to prepare the phycoerythrocyanin fluorescent protein is an environmental-friendly production method. The phycoerythrocyanin fluorescent protein can be applied to the fields of food, health care and medical functional materials and in particular can be used as a fluorescent probe in fields of biological and medical molecular monitoring.

Description

The preparation method of the phycoerythrocyanin fluorescence protein of combined with phycoerythrobilin

Technical field

The invention belongs to chromoprotein material field in the biotechnology, be specifically related to the preparation method of the phycoerythrocyanin fluorescence protein of novel combined with phycoerythrobilin (PEB).

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).CPE, PEC, CPC and APC contain alpha and beta 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 CPE mainly absorb the visible light of about 560nm, the fluorescence of the about 580nm of emission; PEC absorbs the visible light of about 570nm, the fluorescence of the about 630nm of emission; CPC absorbs the visible light of about 620nm, the fluorescence of the about 640nm of emission; APC absorbs the visible light of about 650～660nm, launches the fluorescence of about 660～670nm.CPE bonded prothetic group pigment is phycoerythrobilin (phycoerythrobilin is called for short PEB); The alpha subunit bonded prothetic group pigment of PEC is the purple courage element of algae (phycoviolobilin is called for short PVB); The beta subunit bonded prothetic group pigment of PEC is phycocyanobilin (phycocyanobilin is called for short PCB); CPC and APC bonded prothetic group pigment all are phycocyanobilin PCB.

The alpha subunit of PEC can be produced (Tooley AJ, Glazer AN.Biosynthesis of thecyanobacterial light-harvesting polypeptide phycoerythrocyanin holo-alpha subunit in a heterologous host.JBacteriol.2002 by the bacillus coli gene engineering bacteria; 184 (17): 4666～4671).Different with forefathers' method, we find alr0617 genes encoding beta lyase among the Anabaena sp.PCC7120, also have homologous beta lyase in other blue-green algae.This class beta lyase can not only the catalysis phycocyanobilin PCB and the beta subunit of PEC and 84 cysteine sulfydryls (or homologous cysteine sulfydryl) covalent attachment of homologous protein thereof generate phycoerythrocyanin fluorescence protein, can also catalysis phycoerythrobilin PEB and the beta subunit of PEC and 84 cysteine sulfydryls (or homologous cysteine sulfydryl) covalent attachment of homologous protein thereof generate a kind of novel phycoerythrocyanin fluorescence protein that combines PEB.Phycoerythrobilin PEB can generate (Alvey by HO1, PebA, PebB concerted catalysis, R.M., Karty, J.A.etc.Lesions in Phycoerythrin ChromophoreBiosynthesis in Fremyella diplosiphon Reveal Coordinated Light Regulation of Apoprotein and PigmentBiosynthetic Enzyme Gene Expression.Plant Cell 15 (10), 2448-2463 (2003)).In the blue-green algae and red algae of the overwhelming majority, all have the gene of the required enzyme of coding CPE apoprotein, CPC apoprotein, APC apoprotein, beta lyase and PEB biosynthesizing, wherein some lacks PE and has and have PEC in the blue-green algae of heterocyst; Do not have phycobilisome in the latent algae, do not have the gene of the APC 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 novel phycoerythrocyanin (pec).Phycoerythrocyanin fluorescence protein has good photoluminescent property, and this novel phycoerythrocyanin (pec) in conjunction with PEB is for the development of new fluorescent probe provides more choices.Present method is that phycoerythrocyanin 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 the method for the phycoerythrocyanin fluorescence protein of a kind of combined with phycoerythrobilin (FEB), it is to use beta lyase catalysis PEB and phycoerythrocyanin apoprotein covalent attachment, thereby prepares novel phycoerythrocyanin fluorescence protein (the phycoerythrocyanin apoprotein is to combine with PCB under the native state).The expression plasmid that will contain beta lyase genes, phycoerythrocyanin apoprotein gene and PEB biosynthetic enzyme genes changes the host bacterium successively over to, obtain the corresponding engineering bacterium, produce novel phycoerythrocyanin fluorescence protein by the genetic engineering bacterium of design like this.

Technical scheme of the present invention is such: a kind of preparation method of phycoerythrocyanin fluorescence protein of combined with phycoerythrobilin comprises the steps:

(1) uses gene engineering method, beta lyase genes or its homologous gene are cloned in expression vector, obtain beta lyase expression plasmid;

(2) use gene engineering method, phycoerythrocyanin apoprotein gene or its homologous gene are cloned in second expression vector, obtain the apoprotein expression plasmid;

(3) use gene engineering method, phycoerythrobilin biosynthetic enzyme genes ho1 and pebB or its homologous gene are cloned in the 3rd expression vector, obtain ho1 and pebB expression plasmid;

(4) use gene engineering method, phycoerythrobilin biosynthetic enzyme genes pebA or its homologous gene are cloned in the 4th expression vector, obtain the pebA expression plasmid;

(5) change beta lyase expression plasmid, apoprotein expression plasmid, ho1 and pebB expression plasmid and pebA 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 by the phycoerythrocyanin fluorescence protein of fermentation engineering production in conjunction with PEB; After the fermentation, protein purification techniques routinely, purifying obtains accordingly phycoerythrocyanin fluorescence protein in conjunction with PEB.

Technical scheme of the present invention also can be such: a kind of preparation method of phycoerythrocyanin fluorescence protein of combined with phycoerythrobilin comprises the steps:

(1) uses gene engineering method, beta lyase genes or its homologous gene and phycoerythrocyanin apoprotein gene or its homologous gene are cloned simultaneously in expression vector, obtain beta lyase and apoprotein expression plasmid;

(2) use gene engineering method, phycoerythrobilin biosynthetic enzyme genes ho1 and pebB or its homologous gene are cloned in the 3rd expression vector, obtain ho1 and pebB expression plasmid;

(3) use gene engineering method, phycoerythrobilin biosynthetic enzyme genes pebA or its homologous gene are cloned in the 4th expression vector, obtain the pebA expression plasmid;

(4) change beta lyase expression plasmid, apoprotein expression plasmid, ho1 and pebB expression plasmid and pebA 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 by the phycoerythrocyanin fluorescence protein of fermentation engineering production in conjunction with PEB; After the fermentation, protein purification techniques routinely, purifying obtains accordingly phycoerythrocyanin fluorescence protein in conjunction with PEB.

