CN101948887A - Preparation method and application of recombinant phycocyanobilin - Google Patents

Abstract

The invention discloses a method for preparing recombinant phycocyanobilin, in particular a method for producing the recombinant phycocyanobilin by gene engineering technology. The method comprises the following steps of: cloning phycocyanobilin synthesizing key enzyme genes (hol and pcyA) from algae and cloning the key enzyme genes to proper expression vectors; inoculating a recombinant vector into an Escherichia coli expression system for heterogenous expression, and synthesizing the recombinant phycocyanobilin through enzymatic catalytic reaction; and finally performing extraction, chromatography and other purification steps to prepare the recombinant phycocyanobilin. The prepared recombinant phycocyanobilin has the advantages of higher capacity of removing DPPH radicals, can be used for treating a plurality of diseases caused by oxidative damage, and also can be used as an additive in functional foods.

Description

A kind of preparation method of the phycocyanobilin of recombinating and application thereof

Technical field

The present invention relates to genetically engineered and food and medicine field, specifically a kind of preparation method of the phycocyanobilin of recombinating and application thereof.

Background technology

(Phycocyanobilin PCB) is a kind of photosynthesis accessory pigment that are present in red algae and the blue-green algae to phycocyanobilin.With bilirubinoid seemingly, be to be straight chain by 4 pyrrole rings by methene to connect, molecular weight is about 588.69g/mol.They combine with soluble protein in vivo and are called Phycocyanins, C-(phycocyanin) or allophycocyanin, are dissolved in rare salts solution.They work to absorb and transmit luminous energy in photosynthesis.

As bilirubinic derivative; phycocyanobilin has been proved to be at present has anti-oxidant (Hirata; T.; Tanaka; M.; Ooike; M.; Tsunomura; T.; and Sakaguchi, M.Antioxidant activities of phycocyanobilin prepared from Spirulina platensis J Appl Phycol 12,2000; 435-439.); remove hydroxy radical qiao and oxyradical; the protection microbial film is avoided oxidative damage (Hirata; T., Tanaka, M.; Ooike; M., Tsunomura, T.; and Sakaguchi; M.Radical scavenging activities of phycocyanobilin prepared from a cyanobacterium, Spirulina platensis, Fisheries Sci 1999; 65; 971-974.); and anti-inflammatory (Bhat, V.B., and Madyastha; K.M.Scavenging of peroxynitrite by phycocyanin and phycocyanobilin from Spirulina platensis:protection against oxidative damage to DNA; Biochem Biophys Res Commun 2001,285, multiple efficacies such as 262-266.).The current research evidence is found, phycocyanobilin can be reduced by the protoheme reductase enzyme in cell and be converted into bilirubin, and then the overexpression of the nadph oxidase that causes by oxidative damage of feedback inhibition, for diabetes, the disease that Gilbert syndromes etc. is caused by oxidative damage plays better curative effect (McCarty, M.F., Barroso-Aranda, J., and Contreras, F.NADPH oxidase mediates glucolipotoxicity-induced beta cell dysfunction--clinical implications, Med Hypotheses 2010,74,596-600; McCarty, M.F., Barroso-Aranda, J., and Contreras, F.Oral phycocyanobilin may diminish the pathogenicity of activated brain microglia in neurodegenerative disorders, Med Hypotheses 2010,74,601-605.).U.S. food medicine office (FDA) approved is used for the photosensitive treatment of skin carcinoma, mammary cancer with Phycocyanins, C-(phycocyanobilin and apoprotein combine), and (model is known, the prospect of marine alga chemical research, ocean science, 1996, (2): 24).Phycocyanins, C-is used for also patent applied for (US4886831) of atherosclerotic photosensitive treatment simultaneously.Phycocyanobilin can be used as fluorescent probe and natural pigment simultaneously, has wide purposes (Wu at biological detection and functional food aspect preparing, S.H., McDowell, M.T., and Lagarias, J.C.Phycocyanobilin is the natural precursor of the phytochrome chromophore in the green alga Mesotaenium caldariorum, J Biol Chem 1997,272,25700-25705.).

The extractive technique of existing phycocyanobilin mainly is a natural extract from spirulina.It comprises a series of long period, expensive, complicated technological process.Mainly comprise: the cultivation of frustule, centrifugal collection, the fragmentation of frond and the extracting of phycocyanobilin, refining etc.Wherein the separation purification method of phycocyanobilin mainly contains: hot methanol cracking process and two step of methyl alcohol extraction process.The hot methanol cracking process is to utilize hot methanol (70-75 ℃) slowly to open the thioether bond between phycocyanobilin and the apoprotein, thereby extracting obtains the free phycocyanobilin about 2/3.But owing to also contain multiple pigment such as Chlorophylls and Carotenoids in the spirulina, this method often can only obtain the not high phycocyanobilin of purity.And two step of methyl alcohol extraction process is earlier with cold ethanol extracting, remove most Chlorophylls and Carotenoids, this moment, phycocyanobilin was insoluble to cold ethanol, and then use the hot methanol extracting, can obtain the phycocyanobilin (O ' Carra of purity higher (about 80%) like this, P., Murphy, R.F., and Killilea, S.D.The native forms of the phycobilin chromophores of algal biliproteins.A clarification, Biochem J 1980,187,303-309.).More highly purified phycocyanobilin needs further thin-layer chromatography or preparation high-efficient liquid phase chromatogram purification to obtain.

Although phycocyanobilin content in spirulina abundant (account for frond dry weight about 1%), spirulina large scale culturing technology is comparative maturity also, but because the SPIRULINA CULTIVATION cycle is longer, stand density is lower, need take the big area soil, and the complicated (McCarty of the natural extract technology of the free phycocyanobilin of high purity, M.F.Clinical potential of Spirulina as a source of phycocyanobilin, J Med Food 2007,10,566-570.), cause high purity phycocyanobilin price higher (price as photosensitizers high purity phycocyanobilin is about 150-180 dollar/milligram) on the market, mainly depend on import at present, still be not suitable as the healthcare products widespread use from the U.S..Therefore development technology phycocyanobilin macro preparation technology simple, with low cost will lay the foundation for the widespread use of phycocyanobilin as high-efficiency antioxidant agent and food dye.

Summary of the invention

The object of the invention is to provide a kind of easy, phycocyanobilin preparation method that recombinates fast, cheaply, and in the application of aspects such as anti-oxidation medicine and functional food.

To achieve these goals, the invention provides a kind of reorganization phycocyanobilin preparation method, comprise the steps:

By cloning heme oxidase gene ho1 and ferredoxin oxide-reductase gene pcyA in the algae, above-mentioned ho1 gene and pcyA gene (phycocyanobilin synthesizes key gene) are cloned on the expression vector, then import and carry out heterogenous expression in the intestinal bacteria, produce the reorganization phycocyanobilin.

Also further may further comprise the steps in the aforesaid method: intestinal bacteria fragmentation, chloroform extraction and gel permeation chromatography after will transforming obtain described reorganization phycocyanobilin.