Among the preparation method of the phycoerythrocyanin fluorescence protein of above-mentioned combined with phycoerythrobilin, described host bacterium is intestinal bacteria; Described beta lyase genes be meant with Anabaena sp.PCC7120 in the gene of alr0617 dna homolog; Described phycoerythrocyanin apoprotein gene be meant with Anabaena sp.PCC7120 or M.laminosus sp.PCC7603 in the gene of pecB dna homolog.

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 phycoerythrocyanin 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, generation has the spectral response curve different with natural phycoerythrocyanin (pec) in conjunction with the novel phycoerythrocyanin (pec) of PEB, for development fluorescence phycobiliprotein class chromoprotein matter functional materials provides more more options.

Description of drawings

Absorption and fluorescence spectrum that Fig. 1 generates down for Alr0617 catalysis among the present invention in conjunction with the phycoerythrocyanin fluorescence protein of PEB; Wherein solid line is an absorption spectrum, and dotted line is a fluorescence spectrum.

Embodiment

The present invention is further detailed explanation below in conjunction with embodiment, but do not constitute any limitation of the invention.

Embodiment 1

(1) can find from GeneBank, the complete sequence of algae kind Anabaena sp.PCC7120 is finished after measured, and M.laminosus sp.PCC7603 and Calothrix sp.PCC7601 partial sequence are after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, can find required gene (pecB, alr0617, hol) from finding sequence; The gene order of using is numbered in GeneBank among the M.laminosus sp.PCC7603: M34254, can find required gene (pecB) from finding sequence; The gene order of using is numbered AY363679 among the Calothrix sp.PCC7601 in GeneBank, can find required gene (pebA and pebB) from finding sequence.By gene engineering method, in the pETDuet-1 of Novagen company, the gained plasmid can be expressed the beta lyase pETDuet-alr0617 in intestinal bacteria with the alr0617 gene clone among the Anabaenasp.PCC7120.

According to the gene order of finding among the GeneBank, the design primer utilizes corresponding algae kind to extract total DNA as template, 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 phycoerythrocyanin apoprotein gene pecB among the Anabaena sp.PCC7120 is cloned in the expression vector pET30 of Novagen company, the gained plasmid can be expressed apoprotein phycoerythrocyanin (pec) B pET30-pecB in intestinal bacteria.

(3) phycoerythrobilin biosynthetic enzyme genes (ho1 and pebB) is cloned in the pCDFDuet-1 of Novagen company, the gained plasmid can be expressed HO1 and PebB pCDFDuet-ho1-pebB simultaneously in intestinal bacteria.

(4) phycoerythrobilin biosynthetic enzyme genes (pebA) is cloned in the pACYCDuet-1 of Novagen company, the gained plasmid can be expressed PebA pACYCDuet-pebA in intestinal bacteria.

Be that apoprotein gene pecB is in pET30 between EcoRV and two restriction enzyme sites of Xho I; Lyase genes alr0617 is between second multiple clone site BglII and the Xho I in pETDuet-1; The PEB synthase gene is 3 (ho1, pebA and pebB), ho1 and pebB are in same carrier pCDFDuet-1, ho1 is between first multiple clone site Nco I and Pst I, pebB is between second multiple clone site Nde I and Xho I, and pebA is in pACYCDuet-1 between second multiple clone site BglII and the Xho I.

The step of gene insertion vector is: according to gene order design primer, divide upstream and downstream, and a pair of; The upstream downstream primer has restriction enzyme site respectively; Utilize corresponding algae kind to extract total DNA as template; 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 restriction enzyme of design, simultaneously carrier is carried out enzyme with same restriction enzyme and cuts, and reclaims gene fragment and carrier respectively; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4～6 hours) 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.

(5) change pETDuet-alr0617, pET30-pecB, pCDFDuet-ho1-pebB and pACYCDuet-pebA 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 pH 7.0,37 ℃ 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 about 12 hours, produced phycoerythrocyanin fluorescence protein phycoerythrobilin-phycoerythrocyanin (pec) 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 phycoerythrocyanin fluorescence protein phycoerythrobilin-phycoerythrocyanin (pec) B.Its absorption and fluorescence spectrum are seen shown in Figure 1, and wherein solid line is an absorption spectrum, and dotted line is a fluorescence spectrum.

The transform mode that the combination of used plasmid changes in the intestinal bacteria 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 to OD ₆₀₀=0.3～0.4 o'clock, bacterium liquid is transferred in the aseptic centrifuge tube of 5mL, place 10min on ice; Centrifugal 1min (10000g, 4 ℃) supernatant discarded, the collecting precipitation thalline; 0.1mol/L CaCl with the 1mL precooling ₂The resuspended thalline of sterile solution, centrifugal 30s (10000g, 4 ℃) removes supernatant, and bacterial sediment is with the 0.1mol/L CaCl of 100 μ L precoolings ₂Resuspended, be positioned on ice, add a certain amount of plasmid that builds, ice bath is placed 30min behind the mixing, 42 ℃ of heat-shocked 90s add 300 μ L LB substratum behind the ice bath 5min, 37 ℃ of low-speed oscillations are cultivated 45min, 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.

Extract 3 left and right sides bacterium colonies, be inoculated into 5ml respectively and contain in the corresponding antibiotic LB substratum, shaking culture is to saturated; Therefrom getting 100 μ L respectively is forwarded to 5ml and contains in the corresponding antibiotic LB substratum; 37 ℃ of shaking culture are to OD ₆₀₀During=0.5-0.7, the about 30min of ice bath adds the IPTG abduction delivering; Lucifuge, 20 ℃, 150 rev/mins were expressed about 12 hours.Centrifugal collecting cell, it is resuspended that cell adds damping fluid; Survey its product fluorescence volume by spectrograph, choose the high bacterial strain of fluorescence quantum yield and carry out great expression.

Embodiment 2

(1) can find from GeneBank, the complete sequence of algae kind Anabaena sp.PCC7120 is finished after measured, and M.laminosus sp.PCC7603 and Calothrix sp.PCC7601 partial sequence are after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, can find required gene (pecB, alr0617, ho1) from finding sequence; The gene order of using is numbered in GeneBank among the M.laminosus sp.PCC7603: M34254, can find required gene (pecB) from finding sequence; The gene order of using is numbered AY363679 among the Calothrix sp.PCC7601 in GeneBank, can find required gene (pebA and pebB) from finding sequence.By gene engineering method, in the pETDuet-1 of Novagen company, the gained plasmid can be expressed the beta lyase pETDuet-alr0617 in intestinal bacteria with the alr0617 gene clone among the Anabaenasp.PCC7120.