Described algae is selected from blue-green algae (Cyanobacteria), red algae (Rhodophyta), latent algae (Cryptomonas), dinoflagellate (Pyrrophyta) or proto green algae (Prochloroccus marinus), preferentially is selected from cytoalgae (Synechocystis), synechococcus (Synechococcus), Chlamydomonas reinhardtii (Chlamydomonas reinhardtiii) or thermophilic blue-green algae (Thermosynechococcus elongates).

Described heme oxidase gene ho1 preferably is numbered the ho1 gene of sll1184, syc2236_c, SYNW0171, CHLREDRAFT_152591 or tll0365 in Genebank; Described ferredoxin oxide-reductase gene pcyA preferably is numbered the pcyA gene of slr0116, syc0325_d, SYNW1084, CHLREDRAFT_156256 or tll2308 in Genebank.

Described expression vector is the Bacillus coli communis expression vector, preferably from pETDuet-1, pET28a-1, pCDFDuet-1 or pACYCDuet-1.

Described intestinal bacteria are preferably e. coli bl21.

The present invention also further provides the reorganization phycocyanobilin of method for preparing in medicine or Application in Food, is preferred for anti-oxidation medicine or antioxidant food.

The present invention has following advantage:

1. the present invention is the utilization genetic engineering technique, the synthetic key gene of phycocyanobilin is changed in the coli expression system, and be substrate with intestinal bacteria self protoheme, produce phycocyanobilin by two step enzymic catalytic reactions.This production process has been avoided this long period process of algal culture, and intestinal bacteria are easy to realize the cultivation of high-density scale simultaneously, thereby reduce production costs greatly.

2. compare with algae, Bacillus coli cells is broken easily, and does not contain other photosynthetic pigments such as chlorophyll, carotenoid, makes the downstream purification process become simpler, obtains low-cost, highly purified phycocyanobilin easily.

3. the present invention directly expresses the phycocyanobilin color base, do not contain the apo-Phycocyanins, C-, therefore do not relate to the breakage problem of thioether bond between color base and albumen, so easier acquisition structure is single, the phycocyanobilin of stable in properties, unified quality standard be can set up, mass preparation and application are suitable for.

4, expression vector pETDuet-1, pET28a-1, pCDFDuet-1 or pACYCDuet-1 preferred for this invention can impel heme oxidase gene ho1 and ferredoxin oxide-reductase gene pcyA to efficiently express in intestinal bacteria.

5, the recombinant vectors of Gou Jianing especially can be expressed by high quality at e. coli bl21, and the phycocyanobilin quality of being produced is no less than the phycocyanobilin of wild algae.

6, heme oxidase gene ho1 and the ferredoxin oxide-reductase gene pcyA that derives from cytoalgae, synechococcus, Chlamydomonas reinhardtii or thermophilic blue-green algae shows the high expression level amount in e. coli bl21, is more suitable at expression in escherichia coli.

Description of drawings

Fig. 1 expresses the design of graphics of phycocyanobilin recombinant vectors pETDuet-ho1-pcyA for the present invention.

Fig. 2 expresses the design of graphics of phycocyanobilin recombinant vectors pET28a-ho1-pcyA for the present invention.

Fig. 3 expresses the design of graphics of phycocyanobilin recombinant vectors pCDFDuet-ho1-pcyA for the present invention.

Fig. 4 expresses the design of graphics of phycocyanobilin recombinant vectors pACYCDuet-ho1-pcyA for the present invention.

Fig. 5 is phycocyanobilin antioxidant effect figure of the present invention.

Embodiment

Further describe the present invention below in conjunction with drawings and Examples, but do not limit protection scope of the present invention.

The preparation method of reorganization phycocyanobilin: by cloning the synthetic key gene (heme oxidase gene ho1 and ferredoxin oxide-reductase gene pcyA) of phycocyanobilin in the algae, with ho1 and pcyA gene clone to suitable expression vector, then recombinant expression vector is imported and carry out heterogenous expression in the intestinal bacteria, intestinal bacteria are substrate with self protoheme, by Ho1 and PcyA two steps enzymic catalytic reaction, produce the reorganization phycocyanobilin.Finally, obtain blue elutriant, promptly get above-described reorganization phycocyanobilin through bacterial cell disruption, chloroform extraction and gel permeation chromatography.Described reorganization phycocyanobilin is blue compound, is dissolved in rare salts solution, and molecular weight is about 588.69g/mol.This reorganization phycocyanobilin can directly be used as medicine, reagent, foodstuff additive, health care or functional food effective constituent etc.

Wherein said algae can be blue-green algae, green alga, red algae, latent algae, dinoflagellate or proto green algae, be preferably cytoalgae (described ho1 and pcyA gene are respectively sll1184 and slr0116), synechococcus (described ho1 and pcyA gene are respectively syc2236_c and syc0325_d), Chlamydomonas reinhardtii (described ho1 and pcyA gene are respectively CHLREDRAFT_152591 and CHLREDRAFT_156256) or thermophilic blue-green algae (Thermosynechococcus elongatus, described ho1 and pcyA gene are respectively tll0365 and tll2308).

Various carriers are selected from the carrier series such as pETDuet-1, pET28a-1, pCDFDuet-1 or pACYCDuet-1 of intestinal bacteria (E.coli).

It should be noted especially in the following example, the DNA extraction of using, pcr amplification reaction, expression vector establishment and conversion, colibacillary cultivation, technology such as solvent extraction and chromatogram purification and oxidation-resistance analysis all are well known to those of ordinary skill in the art except that specifying, and can be such as " molecular cloning experiment guide " (J. Sa nurse Brooker etc. work, Huang Peitang etc. translate, Science Press, the third edition in 2002), " fine works molecular biology experiment guide " (F.M. Ao Sibai, R.E. work such as James Kingston, Ma Xuejuns etc. are translated, Science Press, version in 2005), " solvent extraction handbook (Wang Jiading, work such as Chen Jiayong, Chemical Industry Press, calendar year 2001 version), Improved DPPH determination for antioxidant activity spectrophotometric assay, Chemical Papers, 2007,60 (3): find in the reference such as 214-216., here integral body is quoted.

Embodiment 1

(1) cytoalgae Synechocystis sp.PCC 6803 full genomes have checked order and have finished, and can freely obtain from Genebank (Genebank is numbered BA000022).Gene order according to the cytoalgae of announcing among the Genebank 6803 (Synechocystis sp.PCC 6803) heme oxidase gene (ho1) and ferredoxin oxide-reductase gene (pcyA), design suitable primer, with cytoalgae 6803 (Synechocystis sp.PCC 6803) genomic dna is template, adopt gene engineering method, synthetic key gene ho1 of clone's phycocyanobilin and pcyA.Synthetic key gene ho1 of phycocyanobilin and the numbering of pcyA in Genebank are respectively sll1184 and slr0116.