According to the gene order of finding among the GeneBank, the design primer utilizes corresponding algae kind to extract total DNA as template, 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 phycoerythrocyanin apoprotein gene pecB (C155I) among the Anabaena sp.PCC7120 is cloned in the expression vector pET30 of Novagen company, the gained plasmid can be expressed apoprotein phycoerythrocyanin (pec) B pET30-pecB (C155I) in intestinal bacteria.

(3) phycoerythrobilin biosynthetic enzyme genes (ho1 and pebB) is cloned in the pCDFDuet-1 of Novagen company, the gained plasmid can be expressed HO1 and PebB pCDFDuet-ho1-pebB simultaneously in intestinal bacteria.

(4) phycoerythrobilin biosynthetic enzyme genes (pebA) is cloned in the pACYCDuet-1 of Novagen company, the gained plasmid can be expressed PebA pACYCDuet-pebA in intestinal bacteria.

Be that apoprotein gene pecB (C155I) is in pET30 between EcoRV and two restriction enzyme sites of Xho I; Lyase genes alr0617 is between second multiple clone site BglII and the XhoI in pETDuet-1; The PEB synthase gene is 3 (ho1, pebA and pebB), ho1 and pebB are in same carrier pCDFDuet-1, ho1 is between first multiple clone site Nco I and Pst I, pebB is between second multiple clone site Nde I and XhoI, and pebA is in pACYCDuet-1 between second multiple clone site Bgl II and the Xho I.

The step of gene insertion vector is: according to gene order design primer, divide upstream and downstream, and a pair of; The upstream downstream primer has restriction enzyme site respectively; Utilize corresponding algae kind to extract total DNA as template; 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 restriction enzyme of design, simultaneously carrier is carried out enzyme with same restriction enzyme and cuts, and reclaims gene fragment and carrier respectively; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4～6 hours) 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.

(5) change pETDuet-alr0617, pET30-pecB (C155I), pCDFDuet-ho1-pebB and pACYCDuet-pebA 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 pH 7.0,37 ℃ of shaking culture are to OD ₆₀₀Be 0.5 to 0.8, add isopropylthio-(isoprophyl thio-β-D-galactoside, be called for short IPTG) to final concentration be 1mmol/L, 20 ℃ to 37 ℃ vibrations were expressed about 12 hours, produced phycoerythrocyanin fluorescence protein phycoerythrobilin-phycoerythrocyanin (pec) 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 phycoerythrocyanin fluorescence protein phycoerythrobilin-phycoerythrocyanin (pec) B.

The transform mode that the combination of used plasmid is changed in the intestinal bacteria is identical with embodiment 1.

Embodiment 3

(1) can find from GeneBank, the complete sequence of algae kind Anabaena sp.PCC7120 is finished after measured, and M.laminosus sp.PCC7603 and Calothrix sp.PCC7601 partial sequence are after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, can find required gene (pecB, alr0617, ho1) from finding sequence; The gene order of using is numbered in GeneBank among the M.laminosus sp.PCC7603: M34254, can find required gene (pecB) from finding sequence; The gene order of using is numbered AY363679 among the Calothrix sp.PCC7601 in GeneBank, can find required gene (pebA and pebB) from finding sequence.Pass through gene engineering method, alr0617 gene among the Anabaenasp.PCC7120 and phycoerythrocyanin apoprotein gene pecB are cloned in the pETDuet-1 of Novagen company, the gained plasmid can be expressed phycoerythrocyanin apoB and beta lyase pETDuet-pecB-alr0617 in intestinal bacteria.

According to the gene order of finding among the GeneBank, the design primer utilizes corresponding algae kind to extract total DNA as template, 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) phycoerythrobilin biosynthetic enzyme genes (ho1 and pebB) is cloned in the pCDFDuet-1 of Novagen company, the gained plasmid can be expressed HO1 and PebB pCDFDuet-ho1-pebB simultaneously in intestinal bacteria.

(3) phycoerythrobilin biosynthetic enzyme genes (pebA) is cloned in the pACYCDuet-1 of Novagen company, the gained plasmid can be expressed PebA pACYCDuet-pebA in intestinal bacteria.

Be apoprotein gene pecB and lyase genes alr0617 in pETDuet-1, pecB is in first multiple clone site, restriction enzyme site is EcoR I and Pst I, alr0617 is between second multiple clone site BglI and the Xho I; The PEB synthase gene is 3 (ho1, pebA and pebB), ho1 and pebB are in same carrier pCDFDuet-1, ho1 is between first multiple clone site Nco I and Pst I, pebB is between second multiple clone site Nde I and Xho I, and pebA is in pACYCDuet-1 between second multiple clone site BglII and the Xho I.

The step of gene insertion vector is: according to gene order design primer, divide upstream and downstream, and a pair of; The upstream downstream primer has restriction enzyme site respectively; Utilize corresponding algae kind to extract total DNA as template; 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 restriction enzyme of design, simultaneously carrier is carried out enzyme with same restriction enzyme and cuts, and reclaims gene fragment and carrier respectively; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4～6 hours) 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) change pETDuet-pecB-alr0617, pCDFDuet-hol-pebB and pACYCDuet-pebA 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 pH 7.0,37 ℃ 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 about 12 hours, produced phycoerythrocyanin fluorescence protein phycoerythrobilin-phycoerythrocyanin (pec) 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 phycoerythrocyanin fluorescence protein phycoerythrobilin-phycoerythrocyanin (pec) B.

The transform mode that the combination of used plasmid is changed in the intestinal bacteria is identical with embodiment 1.

Embodiment 4

(1) can find from GeneBank, the complete sequence of algae kind Anabaena sp.PCC7120 is finished after measured, and M.laminosus sp.PCC7603 and Calothrixsp.PCC7601 partial sequence are after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, can find required gene (pecB, alr0617, ho1) from finding sequence; The gene order of using is numbered in GeneBank among the M.laminosus sp.PCC7603: M34254, can find required gene (pecB) from finding sequence; The gene order of using is numbered AY363679 among the Calothrix sp.PCC7601 in GeneBank, can find required gene (pebA and pebB) from finding sequence.Pass through gene engineering method, alr0617 gene among the Anabaenasp.PCC7120 and phycoerythrocyanin apoprotein gene pecB (C155I) are cloned in the pETDuet-1 of Novagen company, the gained plasmid can be expressed phycoerythrocyanin apoB and beta lyase pETDuet-pecB (C155I)-alr0617 in intestinal bacteria.

According to the gene order of finding among the GeneBank, the design primer utilizes corresponding algae kind to extract total DNA as template, 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) phycoerythrobilin biosynthetic enzyme genes (ho1 and pebB) is cloned in the pCDFDuet-1 of Novagen company, the gained plasmid can be expressed HO1 and PebB pCDFDuet-ho1-pebB simultaneously in intestinal bacteria.

(3) phycoerythrobilin biosynthetic enzyme genes (pebA) is cloned in the pACYCDuet-1 of Novagen company, the gained plasmid can be expressed PebA pACYCDuet-pebA in intestinal bacteria.

Be apoprotein gene pecB (C155I) and lyase genes alr0617 in pETDuet-1, pecB is in first multiple clone site, restriction enzyme site is EcoR I and Pst I, alr0617 is between second multiple clone site BglII and the XhoI; The PEB synthase gene is 3 (ho1, pebA and pebB), ho1 and pebB are in same carrier pCDFDuet-1, ho1 is between first multiple clone site Nco I and Pst I, pebB is between second multiple clone site Nde I and Xho I, and pebA is in pACYCDuet-1 between second multiple clone site BglII and the Xho I.

The step of gene insertion vector is: according to gene order design primer, divide upstream and downstream, and a pair of; The upstream downstream primer has restriction enzyme site respectively; Utilize corresponding algae kind to extract total DNA as template; 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 restriction enzyme of design, simultaneously carrier is carried out enzyme with same restriction enzyme and cuts, and reclaims gene fragment and carrier respectively; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4～6 hours) 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) change pETDuet-pecB (C155I)-alr0617, pCDFDuet-ho1-pebB and pACYCDuet-pebA 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 pH 7.0,37 ℃ 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 about 12 hours, produced phycoerythrocyanin fluorescence protein phycoerythrobilin-phycoerythrocyanin (pec) 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 phycoerythrocyanin fluorescence protein phycoerythrobilin-phycoerythrocyanin (pec) B.

The transform mode that the combination of used plasmid is changed in the intestinal bacteria is identical with embodiment 1.

Embodiment 5

(1) can find from GeneBank, the complete sequence of algae kind Anabaena sp.PCC7120 is finished after measured, and M.laminosus sp.PCC7603 and Calothrix sp.PCC7601 partial sequence are after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, can find required gene (pecB, alr0617, ho1) from finding sequence; The gene order of using is numbered in GeneBank among the M.laminosus sp.PCC7603: M34254, can find required gene (pecB) from finding sequence; The gene order of using is numbered AY363679 among the Calothrix sp.PCC7601 in GeneBank, can find required gene (pebA and pebB) from finding sequence.By gene engineering method, in the pETDuet-1 of Novagen company, the gained plasmid can be expressed the beta lyase pETDuet-alr0617 in intestinal bacteria with the alr0617 gene clone among the Anabaenasp.PCC7120.

According to the gene order of finding among the GeneBank, the design primer utilizes corresponding algae kind to extract total DNA as template, 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 phycoerythrocyanin apoprotein gene pecB among the Anabaena sp.PCC7120 is cloned in the expression vector pCOLADuet-1 of Novagen company, the gained plasmid can be expressed apoprotein phycoerythrocyanin (pec) B pCOLADuet-pecB in intestinal bacteria.

(3) phycoerythrobilin biosynthetic enzyme genes (ho1 and pebB) is cloned in the pCDFDuet-1 of Novagen company, the gained plasmid can be expressed HO1 and PebB pCDFDuet-ho1-pebB simultaneously in intestinal bacteria.

(4) phycoerythrobilin biosynthetic enzyme genes (pebA) is cloned in the pACYCDuet-1 of Novagen company, the gained plasmid can be expressed PebA pACYCDuet-pebA in intestinal bacteria.

Be that apoprotein gene pecB is between first multiple clone site of pCOLADuet-1 EcoR I and two restriction enzyme sites of Pst I; Lyase genes alr0617 is between second multiple clone site BglII and the Xho I in pETDuet-1; The PEB synthase gene is 3 (ho1, pebA and pebB), ho1 and pebB are in same carrier pCDFDuet-1, ho1 is between first multiple clone site Nco I and Pst I, pebB is between second multiple clone site Nde I and Xho I, and pebA is in pACYCDuet-1 between second multiple clone site BglII and the Xho I.

The step of gene insertion vector is: according to gene order design primer, divide upstream and downstream, and a pair of; The upstream downstream primer has restriction enzyme site respectively; Utilize corresponding algae kind to extract total DNA as template; 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 restriction enzyme of design, simultaneously carrier is carried out enzyme with same restriction enzyme and cuts, and reclaims gene fragment and carrier respectively; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4～6 hours) 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.

(5) change pETDuet-alr0617, pCOLADuet-pecB, pCDFDuet-ho1-pebB and pACYCDuet-pebA 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 pH 7.0,37 ℃ of shaking culture are to OD ₆₀₀Be 0.5 to 0.8, add isopropylthio-(isoprophyl thio-β-D-galactoside, be called for short IPTG) to final concentration be 1mmol/L, 20 ℃ to 37 ℃ vibrations were expressed about 12 hours, produced phycoerythrocyanin fluorescence protein phycoerythrobilin-phycoerythrocyanin (pec) 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 phycoerythrocyanin fluorescence protein phycoerythrobilin-phycoerythrocyanin (pec) B.

The transform mode that the combination of used plasmid is changed in the intestinal bacteria is identical with embodiment 1.

Embodiment 6

(1) can find from GeneBank, the complete sequence of algae kind Anabaena sp.PCC7120 is finished after measured, and M.laminosus sp.PCC7603 and Calothrix sp.PCC7601 partial sequence are after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, can find required gene (pecB, alr0617, ho1) from finding sequence; The gene order of using is numbered in GeneBank among the M.laminosus sp.PCC7603: M34254, can find required gene (pecB) from finding sequence; The gene order of using is numbered AY363679 among the Calothrix sp.PCC7601 in GeneBank, can find required gene (pebA and pebB) from finding sequence.By gene engineering method, in the pETDuet-1 of Novagen company, the gained plasmid can be expressed the beta lyase pETDuet-alr0617 in intestinal bacteria with the alr0617 gene clone among the Anabaenasp.PCC7120.

According to the gene order of finding among the GeneBank, the design primer utilizes corresponding algae kind to extract total DNA as template, 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 phycoerythrocyanin apoprotein gene pecB (C155I) among the Anabaena sp.PCC7120 is cloned in the expression vector pCOLADuet-1 of Novagen company, the gained plasmid can be expressed apoprotein phycoerythrocyanin (pec) B pCOLADuet-pecB (C155I) in intestinal bacteria.