(2) using gene engineering method, add BamH I and Sac I restriction enzyme site respectively at ho1 gene two ends, and be inserted into the pETDuet-1 plasmid (available from Novagen, Germany) first multiple clone site place (BamH I and Sac I restriction enzyme site, hereinafter express with identical form of presentation), obtain recombinant vectors pETDuet-ho1.Add Nde I and Xho I restriction enzyme site at gene pcyA two ends respectively, and be cloned into another multiple clone site place of pETDuet-ho1, obtain recombinant vectors pETDuet-ho1-pcyA (as shown in Figure 1).Concrete operation method is as follows:

According to ho1 that is announced among the Genebank and pcyA gene order design primer.Forward primer P1:5 '-TA of ho1 GGATCCTATGAGTGTCAACTTAGCTTCCCAG-3 ', reverse primer P2:5 '-TT GAGCTCCTAGCCTTCGGAGGTGGCGAG-3 '.Forward primer P3:5 '-TT of pcyA CATATGATGGCCGTCACTGATTTAAGTTTGACC-3 ', reverse primer P4:5 '-TG CTCGAGTTATTGGATAACATCAAATAAGAC-3 '.React amplification template with Synechocystis sp.PCC 6803 genomic dnas as PCR.Obtain required gene fragment through pcr amplification.The PCR product is through Cycle pure test kit (Omega company) purifying.With gained ho1 and pcyA gene fragment and pETDuet-1 carrier respectively with 37 ℃ of digestion of corresponding suitable endonuclease 6h.Enzyme is cut product behind agarose gel electrophoresis, reclaims from glue, and gene fragment is mixed with carrier at 3: 1, and 16 ℃ of connections are spent the night under the effect of T4DNA ligase enzyme, connects product transformed into escherichia coli DH5 α.Utilization contains the LB plate screening of 100ug/ml penbritin, and the picking positive colony utilizes PCR and dna sequencing to verify, obtains recombinant expression vector pETDuet-ho1-pcyA.Extract the expression plasmid pETDuet-ho1-pcyA that builds with alkaline denaturation, transformed competence colibacillus e. coli bl21 (DE3) (Suo Laibao Science and Technology Ltd., down together), obtaining can be at T ₇Efficiently express the coli strain P1 of active phycocyanobilin under the promoters driven.The method that the screening of above-mentioned plasmid extraction, connection, conversion and positive recombinant and evaluation are all introduced with reference to " molecular cloning experiment guide " (work such as J. Sa nurse Brooker, Huang Peitang etc. translate, Science Press, the third edition in 2002).

(3) the single bacterium colony of picking genetically engineered bacteria strain P1 contains the liquid LB substratum of 100ug/mL penbritin in 5mL from LB solid (the LB substratum that contains 1.5% agar) flat board, in the constant-temperature shaking culture case, cultivate (temperature: 37 ℃, rotating speed: about 200r/min).Overnight culture is inoculated in the TB substratum (containing penbritin 100ug/mL) of 1L with 1: 100 ratio.Be cultured to OD under the culture condition with 37 ℃ of temperature, rotating speed 200r/min in the constant-temperature shaking culture case ₆₀₀(IPTG, Isopropyl-β-D-thiogalactopyranoside) is that 0.5mmol/L induces to final concentration to add isopropyl-at=0.5～0.7 o'clock.After adding inductor, after continuing to cultivate 24h, the culture condition of 30 ℃ of temperature, rotating speed 200r/min stops.

With results bacterium liquid 10, centrifugal 10min abandons supernatant liquor under the 000g condition.The thalline of collecting is resuspended in thalline in the 1 * PBS damping fluid that is equivalent to stock culture 1/50 volume precooling after washing 2 times with 1 * PBS damping fluid (biological test reagent commonly used).The ultrasonic disruption cell, power 30W, interval 5s amounts to 30min behind every broken 15s, keeps cell low temperature (0 ℃) in the shattering process.Broken back in 4 ℃ with 12, the centrifugal 30min of 000g, supernatant liquor with 0.45 μ m nitrocellulose membrane filtration after, add isopyknic chloroform, put upside down even 1h.At the centrifugal 30min of 10000g, the careful blueness precipitation that is rich in phycocyanobilin of drawing.To precipitate dissolving again with DMSO (dimethyl sulfoxide (DMSO), down together).Insolubles is removed through the centrifugal 10min of 10000g.

With the blue solution that above-mentioned processing obtains, last sample to the Desalting column that crosses with PBS damping fluid balance in advance (GE healthcare, Sweden), last sample volume is no more than 1/3 of column volume, flow velocity is 2mL/min; Then with PBS damping fluid flush away foreign protein, last phycocyanobilin elutes with 20% ethanolic soln then.Monitor A during wash-out simultaneously ₂₈₀And A ₃₆₉, collect elution peak, thereby obtain the higher phycocyanobilin of purity.

Embodiment 2

(1) cytoalgae Synechocystis sp.PCC 6803 full genomes have checked order and have finished, and can freely obtain from Genebank (Genebank is numbered BA000022).Gene order according to the cytoalgae of announcing among the Genebank 6803 (Synechocystis sp.PCC 6803) heme oxidase gene (ho1) and ferredoxin oxide-reductase gene (pcyA), design suitable primer, with cytoalgae 6803 (Synechocystis sp.PCC 6803) genomic dna is template, adopt gene engineering method, synthetic key gene ho1 of clone's phycocyanobilin and pcyA.Synthetic key gene ho1 of phycocyanobilin and the numbering of pcyA in Genebank are respectively sll1184 and slr0116.

(2) using gene engineering method adds Nde I and EcoR V restriction enzyme site respectively at ho1 gene two ends, and (Novagen, multiple clone site place Germany) obtains recombinant vectors pET28a-ho1 to be inserted into the pET28a-1 plasmid.Add EcoR V and Xho I restriction enzyme site at gene pcyA two ends respectively, and be cloned into the downstream site of pET28a-ho1, obtain recombinant vectors pET28a-ho1-pcyA (as shown in Figure 2).Concrete operation method is as follows:

According to ho1 that is announced among the Genebank and pcyA gene order design primer.Forward primer P1:5 '-TA of ho1 CATATGTATGAGTGTCAACTTAGCTTCCCAG-3 ', reverse primer P2:5 '-TT GATATCCTAGCCTTCGGAGGTGGCGAG-3 '.Forward primer P3:5 '-TT of pcyA GATATCATGGCCGTCACTGATTTAAGTTTGACC-3 ', reverse primer P4:5 '-TG CTCGAGTTATTGGATAACATCAAATAAGAC-3 '.React amplification template with Synechocystis sp.PCC 6803 genomic dnas as PCR.Obtain required gene fragment through pcr amplification.The PCR product is through Cycle pure test kit (Omega company) purifying.With gained ho1 and pcyA gene fragment and pET28a-1 carrier respectively with 37 ℃ of digestion of corresponding suitable endonuclease 6h.Enzyme is cut product behind agarose gel electrophoresis, reclaims from glue, and gene fragment is mixed with carrier at 3: 1, and 16 ℃ of connections are spent the night under the effect of T4 dna ligase, connects product transformed into escherichia coli DH5 α.Utilization contains the LB plate screening of 50ug/ml kantlex, and the picking positive colony utilizes PCR and dna sequencing to verify, obtains recombinant expression vector pET28a-ho1-pcyA.Extract the expression plasmid pET28a-ho1-pcyA that builds with alkaline denaturation, transformed competence colibacillus e. coli bl21 (DE3), obtaining can be at T ₇Efficiently express the coli strain P2 of active phycocyanobilin under the promoters driven.The method that the screening of above-mentioned plasmid extraction, connection, conversion and positive recombinant and evaluation are all introduced with reference to " molecular cloning experiment guide " (work such as J. Sa nurse Brooker, Huang Peitang etc. translate, Science Press, the third edition in 2002).