(3) phycoerythrobilin biosynthetic enzyme genes (ho1 and pebB) is cloned in the pCDFDuet-1 of Novagen company, the gained plasmid can be expressed HO1 and PebB pCDFDuet-ho1-pebB simultaneously in intestinal bacteria.

(4) phycoerythrobilin biosynthetic enzyme genes (pebA) is cloned in the pACYCDuet-1 of Novagen company, the gained plasmid can be expressed PebA pACYCDuet-pebA in intestinal bacteria.

Be that apoprotein gene pecB (C155I) is between first multiple clone site of pCOLADuet-1 EcoR I and two restriction enzyme sites of Pst I; Lyase genes alr0617 is between second multiple clone site BglII and the XhoI in pETDuet-1; The PEB synthase gene is 3 (ho1, pebA and pebB), ho1 and pebB are in same carrier pCDFDuet-1, ho1 is between first multiple clone site Nco I and Pst I, pebB is between second multiple clone site Nde I and Xho I, and pebA is in pACYCDuet-1 between second multiple clone site BglII and the Xho I.

The step of gene insertion vector is: according to gene order design primer, divide upstream and downstream, and a pair of; The upstream downstream primer has restriction enzyme site respectively; Utilize corresponding algae kind to extract total DNA as template; 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 restriction enzyme of design, simultaneously carrier is carried out enzyme with same restriction enzyme and cuts, and reclaims gene fragment and carrier respectively; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4～6 hours) 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.

(5) change pETDuet-alr0617, pCOLADuet-pecB (C155I), pCDFDuet-ho1-pebB and pACYCDuet-pebA 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 pH 7.0,37 ℃ of shaking culture are to OD ₆₀₀Be 0.5 to 0.8, add isopropylthio-(isoprophyl thio-β-D-galactoside, be called for short IPTG) to final concentration be 1mmol/L, 20 ℃ to 37 ℃ vibrations were expressed about 12 hours, produced phycoerythrocyanin fluorescence protein phycoerythrobilin-phycoerythrocyanin (pec) 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 phycoerythrocyanin fluorescence protein phycoerythrobilin-phycoerythrocyanin (pec) B.

The transform mode that the combination of used plasmid is changed in the intestinal bacteria is identical with embodiment 1.

Embodiment 7

(1) can find from GeneBank, the complete sequence of algae kind Anabaena sp.PCC7120 is finished after measured, and M.laminosus sp.PCC7603 and Calothrix sp.PCC7601 partial sequence are after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, can find required gene (pecB, alr0617, ho1) from finding sequence; The gene order of using is numbered in GeneBank among the M.laminosus sp.PCC7603: M34254, can find required gene (pecB) from finding sequence; The gene order of using is numbered AY363679 among the Calothrix sp.PCC7601 in GeneBank, can find required gene (pebA and pebB) from finding sequence.By gene engineering method, in the pETDuet-1 of Novagen company, the gained plasmid can be expressed the beta lyase pETDuet-alr0617 in intestinal bacteria with the alr0617 gene clone among the Anabaenasp.PCC7120.

According to the gene order of finding among the GeneBank, the design primer utilizes corresponding algae kind to extract total DNA as template, 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 phycoerythrocyanin apoprotein gene pecB among the M.laminosus sp.PCC7603 is cloned in the expression vector pET30 of Novagen company, the gained plasmid can be expressed apoprotein phycoerythrocyanin (pec) B pET30-pecB in intestinal bacteria.

(3) phycoerythrobilin biosynthetic enzyme genes (ho1 and pebB) is cloned in the pCDFDuet-1 of Novagen company, the gained plasmid can be expressed HO1 and PebB pCDFDuet-ho1-pebB simultaneously in intestinal bacteria.

(4) phycoerythrobilin biosynthetic enzyme genes (pebA) is cloned in the pACYCDuet-1 of Novagen company, the gained plasmid can be expressed PebA pACYCDuet-pebA in intestinal bacteria.

Be that apoprotein gene pecB is in pET30 between EcoRV and two restriction enzyme sites of Xho I; Lyase genes alr0617 is between second multiple clone site BglII and the Xho I in pETDuet-1; The PEB synthase gene is 3 (ho1, pebA and pebB), ho1 and pebB are in same carrier pCDFDuet-1, ho1 is between first multiple clone site Nco I and Pst I, pebB is between second multiple clone site Nde I and Xho I, and pebA is in pACYCDuet-1 between second multiple clone site BglII and the Xho I.

The step of gene insertion vector is: according to gene order design primer, divide upstream and downstream, and a pair of; The upstream downstream primer has restriction enzyme site respectively; Utilize corresponding algae kind to extract total DNA as template; 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 restriction enzyme of design, simultaneously carrier is carried out enzyme with same restriction enzyme and cuts, and reclaims gene fragment and carrier respectively; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4～6 hours) 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.

(5) change pETDuet-alr0617, pET30-pecB, pCDFDuet-ho1-pebB and pACYCDuet-pebA 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 pH 7.0,37 ℃ of shaking culture are to OD ₆₀₀Be 0.5 to 0.8, add isopropylthio-(i soprophylthio-β-D-galactoside, be called for short IPTG) to final concentration be 1mmol/L, 20 ℃ to 37 ℃ vibrations were expressed about 12 hours, produced phycoerythrocyanin fluorescence protein phycoerythrobilin-phycoerythrocyanin (pec) 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 phycoerythrocyanin fluorescence protein phycoerythrobilin-phycoerythrocyanin (pec) B.

The transform mode that the combination of used plasmid is changed in the intestinal bacteria is identical with embodiment 1.

Embodiment 8

(1) can find from GeneBank, the complete sequence of algae kind Anabaena sp.PCC7120 is finished after measured, and M.laminosus sp.PCC7603 and Calothrix sp.PCC7601 partial sequence are after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, can find required gene (pecB, alr0617, hol) from finding sequence; The gene order of using is numbered in GeneBank among the M.laminosus sp.PCC7603: M34254, can find required gene (pecB) from finding sequence; The gene order of using is numbered AY363679 among the Calothrix sp.PCC7601 in GeneBank, can find required gene (pebA and pebB) from finding sequence.By gene engineering method, in the pETDuet-1 of Novagen company, the gained plasmid can be expressed the beta lyase pETDuet-alr0617 in intestinal bacteria with the alr0617 gene clone among the Anabaenasp.PCC7120.

According to the gene order of finding among the GeneBank, the design primer utilizes corresponding algae kind to extract total DNA as template, 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 phycoerythrocyanin apoprotein gene pecB (C155I) among the M.laminosus sp.PCC7603 is cloned in the expression vector pET30 of Novagen company, the gained plasmid can be expressed apoprotein phycoerythrocyanin (pec) B pET30-pecB (C155I) in intestinal bacteria.

(3) phycoerythrobilin biosynthetic enzyme genes (ho1 and pebB) is cloned in the pCDFDuet-1 of Novagen company, the gained plasmid can be expressed HO1 and PebB pCDFDuet-ho1-pebB simultaneously in intestinal bacteria.

(4) phycoerythrobilin biosynthetic enzyme genes (pebA) is cloned in the pACYCDuet-1 of Novagen company, the gained plasmid can be expressed PebA pACYCDuet-pebA in intestinal bacteria.

Be that apoprotein gene pecB (C155I) is in pET30 between EcoRV and two restriction enzyme sites of Xho I; Lyase genes alr0617 is between second multiple clone site BglII and the Xho I in pETDuet-1; The PEB synthase gene is 3 (ho1, pebA and pebB), ho1 and pebB are in same carrier pCDFDuet-1, ho1 is between first multiple clone site Nco I and Pst I, pebB is between second multiple clone site Nde I and Xho I, and pebA is in pACYCDuet-1 between second multiple clone site BglII and the Xho I.

The step of gene insertion vector is: according to gene order design primer, divide upstream and downstream, and a pair of; The upstream downstream primer has restriction enzyme site respectively; Utilize corresponding algae kind to extract total DNA as template; 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 restriction enzyme of design, simultaneously carrier is carried out enzyme with same restriction enzyme and cuts, and reclaims gene fragment and carrier respectively; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4～6 hours) 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.

(5) change pETDuet-alr0617, pET30-pecB (C155I), pCDFDuet-ho1-pebB and pACYCDuet-pebA 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 pH 7.0,37 ℃ of shaking culture are to OD ₆₀₀Be 0.5 to 0.8, add isopropylthio-(isoprophyl thio-β-D-galactoside, be called for short IPTG) to final concentration be 1mmol/L, 20 ℃ to 37 ℃ vibrations were expressed about 12 hours, produced phycoerythrocyanin fluorescence protein phycoerythrobilin-phycoerythrocyanin (pec) 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 phycoerythrocyanin fluorescence protein phycoerythrobilin-phycoerythrocyanin (pec) B.

The transform mode that the combination of used plasmid is changed in the intestinal bacteria is identical with embodiment 1.

Embodiment 9

(1) can find from GeneBank, the complete sequence of algae kind Anabaena sp.PCC7120 is finished after measured, and M.laminosus sp.PCC7603 and Calothrix sp.PCC7601 partial sequence are after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, can find required gene (pecB, air0617, ho1) from finding sequence; The gene order of using is numbered in GeneBank among the M.laminosus sp.PCC7603: M34254, can find required gene (pecB) from finding sequence; The gene order of using is numbered AY363679 among the Calothrix sp.PCC7601 in GeneBank, can find required gene (pebA and pebB) from finding sequence.Pass through gene engineering method, alr0617 gene among the Anabaenasp.PCC7120 and the phycoerythrocyanin apoprotein gene pecB among the M.laminosus sp.PCC7603 are cloned in the pETDuet-1 of Novagen company, the gained plasmid can be expressed phycoerythrocyanin apoB and beta lyase pETDuet-pecB-alr0617 in intestinal bacteria.

According to the gene order of finding among the GeneBank, the design primer utilizes corresponding algae kind to extract total DNA as template, 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) phycoerythrobilin biosynthetic enzyme genes (ho1 and pebB) is cloned in the pCDFDuet-1 of Novagen company, the gained plasmid can be expressed HO1 and PebB pCDFDuet-ho1-pebB simultaneously in intestinal bacteria.

(3) phycoerythrobilin biosynthetic enzyme genes (pebA) is cloned in the pACYCDuet-1 of Novagen company, the gained plasmid can be expressed PebA pACYCDuet-pebA in intestinal bacteria.

Be apoprotein gene pecB and lyase genes alr0617 in pETDuet-1, pecB is in first multiple clone site, restriction enzyme site is EcoR I and Pst I, alr0617 is between second multiple clone site BglII and the Xho I; The PEB synthase gene is 3 (ho1, pebA and pebB), ho1 and pebB are in same carrier pCDFDuet-1, ho1 is between first multiple clone site Nco I and Pst I, pebB is between second multiple clone site Nde I and Xho I, and pebA is in pACYCDuet-1 between second multiple clone site BglII and the Xho I.

The step of gene insertion vector is: according to gene order design primer, divide upstream and downstream, and a pair of; The upstream downstream primer has restriction enzyme site respectively; Utilize corresponding algae kind to extract total DNA as template; 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 restriction enzyme of design, simultaneously carrier is carried out enzyme with same restriction enzyme and cuts, and reclaims gene fragment and carrier respectively; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4～6 hours) 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) change pETDuet-pecB-alr0617, pCDFDuet-ho1-pebB and pACYCDuet-pebA 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 pH 7.0,37 ℃ 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 about 12 hours, produced phycoerythrocyanin fluorescence protein phycoerythrobilin-phycoerythrocyanin (pec) 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 phycoerythrocyanin fluorescence protein phycoerythrobilin-phycoerythrocyanin (pec) B.

The transform mode that the combination of used plasmid is changed in the intestinal bacteria is identical with embodiment 1.

Embodiment 10

(1) can find from GeneBank, the complete sequence of algae kind Anabaena sp.PCC7120 is finished after measured, and M.laminosus sp.PCC7603 and Calothrix sp.PCC7601 partial sequence are after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, can find required gene (pecB, alr0617, ho1) from finding sequence; The gene order of using is numbered in GeneBank among the M.laminosus sp.PCC7603: M34254, can find required gene (pecB) from finding sequence; The gene order of using is numbered AY363679 among the Calothrix sp.PCC7601 in GeneBank, can find required gene (pebA and pebB) from finding sequence.Pass through gene engineering method, alr0617 gene among the Anabaenasp.PCC7120 and the phycoerythrocyanin apoprotein gene pecB (C155I) among the M.laminosus sp.PCC7603 are cloned in the pETDuet-1 of Novagen company, the gained plasmid can be expressed phycoerythrocyanin apoB and beta lyase pETDuet-pecB (C155I)-alr0617 in intestinal bacteria.