(3) the single bacterium colony of picking genetically engineered bacteria strain P2 contains the liquid LB substratum of 50ug/mL kantlex in 5mL from LB solid (the LB substratum that contains 1.5% agar) flat board, in the constant-temperature shaking culture case, cultivate (temperature: 37 ℃, rotating speed: about 200r/min).Overnight culture is inoculated in the TB substratum (containing kantlex 50ug/mL) of 1L with 1: 100 ratio.Be cultured to OD under the culture condition with 37 ℃ of temperature, rotating speed 200r/min in the constant-temperature shaking culture case ₆₀₀(IPTG, Isopropyl-β-D-thiogalactopyranoside) is that 0.5mmol/L induces to final concentration to add isopropyl-at=0.5～0.7 o'clock.After adding inductor, after continuing to cultivate 24h, the culture condition of 30 ℃ of temperature, rotating speed 200r/min stops.

With results bacterium liquid 10, centrifugal 10min abandons supernatant liquor under the 000g condition.After thalline 1 * PBS damping fluid washing of collecting 2 times, thalline is resuspended in the 1 * PBS damping fluid that is equivalent to stock culture 1/50 volume precooling.The ultrasonic disruption cell, power 30W, interval 5s amounts to 30min behind every broken 15s, keeps cell low temperature (0 ℃) in the shattering process.Broken back in 4 ℃ with 12, the centrifugal 30min of 000g, supernatant liquor with 0.45 μ m nitrocellulose membrane filtration after, add isopyknic chloroform, put upside down even 1h.At the centrifugal 30min of 10000g, the careful blueness precipitation that is rich in PCB of drawing.To precipitate and dissolve again with DMSO.Insolubles is removed through the centrifugal 10min of 10000g.

With the blue solution that above-mentioned processing obtains, last sample is to the Desalting column that crosses with PBS damping fluid balance in advance (GE healthcare), and last sample volume is no more than 1/3 of column volume, and flow velocity is 2mL/min; Then with PBS damping fluid flush away foreign protein, last phycocyanobilin elutes with 20% ethanolic soln then.Monitor A during wash-out simultaneously ₂₈₀And A ₃₆₉, collect elution peak, thereby obtain the higher phycocyanobilin of purity.

Embodiment 3

(1) cytoalgae Synechocystis sp.PCC 6803 full genomes have checked order and have finished, and can freely obtain from Genebank (Genebank is numbered BA000022).Gene order according to the cytoalgae of announcing among the Genebank 6803 (Synechocystis sp.PCC 6803) heme oxidase gene (ho1) and ferredoxin oxide-reductase gene (pcyA), design suitable primer, with cytoalgae 6803 (Synechocystis sp.PCC 6803) genomic dna is template, adopt gene engineering method, synthetic key gene ho1 of clone's phycocyanobilin and pcyA.Synthetic key gene ho1 of phycocyanobilin and the numbering of pcyA in Genebank are respectively sll1184 and slr0116.

(2) using gene engineering method adds BamH I and Sac I restriction enzyme site respectively at ho1 gene two ends, and (Novagen, first multiple clone site place Germany) obtains recombinant vectors pCDFDuet-ho1 to be inserted into the pCDFDuet-1 plasmid.Add Nde I and Xho I restriction enzyme site at gene pcyA two ends respectively, and be cloned into another multiple clone site place of pCDFDuet-ho1, obtain recombinant vectors pCDFDuet-ho1-pcyA (as shown in Figure 3).Concrete operation method is as follows:

According to ho1 that is announced among the Genebank and pcyA gene order design primer.Forward primer P1:5 '-TA of ho1 GGATCCTATGAGTGTCAACTTAGCTTCCCAG-3 ', reverse primer P2:5 '-TT GAGCTCCTAGCCTTCGGAGGTGGCGAG-3 '.Forward primer P3:5 '-TT of pcyA CATATGATGGCCGTCACTGATTTAAGTTTGACC-3 ', reverse primer P4:5 '-TG CTCGAGTTATTGGATAACATCAAATAAGAC-3 '.React amplification template with Synechocystis sp.PCC 6803 genomic dnas as PCR.Obtain required gene fragment through pcr amplification.The PCR product is through Cycle pure test kit (Omega company) purifying.With gained ho1 and pcyA gene fragment and pCDFDuet-1 carrier respectively with 37 ℃ of digestion of corresponding suitable endonuclease 6h.Enzyme is cut product behind agarose gel electrophoresis, reclaims from glue, and gene fragment is mixed with carrier 3:1, and 16 ℃ of connections are spent the night under the effect of T4DNA ligase enzyme, connects product transformed into escherichia coli DH5 α.Utilization contains the LB plate screening of 50ug/ml spectinomycin, and the picking positive colony utilizes PCR and dna sequencing to verify, obtains recombinant expression vector pCDFDuet-ho1-pcyA.Extract the expression plasmid pCDFDuet-ho1-pcyA that builds with alkaline denaturation, transformed competence colibacillus e. coli bl21 (DE3), obtaining can be at T ₇Efficiently express the coli strain P3 of active phycocyanobilin under the promoters driven.The method that the screening of above-mentioned plasmid extraction, connection, conversion and positive recombinant and evaluation are all introduced with reference to " molecular cloning experiment guide " (work such as J. Sa nurse Brooker, Huang Peitang etc. translate, Science Press, the third edition in 2002).

(3) the single bacterium colony of picking genetically engineered bacteria strain P3 contains the liquid LB substratum of 50ug/mL spectinomycin in 5mL from LB solid (the LB substratum that contains 1.5% agar) flat board, in the constant-temperature shaking culture case, cultivate (temperature: 37 ℃, rotating speed: about 200r/min).Overnight culture is inoculated in the TB substratum (containing spectinomycin 50ug/mL) of 1L with 1: 100 ratio.Be cultured to OD under the culture condition with 37 ℃ of temperature, rotating speed 200r/min in the constant-temperature shaking culture case ₆₀₀(IPTG, Isopropyl-β-D-thiogalactopyranoside) is that 0.5mmol/L induces to final concentration to add isopropyl-at=0.5～0.7 o'clock.After adding inductor, after continuing to cultivate 24h, the culture condition of 30 ℃ of temperature, rotating speed 200r/min stops.