According to the gene order of finding among the GeneBank, the design primer utilizes corresponding algae kind to extract total DNA as template, 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) phycoerythrobilin biosynthetic enzyme genes (ho1 and pebB) is cloned in the pCDFDuet-1 of Novagen company, the gained plasmid can be expressed HO1 and PebB pCDFDuet-ho1-pebB simultaneously in intestinal bacteria.

(3) phycoerythrobilin biosynthetic enzyme genes (pebA) is cloned in the pACYCDuet-1 of Novagen company, the gained plasmid can be expressed PebA pACYCDuet-pebA in intestinal bacteria.

Be apoprotein gene pecB (C155I) and lyase genes alr0617 in pETDuet-1, pecB is in first multiple clone site, restriction enzyme site is EcoR I and Pst I, alr0617 is in second multiple clone site BglII and Xho

Between the I; The PEB synthase gene is 3 (ho1, pebA and pebB), ho1 and pebB are in same carrier pCDFDuet-1, ho1 is between first multiple clone site Nco I and Pst I, pebB is between second multiple clone site Nde I and Xho I, and pebA is in pACYCDuet-1 between second multiple clone site BglII and the Xho I.

The step of gene insertion vector is: according to gene order design primer, divide upstream and downstream, and a pair of; The upstream downstream primer has restriction enzyme site respectively; Utilize corresponding algae kind to extract total DNA as template; 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 restriction enzyme of design, simultaneously carrier is carried out enzyme with same restriction enzyme and cuts, and reclaims gene fragment and carrier respectively; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4～6 hours) 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) change pETDuet-pecB (C155I)-alr0617, pCDFDuet-ho1-pebB and pACYCDuet-pebA 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 pH 7.0,37 ℃ 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 about 12 hours, produced phycoerythrocyanin fluorescence protein phycoerythrobilin-phycoerythrocyanin (pec) 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 phycoerythrocyanin fluorescence protein phycoerythrobilin-phycoerythrocyanin (pec) B.

The transform mode that the combination of used plasmid is changed in the intestinal bacteria is identical with embodiment 1.

Embodiment 11

(1) can find from GeneBank, the complete sequence of algae kind Anabaena sp.PCC7120 is finished after measured, and M.laminosus sp.PCC7603 and Calothrix sp.PCC7601 partial sequence are after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, can find required gene (pecB, alr0617, hol) from finding sequence; The gene order of using is numbered in GeneBank among the M.laminosus sp.PCC7603: M34254, can find required gene (pecB) from finding sequence; The gene order of using is numbered AY363679 among the Calothrix sp.PCC7601 in GeneBank, can find required gene (pebA and pebB) from finding sequence.By gene engineering method, in the pETDuet-1 of Novagen company, the gained plasmid can be expressed the beta lyase pETDuet-alr0617 in intestinal bacteria with the alr0617 gene clone among the Anabaenasp.PCC7120.

According to the gene order of finding among the GeneBank, the design primer utilizes corresponding algae kind to extract total DNA as template, 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 phycoerythrocyanin apoprotein gene pecB among the M.laminosus sp.PCC7603 is cloned in the expression vector pCOLADuet-1 of Novagen company, the gained plasmid can be expressed apoprotein phycoerythrocyanin (pec) B pCOLADuet-pecB in intestinal bacteria.

(3) phycoerythrobilin biosynthetic enzyme genes (ho1 and pebB) is cloned in the pCDFDuet-1 of Novagen company, the gained plasmid can be expressed HO1 and PebB pCDFDuet-ho1-pebB simultaneously in intestinal bacteria.

(4) phycoerythrobilin biosynthetic enzyme genes (pebA) is cloned in the pACYCDuet-1 of Novagen company, the gained plasmid can be expressed PebA pACYCDuet-pebA in intestinal bacteria.

Be that apoprotein gene pecB is between first multiple clone site of pCOLADuet-1 EcoR I and two restriction enzyme sites of Pst I; Lyase genes alr0617 is between second multiple clone site BglII and the Xho I in pETDuet-1; The PEB synthase gene is 3 (ho1, pebA and pebB), ho1 and pebB are in same carrier pCDFDuet-1, ho1 is between first multiple clone site Nco I and Pst I, pebB is between second multiple clone site Nde I and Xho I, and pebA is in pACYCDuet-1 between second multiple clone site BglII and the Xho I.

The step of gene insertion vector is: according to gene order design primer, divide upstream and downstream, and a pair of; The upstream downstream primer has restriction enzyme site respectively; Utilize corresponding algae kind to extract total DNA as template; 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 restriction enzyme of design, simultaneously carrier is carried out enzyme with same restriction enzyme and cuts, and reclaims gene fragment and carrier respectively; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4～6 hours) 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.

(5) change pETDuet-alr0617, pCOLADuet-pecB, pCDFDuet-ho1-pebB and pACYCDuet-pebA 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 pH 7.0,37 ℃ of shaking culture are to OD ₆₀₀Be 0.5 to 0.8, add isopropylthio-(isoprophyl thio-β-D-galactoside, be called for short IPTG) to final concentration be 1mmol/L, 20 ℃ to 37 ℃ vibrations were expressed about 12 hours, produced phycoerythrocyanin fluorescence protein phycoerythrobilin-phycoerythrocyanin (pec) 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 phycoerythrocyanin fluorescence protein phycoerythrobilin-phycoerythrocyanin (pec) B.

The transform mode that the combination of used plasmid is changed in the intestinal bacteria is identical with embodiment 1.

Embodiment 12

(1) can find from GeneBank, the complete sequence of algae kind Anabaena sp.PCC7120 is finished after measured, and M.laminosus sp.PCC7603 and Calothrix sp.PCC7601 partial sequence are after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, can find required gene (pecB, alr0617, ho1) from finding sequence; The gene order of using is numbered in GeneBank among the M.laminosus sp.PCC7603: M34254, can find required gene (pecB) from finding sequence; The gene order of using is numbered AY363679 among the Calothrix sp.PCC7601 in GeneBank, can find required gene (pebA and pebB) from finding sequence.By gene engineering method, in the pETDuet-1 of Novagen company, the gained plasmid can be expressed the beta lyase pETDuet-alr0617 in intestinal bacteria with the alr0617 gene clone among the Anabaenasp.PCC7120.