With results bacterium liquid 10, centrifugal 10min abandons supernatant liquor under the 000g condition.After thalline 1 * PBS damping fluid washing of collecting 2 times, thalline is resuspended in the 1 * PBS damping fluid that is equivalent to stock culture 1/50 volume precooling.The ultrasonic disruption cell, power 30W, interval 5s amounts to 30min behind every broken 15s, keeps cell low temperature (0 ℃) in the shattering process.Broken back in 4 ℃ with 12, the centrifugal 30min of 000g, supernatant liquor with 0.45 μ m nitrocellulose membrane filtration after, add isopyknic chloroform, put upside down even 1h.At the centrifugal 30min of 10000g, the careful blueness precipitation that is rich in PCB of drawing.To precipitate and dissolve again with DMSO.Insolubles is removed through the centrifugal 10min of 10000g.

With the blue solution that above-mentioned processing obtains, last sample is to the Desalting column that crosses with PBS damping fluid balance in advance (GE healthcare), and last sample volume is no more than 1/3 of column volume, and flow velocity is 2mL/min; Then with PBS damping fluid flush away foreign protein, last phycocyanobilin elutes with 20% ethanolic soln then.Monitor A during wash-out simultaneously ₂₈₀And A ₃₆₉, collect elution peak, thereby obtain the higher phycocyanobilin of purity.

Embodiment 4

(1) cytoalgae Synechocystis sp.PCC 6803 full genomes have checked order and have finished, and can freely obtain from Genebank (Genebank is numbered BA000022).Gene order according to the cytoalgae of announcing among the Genebank 6803 (Synechocystis sp.PCC 6803) heme oxidase gene (ho1) and ferredoxin oxide-reductase gene (pcyA), design suitable primer, with cytoalgae 6803 (Synechocystis sp.PCC 6803) genomic dna is template, adopt gene engineering method, synthetic key gene ho1 of clone's phycocyanobilin and pcyA.Synthetic key gene ho1 of phycocyanobilin and the numbering of pcyA in Genebank are respectively sll1184 and slr0116.

(2) using gene engineering method adds BamH I and Sac I restriction enzyme site respectively at ho1 gene two ends, and (Novagen, first multiple clone site place Germany) obtains recombinant vectors pACYCDuet-ho1 to be inserted into the pACYCDuet-1 plasmid.Add Nde I and Xho I restriction enzyme site at gene pcyA two ends respectively, and be cloned into another multiple clone site place of pACYCDuet-ho1, obtain recombinant vectors pACYCDuet-ho1-pcyA (as shown in Figure 4).Concrete operation method is as follows:

According to ho1 that is announced among the Genebank and pcyA gene order design primer.Forward primer P1:5 ' the TA of ho1 GGATCCTATGAGTGTCAACTTAGCTTCCCAG-3 ', reverse primer P2:5 '-TT GAGCTCCTAGCCTTCGGAGGTGGCGAG-3 '.Forward primer P3:5 '-TT of pcyA CATATGATGGCCGTCACTGATTTAAGTTTGACC-3 ', reverse primer P4:5 '-TG CTCGAGTTATTGGATAACATCAAATAAGAC-3 '.React amplification template with Synechocystis sp.PCC 6803 genomic dnas as PCR.Obtain required gene fragment through pcr amplification.The PCR product is through Cycle pure test kit (Omega company) purifying.With gained ho1 and pcyA gene fragment and pACYCDuet-1 carrier respectively with 37 ℃ of digestion of corresponding suitable endonuclease 6h.Enzyme is cut product behind agarose gel electrophoresis, reclaims from glue, and gene fragment is mixed with carrier at 3: 1, and 16 ℃ of connections are spent the night under the effect of T4 dna ligase, connects product transformed into escherichia coli DH5 α.Utilization contains the LB plate screening of 50ug/ml clarithromycin, and the picking positive colony utilizes PCR and dna sequencing to verify, obtains recombinant expression vector pACYCDuet-ho1-pcyA.Extract the expression plasmid pACYCDuet-ho1-pcyA that builds with alkaline denaturation, transformed competence colibacillus e. coli bl21 (DE3), obtaining can be at T ₇Efficiently express the coli strain P4 of active phycocyanobilin under the promoters driven.The method that the screening of above-mentioned plasmid extraction, connection, conversion and positive recombinant and evaluation are all introduced with reference to " molecular cloning experiment guide " (work such as J. Sa nurse Brooker, Huang Peitang etc. translate, Science Press, the third edition in 2002).

(3) the single bacterium colony of picking genetically engineered bacteria strain P4 contains the liquid LB substratum of 50ug/mL clarithromycin in 5mL from LB solid (the LB substratum that contains 1.5% agar) flat board, in the constant-temperature shaking culture case, cultivate (temperature: 37 ℃, rotating speed: about 200r/min).Overnight culture is inoculated in the TB substratum (containing clarithromycin 50ug/mL) of 1L with 1: 100 ratio.Be cultured to OD under the culture condition with 37 ℃ of temperature, rotating speed 200r/min in the constant-temperature shaking culture case ₆₀₀(IPTG, Isopropyl-β-D-thiogalactopyranoside) is that 0.5mmol/L induces to final concentration to add isopropyl-at=0.5～0.7 o'clock.After adding inductor, after continuing to cultivate 24h, the culture condition of 30 ℃ of temperature, rotating speed 200r/min stops.

With results bacterium liquid 10, centrifugal 10min abandons supernatant liquor under the 000g condition.After thalline 1 * PBS damping fluid washing of collecting 2 times, thalline is resuspended in the 1 * PBS damping fluid that is equivalent to stock culture 1/50 volume precooling.The ultrasonic disruption cell, power 30W, interval 5s amounts to 30min behind every broken 15s, keeps cell low temperature (0 ℃) in the shattering process.Broken back in 4 ℃ with 12, the centrifugal 30min of 000g, supernatant liquor with 0.45 μ m nitrocellulose membrane filtration after, add isopyknic chloroform, put upside down even 1h.At the centrifugal 30min of 10000g, the careful blueness precipitation that is rich in PCB of drawing.To precipitate and dissolve again with DMSO.Insolubles is removed through the centrifugal 10min of 10000g.

With the blue solution that above-mentioned processing obtains, last sample is to the Desalting column that crosses with PBS damping fluid balance in advance (GE healthcare), and last sample volume is no more than 1/3 of column volume, and flow velocity is 2mL/min; Then with PBS damping fluid flush away foreign protein, last phycocyanobilin elutes with 20% ethanolic soln then.Monitor A during wash-out simultaneously ₂₈₀And A ₃₆₉, collect elution peak, thereby obtain the higher phycocyanobilin of purity.

Embodiment 5

(1) synechococcus Synechococcus elongatus PCC 6301 full genomes have checked order and have finished, and (Genebank is numbered can freely to obtain its gene order from Genebank AP008231).Gene order according to synechococcus Synechococcus elongatus PCC 6301 heme oxidase genes of announcing among the Genebank (ho1) and ferredoxin oxide-reductase gene (pcyA), design suitable primer, with synechococcus Synechococcus elongatus PCC 6301 genomic dnas is template, adopt gene engineering method, synthetic key gene ho1 of clone's phycocyanobilin and pcyA.Wherein synthetic key gene ho1 of phycocyanobilin and the numbering of pcyA in Genebank are respectively syc2236_c and syc0325_d.