According to the gene order of finding among the GeneBank, the design primer utilizes corresponding algae kind to extract total DNA as template, 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 phycoerythrocyanin apoprotein gene pecB (C155I) among the M.laminosus sp.PCC7603 is cloned in the expression vector pCOLADuet-1 of Novagen company, the gained plasmid can be expressed apoprotein phycoerythrocyanin (pec) B pCOLADuet-pecB (C155I) in intestinal bacteria.

(3) phycoerythrobilin biosynthetic enzyme genes (ho1 and pebB) is cloned in the pCDFDuet-1 of Novagen company, the gained plasmid can be expressed HO1 and PebB pCDFDuet-ho1-pebB simultaneously in intestinal bacteria.

(4) phycoerythrobilin biosynthetic enzyme genes (pebA) is cloned in the pACYCDuet-1 of Novagen company, the gained plasmid can be expressed PebA pACYCDuet-pebA in intestinal bacteria.

Be that apoprotein gene pecB (C155I) is between first multiple clone site of pCOLADuet-1 EcoR I and two restriction enzyme sites of Pst I; Lyase genes alr0617 is between second multiple clone site BglII and the XhoI in pETDuet-1; The PEB synthase gene is 3 (ho1, pebA and pebB), ho1 and pebB are in same carrier pCDFDuet-1, ho1 is between first multiple clone site Nco I and Pst I, pebB is between second multiple clone site Nde I and Xho I, and pebA is in pACYCDuet-1 between second multiple clone site Bgl II and the Xho I.

The step of gene insertion vector is: according to gene order design primer, divide upstream and downstream, and a pair of; The upstream downstream primer has restriction enzyme site respectively; Utilize corresponding algae kind to extract total DNA as template; 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 restriction enzyme of design, simultaneously carrier is carried out enzyme with same restriction enzyme and cuts, and reclaims gene fragment and carrier respectively; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4～6 hours) 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.

(5) change pETDuet-alr0617, pCOLADuet-pecB (C155I), pCDFDuet-ho1-pebB and pACYCDuet-pebA 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 pH 7.0,37 ℃ of shaking culture are to OD ₆₀₀Be 0.5 to 0.8, add isopropylthio-(isoprophyl thio-β-D-galactoside, be called for short IPTG) to final concentration be 1mmol/L, 20 ℃ to 37 ℃ vibrations were expressed about 12 hours, produced phycoerythrocyanin fluorescence protein phycoerythrobilin-phycoerythrocyanin (pec) 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 phycoerythrocyanin fluorescence protein phycoerythrobilin-phycoerythrocyanin (pec) B.

The transform mode that the combination of used plasmid is changed in the intestinal bacteria is identical with embodiment 1.

Above-mentioned preparation method is applicable to the beta lyase that adopts in the various blue-green algaes, the phycoerythrocyanin apoprotein, phycoerythrobilin biosynthetic enzyme genes or its homologous gene prepare phycoerythrocyanin fluorescence protein, but relate to the disclosed problem of microbial strains, it is that example is illustrated the inventive method that the present invention has only selected the M.laminosus sp.PCC7603 and the Calothrix sp.PCC7601 that disclose the blue-green algae Anabaena sp.PCC7120 of complete sequence and part order-checking among the GenBank for use, and those skilled in the art can adopt other raw material to implement the present invention according to above-mentioned disclosed content.

Claims (3)

1. the preparation method of the phycoerythrocyanin fluorescence protein of a combined with phycoerythrobilin is characterized in that comprising the steps:

(1) uses gene engineering method, the beta lyase genes is cloned in expression vector, obtain beta lyase expression plasmid;

(2) use gene engineering method, the gene clone of phycoerythrocyanin apoprotein in second expression vector, is obtained the apoprotein expression plasmid;

(3) use gene engineering method, phycoerythrobilin biosynthetic enzyme genes hol and pebB are cloned in the 3rd expression vector, obtain hol and pebB expression plasmid;

(4) use gene engineering method, phycoerythrobilin biosynthetic enzyme genes pebA is cloned in the 4th expression vector, obtain the pebA expression plasmid;

(5) change beta lyase expression plasmid, apoprotein expression plasmid, hol and pebB expression plasmid and pebA expression plasmid over to supporting same host bacterium successively, after these expression plasmids all change the host bacterium over to, obtain the corresponding engineering bacterium, use this engineering bacteria by the phycoerythrocyanin fluorescence protein of fermentation engineering production in conjunction with PEB; After the fermentation, protein purification techniques routinely, purifying obtains accordingly phycoerythrocyanin fluorescence protein in conjunction with PEB;

Described beta lyase genes refers to the alr0617 among the Anabaena sp.PCC7120, phycoerythrocyanin apoprotein gene refers to the pecB gene among Anabaena sp.PCC7120 or the M.Laminosus sp.PCC7603, and the phycoerythrobilin biosynthetic enzyme genes is meant pebA and pebB gene among hol, the Calothrix sp.PCC7601 among the Anabaena sp.PCC7120.

2. the preparation method of the phycoerythrocyanin fluorescence protein of a combined with phycoerythrobilin is characterized in that comprising the steps:

(1) uses gene engineering method, beta lyase genes and phycoerythrocyanin apoprotein gene are cloned simultaneously in expression vector, obtain beta lyase and apoprotein expression plasmid;

(2) use gene engineering method, phycoerythrobilin biosynthetic enzyme genes hol and pebB are cloned in second expression vector, obtain hol and pebB expression plasmid;

(3) use gene engineering method, phycoerythrobilin biosynthetic enzyme genes pebA is cloned in the 3rd expression vector, obtain the pebA expression plasmid;

(4) change beta lyase and apoprotein expression plasmid, hol and pebB expression plasmid and pebA expression plasmid over to supporting same host bacterium successively, after these expression plasmids all change the host bacterium over to, obtain the corresponding engineering bacterium, use this engineering bacteria by the phycoerythrocyanin fluorescence protein of fermentation engineering production in conjunction with PEB; After the fermentation, protein purification techniques routinely, purifying obtains accordingly phycoerythrocyanin fluorescence protein in conjunction with PEB;

Described beta lyase genes refers to the alr0617 among the Anabaena sp.PCC7120, phycoerythrocyanin apoprotein gene refers to the pecB gene among Anabaena sp.PCC7120 or the M.Laminosus sp.PCC7603, and the phycoerythrobilin biosynthetic enzyme genes is meant pebA and pebB gene among hol, the Calothrix sp.PCC7601 among the Anabaena sp.PCC7120.

3. method according to claim 1 and 2 is characterized in that, described host bacterium is intestinal bacteria.

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