(2) using gene engineering method adds BamH I and Sac I restriction enzyme site respectively at ho1 gene two ends, and is inserted into first multiple clone site place of pETDuet-1 plasmid, obtains recombinant vectors pETDuet-ho1.Add Nde I and Xho I restriction enzyme site at gene pcyA two ends respectively, and be cloned into another multiple clone site place of pETDuet-ho1, obtain recombinant vectors pETDuet-ho1-pcyA (as shown in Figure 1).Concrete operation method is as follows:

According to ho1 that is announced among the Genebank and pcyA gene order design primer.Forward primer P5:5 '-TA of ho1 GGATCCTATGGGCGCGAATCTAGCA-3 ', reverse primer P6:5 '-TT GAGCTCTTATTCGGCTGCAGTTG-3 '.Forward primer P7:5 '-TT of pcyA CATATGATGTCCTCCAGCACGCGAG-3 ', reverse primer P8:5 '-TG CTCGAGTCAATCACTGGGCAAATC-3 '.React amplification template with Synechocystis sp.PCC 6301 genomic dnas as PCR.Obtain required gene fragment through pcr amplification.The PCR product is through Cycle pure test kit (Omega company) purifying.With gained ho1 and pcyA gene fragment and pETDuet-1 carrier respectively with 37 ℃ of digestion of corresponding suitable endonuclease 6h.Enzyme is cut product behind agarose gel electrophoresis, reclaims from glue, and gene fragment is mixed with carrier at 3: 1, and 16 ℃ of connections are spent the night under the effect of T4DNA ligase enzyme, connects product transformed into escherichia coli DH5 α.Utilization contains the LB plate screening of penbritin, and the picking positive colony utilizes PCR and dna sequencing to verify, obtains recombinant expression vector pETDuet-ho1-pcyA.Extract the expression plasmid pETDuet-ho1-pcyA that builds with alkaline denaturation, transformed competence colibacillus e. coli bl21 (DE3), obtaining can be at T ₇Efficiently express the coli strain P5 of active phycocyanobilin under the promoters driven.The method that the screening of above-mentioned plasmid extraction, connection, conversion and positive recombinant and evaluation are all introduced with reference to " molecular cloning experiment guide " (work such as J. Sa nurse Brooker, Huang Peitang etc. translate, Science Press, the third edition in 2002).

(3) the single bacterium colony of picking genetically engineered bacteria strain P5 contains the liquid LB substratum of 100ug/mL penbritin in 5mL from LB solid (the LB substratum that contains 1.5% agar) flat board, in the constant-temperature shaking culture case, cultivate (temperature: 37 ℃, rotating speed: about 200r/min).Overnight culture is inoculated in the TB substratum (containing penbritin 100ug/mL) of 1L with 1: 100 ratio.Be cultured to OD under the culture condition with 37 ℃ of temperature, rotating speed 200r/min in the constant-temperature shaking culture case ₆₀₀(IPTG, Isopropyl-β-D-thiogalactopyranoside) is that 0.5mmol/L induces to final concentration to add isopropyl-at=0.5～0.7 o'clock.After adding inductor, after continuing to cultivate 24h, the culture condition of 30 ℃ of temperature, rotating speed 200r/min stops.

With results bacterium liquid 10, centrifugal 10min abandons supernatant liquor under the 000g condition.After thalline 1 * PBS damping fluid washing of collecting 2 times, thalline is resuspended in the 1 * PBS damping fluid that is equivalent to stock culture 1/50 volume precooling.The ultrasonic disruption cell, power 30W, interval 5s amounts to 30min behind every broken 15s, keeps cell low temperature (0 ℃) in the shattering process.Broken back in 4 ℃ with 12, the centrifugal 30min of 000g, supernatant liquor with 0.45 μ m nitrocellulose membrane filtration after, add isopyknic chloroform, put upside down even 1h.At the centrifugal 30min of 10000g, the careful blueness precipitation that is rich in PCB of drawing.To precipitate and dissolve again with DMSO.Insolubles is removed through the centrifugal 10min of 10000g.

With the blue solution that above-mentioned processing obtains, last sample is to the Desalting column that crosses with the PBS balance in advance (GE healthcare), and last sample volume is no more than 1/3 of column volume, and flow velocity is 2mL/min; Then with PBS damping fluid flush away foreign protein, last phycocyanobilin elutes with 20% ethanolic soln then.Monitor A during wash-out simultaneously ₂₈₀And A ₃₆₉, collect elution peak, thereby obtain the higher phycocyanobilin of purity.

Embodiment 6

(1) synechococcus Synechococcus sp.WH 8102 full genomes have checked order and have finished, and can freely obtain from Genebank (Genebank is numbered BX548020).Gene order according to heme oxidase gene of announcing among the Genebank (ho1) and ferredoxin oxide-reductase gene (pcyA), design suitable primer, with Synechococcus sp.WH 8102 genomic dnas is template, adopt gene engineering method, synthetic key gene ho1 of clone's phycocyanobilin and pcyA.Synthetic key gene ho1 of phycocyanobilin and the numbering of pcyA in Genebank are respectively SYNW0171 and SYNW1084.

(2) using gene engineering method adds BamH I and Sac I restriction enzyme site respectively at ho1 gene two ends, and is inserted into the multiple clone site place of pETDuet-1 plasmid, obtains recombinant vectors pETDuet-ho1.Add Nde I and Xho I restriction enzyme site at gene pcyA two ends respectively, and be cloned into the downstream site of pETDuet-ho1, obtain recombinant vectors pETDuet-ho1-pcyA (as shown in Figure 1).Concrete operation method is as follows:

According to ho1 that is announced among the Genebank and pcyA gene order design primer.Forward primer P9:5 '-TA of ho1 GGATCCTATGTCTGTCGCTCTGGCTAC-3 ', reverse primer P10:5 '-TT GAGCTCTCAGGCCGCCGCAG-3 '.Forward primer P11:5 '-TT of pcyA CATATGATGCAGTCCCCGCCGTC-3 ', reverse primer P12:5 '-TG CTCGAGTCAACCTGGAGGCAAAGG-3 '.React amplification template with Synechococcus sp.WH 8102 genomic dnas as PCR.Obtain required gene fragment through pcr amplification.The PCR product is through Cycle pure test kit (Omega company) purifying.With gained ho1 and pcyA gene fragment and pETDuet-1 carrier respectively with 37 ℃ of digestion of corresponding suitable endonuclease 6h.Enzyme is cut product behind agarose gel electrophoresis, reclaims from glue, and gene fragment is mixed with carrier at 3: 1, and 16 ℃ of connections are spent the night under the effect of T4DNA ligase enzyme, connects product transformed into escherichia coli DH5 α.Utilize the LB plate screening of penbritin, the picking positive colony utilizes PCR and dna sequencing to verify, obtains recombinant expression vector pETDuet-ho1-pcyA.Extract the expression plasmid pETDuet-ho1-pcyA that builds with alkaline denaturation, transformed competence colibacillus e. coli bl21 (DE3), obtaining can be at T ₇Efficiently express the coli strain P6 of active phycocyanobilin under the promoters driven.The method that the screening of above-mentioned plasmid extraction, connection, conversion and positive recombinant and evaluation are all introduced with reference to " molecular cloning experiment guide " (work such as J. Sa nurse Brooker, Huang Peitang etc. translate, Science Press, the third edition in 2002).

(3) the single bacterium colony of picking genetically engineered bacteria strain P6 contains the liquid LB substratum of 50ug/mL penbritin in 5mL from LB solid (the LB substratum that contains 1.5% agar) flat board, in the constant-temperature shaking culture case, cultivate (temperature: 37 ℃, rotating speed: about 200r/min).Overnight culture is inoculated in the TB substratum (containing penbritin 50ug/mL) of 1L with 1: 100 ratio.Be cultured to OD under the culture condition with 37 ℃ of temperature, rotating speed 200r/min in the constant-temperature shaking culture case ₆₀₀(IPTG, Isopropyl-β-D-thiogalactopyranoside) is that 0.5mmol/L induces to final concentration to add isopropyl-at=0.5～0.7 o'clock.After adding inductor, after continuing to cultivate 24h, the culture condition of 30 ℃ of temperature, rotating speed 200r/min stops.

With results bacterium liquid 10, centrifugal 10min abandons supernatant liquor under the 000g condition.After thalline 1 * PBS damping fluid washing of collecting 2 times, thalline is resuspended in the 1 * PBS damping fluid that is equivalent to stock culture 1/50 volume precooling.The ultrasonic disruption cell, power 30W, interval 5s amounts to 30min behind every broken 15s, keeps cell low temperature (0 ℃) in the shattering process.Broken back in 4 ℃ with 12, the centrifugal 30min of 000g, supernatant liquor with 0.45 μ m nitrocellulose membrane filtration after, add isopyknic chloroform, put upside down even 1h.At the centrifugal 30min of 10000g, the careful blueness precipitation that is rich in PCB of drawing.To precipitate and dissolve again with DMSO.Insolubles is removed through the centrifugal 10min of 10000g.

With the blue solution that above-mentioned processing obtains, last sample is to the Desalting column that crosses with PBS damping fluid balance in advance (GE healthcare), and last sample volume is no more than 1/3 of column volume, and flow velocity is 2mL/min; Then with PBS damping fluid flush away foreign protein, last phycocyanobilin elutes with 20% ethanolic soln then.Monitor A during wash-out simultaneously ₂₈₀And A ₃₆₉, collect elution peak, thereby obtain the higher phycocyanobilin of purity.

Embodiment 7

(1) the full genome of thermophilic blue-green algae Thermosynechococcus elongatus BP-1 has checked order and has finished, and can freely obtain from Genebank (Genebank is numbered BA000039).Gene order according to heme oxidase gene of announcing among the Genebank (ho1) and ferredoxin oxide-reductase gene (pcyA), design suitable primer, with Thermosynechococcus elongatus BP-1 genomic dna is template, adopt gene engineering method, synthetic key gene ho1 of clone's phycocyanobilin and pcyA.Synthetic key gene ho1 of phycocyanobilin and the numbering of pcyA in Genebank are respectively tll0365 and tll2308.

(2) using gene engineering method adds BamH I and Sac I restriction enzyme site respectively at ho1 gene two ends, and is inserted into first multiple clone site place of pETDuet-1 plasmid, obtains recombinant vectors pETDuet-ho1.Add Nde I and Xho I restriction enzyme site at gene pcyA two ends respectively, and be cloned into another multiple clone site place of pETDuet-ho1, obtain recombinant vectors pETDuet-ho1-pcyA (as shown in Figure 1).Concrete operation method is as follows:

According to ho1 that is announced among the Genebank and pcyA gene order design primer.Forward primer P13:5 '-TA of ho1 GGATCCTATGACAACGTCTCTAGC-3 ', reverse primer P14:5 '-TT GAGCTCTTAGTCGGCGGTGG-3 '.Forward primer P15:5 '-TT of pcyA CATATGATGTCTTTGCGTCAACAC-3 ', reverse primer P16:5 '-TG CTCGAGCTACACCGGGGGGACAT-3 '.React amplification template with Thermosynechococcus elongatus BP-1 genomic dna as PCR.Obtain required gene fragment through pcr amplification.The PCR product is through Cycle pure test kit (Omega company) purifying.With gained ho1 and pcyA gene fragment and pETDuet-1 carrier respectively with 37 ℃ of digestion of corresponding suitable endonuclease 6h.Enzyme is cut product behind agarose gel electrophoresis, reclaims from glue, and gene fragment is mixed with carrier at 3: 1, and 16 ℃ of connections are spent the night under the effect of T4DNA ligase enzyme, connects product transformed into escherichia coli DH5 α.Utilization contains the LB plate screening of penbritin, and the picking positive colony utilizes PCR and dna sequencing to verify, obtains recombinant expression vector pETDuet-ho1-pcyA.Extract the expression plasmid pETDuet-ho1-pcyA that builds with alkaline denaturation, transformed competence colibacillus e. coli bl21 (DE3), obtaining can be at T ₇Efficiently express the coli strain P7 of active phycocyanobilin under the promoters driven.The method that the screening of above-mentioned plasmid extraction, connection, conversion and positive recombinant and evaluation are all introduced with reference to " molecular cloning experiment guide " (work such as J. Sa nurse Brooker, Huang Peitang etc. translate, Science Press, the third edition in 2002).

(3) the single bacterium colony of picking genetically engineered bacteria strain P7 contains the liquid LB substratum of 100ug/mL penbritin in 5mL from LB solid (the LB substratum that contains 1.5% agar) flat board, in the constant-temperature shaking culture case, cultivate (temperature: 37 ℃, rotating speed: about 200r/min).Overnight culture is inoculated in the TB substratum (containing penbritin 100ug/mL) of 1L with 1: 100 ratio.Be cultured to OD under the culture condition with 37 ℃ of temperature, rotating speed 200r/min in the constant-temperature shaking culture case ₆₀₀(IPTG, Isopropyl-β-D-thiogalactopyranoside) is that 0.5mmol/L induces to final concentration to add isopropyl-at=0.5～0.7 o'clock.After adding inductor, after continuing to cultivate 24h, the culture condition of 30 ℃ of temperature, rotating speed 200r/min stops.

With results bacterium liquid 10, centrifugal 10min abandons supernatant liquor under the 000g condition.After thalline 1 * PBS wash buffer of collecting is washed 2 times, thalline is resuspended in the 1 * PBS damping fluid that is equivalent to stock culture 1/50 volume precooling.The ultrasonic disruption cell, power 30W, interval 5s amounts to 30min behind every broken 15s, keeps cell low temperature (0 ℃) in the shattering process.Broken back in 4 ℃ with 12, the centrifugal 30min of 000g, supernatant liquor with 0.45 μ m nitrocellulose membrane filtration after, add isopyknic chloroform, put upside down even 1h.At the centrifugal 30min of 10000g, the careful blueness precipitation that is rich in PCB of drawing.To precipitate and dissolve again with DMSO.Insolubles is removed through the centrifugal 10min of 10000g.

With the blue solution that above-mentioned processing obtains, last sample is to the Desalting column that crosses with PBS damping fluid balance in advance (GE healthcare), and last sample volume is no more than 1/3 of column volume, and flow velocity is 2mL/min; Then with PBS damping fluid flush away foreign protein, last phycocyanobilin elutes with 20% ethanolic soln then.Monitor A during wash-out simultaneously ₂₈₀And A ₃₆₉, collect elution peak, thereby obtain the higher phycocyanobilin of purity.

Embodiment 8

(1) the full genome of Chlamydomonas reinhardtii Chlamydomonas reinhardtii has checked order and has finished, and can freely obtain that (Genebank is numbered from Genebank ABCN00000000).Gene order according to heme oxidase gene of announcing among the Genebank (ho1) and ferredoxin oxide-reductase gene (pcyA), design suitable primer, with Chlamydomonas reinhardtii genomic dna is template, adopt gene engineering method, synthetic key gene ho1 of clone's phycocyanobilin and pcyA.Synthetic key gene ho1 of phycocyanobilin and the numbering of pcyA in Genebank are respectively CHLREDRAFT_152591 and CHLREDRAFT_156256.

(2) using gene engineering method adds Nde I and EcoR V restriction enzyme site respectively at ho1 gene two ends, and is inserted into the multiple clone site place of pET28a-1 plasmid, obtains recombinant vectors pET28a-ho1.Add EcoR V and Xho I restriction enzyme site at gene pcyA two ends respectively, and be cloned into the downstream site (as shown in Figure 2) of pET28a-ho1, obtain recombinant vectors pET28a-ho1-pcyA.Concrete operation method is as follows:

According to ho1 that is announced among the Genebank and pcyA gene order design primer.Forward primer P17:5 '-TA of ho1 CATATGTATGTCGGATGCA-3 ', reverse primer P18:5 '-TT GATATCCGTGTGCGGCAAAGCAGCA-3 '.Forward primer P19:5 '-TT of pcyA GATATCATGCGCTGCTGAGGCGCATT-3 ', reverse primer P20:5 '-TG CTCGAGTGCCGACAGCGGAGCTAA-3 '.React amplification template with Chlamydomonas reinhardtii Chlamydomonas reinhardtii genomic dna as PCR.Obtain required gene fragment through pcr amplification.The PCR product is through Cycle pure test kit (Omega company) purifying.With gained ho1 and pcyA gene fragment and pET28a-1 carrier respectively with 37 ℃ of digestion of corresponding suitable endonuclease 6h.Enzyme is cut product behind agarose gel electrophoresis, reclaims from glue, and gene fragment is mixed with carrier at 3: 1, and 16 ℃ of connections are spent the night under the effect of T4DNA ligase enzyme, connects product transformed into escherichia coli DH5 α.Utilization contains the LB plate screening of kantlex, and the picking positive colony utilizes PCR and dna sequencing to verify, obtains recombinant expression vector pET28a-ho1-pcyA.Extract the expression plasmid pET28a-ho1-pcyA that builds with alkaline denaturation, transformed competence colibacillus e. coli bl21 (DE3), obtaining can be at T ₇Efficiently express the coli strain P8 of active phycocyanobilin under the promoters driven.The method that the screening of above-mentioned plasmid extraction, connection, conversion and positive recombinant and evaluation are all introduced with reference to " molecular cloning experiment guide " (work such as J. Sa nurse Brooker, Huang Peitang etc. translate, Science Press, the third edition in 2002).

(3) the single bacterium colony of picking genetically engineered bacteria strain P8 contains the liquid LB substratum of 50ug/mL kantlex in 5mL from LB solid (the LB substratum that contains 1.5% agar) flat board, in the constant-temperature shaking culture case, cultivate (temperature: 37 ℃, rotating speed: about 200r/min).Overnight culture is inoculated in the TB substratum (containing kantlex 50ug/mL) of 1L with 1: 100 ratio.Be cultured to OD under the culture condition with 37 ℃ of temperature, rotating speed 200r/min in the constant-temperature shaking culture case ₆₀₀(IPTG, Isopropyl-β-D-thiogalactopyranoside) is that 0.5mmol/L induces to final concentration to add isopropyl-at=0.5～0.7 o'clock.After adding inductor, after continuing to cultivate 24h, the culture condition of 30 ℃ of temperature, rotating speed 200r/min stops.

With results bacterium liquid 10, centrifugal 10min abandons supernatant liquor under the 000g condition.After thalline 1 * PBS damping fluid washing of collecting 2 times, thalline is resuspended in the 1 * PBS damping fluid that is equivalent to stock culture 1/50 volume precooling.The ultrasonic disruption cell, power 30W, interval 5s amounts to 30min behind every broken 15s, keeps cell low temperature (0 ℃) in the shattering process.Broken back in 4 ℃ with 12, the centrifugal 30min of 000g, supernatant liquor with 0.45 μ m nitrocellulose membrane filtration after, add isopyknic chloroform, put upside down even 1h.At the centrifugal 30min of 10000g, the careful blueness precipitation that is rich in PCB of drawing.To precipitate and dissolve again with DMSO.Insolubles is removed through the centrifugal 10min of 10000g.

With the blue solution that above-mentioned processing obtains, last sample is to the Desalting column that crosses with PBS damping fluid balance in advance (GE healthcare), and last sample volume is no more than 1/3 of column volume, and flow velocity is 2mL/min; Then with PBS damping fluid flush away foreign protein, last phycocyanobilin elutes with 20% ethanolic soln then.Monitor A during wash-out simultaneously ₂₈₀And A ₃₆₉, collect elution peak, thereby obtain the higher phycocyanobilin of purity.

The antioxygenation of embodiment 9 phycocyanobilins

Adopt DPPH free radical scavenging method (Hirata, T., Tanaka, M., Ooike, M., Tsunomura, T., and Sakaguchi, M.Antioxidant activities of phycocyanobilin prepared from Spirulina platensis J Appl Phycol 12,2000,435-439. indicates reference).Measuring method is as follows: the tested liquid (A) of getting the 200 μ M DPPH ethanolic solns (each moiety as table 1-1 shown in) of 100 μ L and 100 μ L is in 96 orifice plates, mixing, after placing 30min under the room temperature, survey absorbancy at the 517nm place, measure 100 μ L DPPH solution+100 μ L dehydrated alcohol mixed solutions (B) and the tested liquid of the 100 μ L+absorbancy of 100 μ L dehydrated alcohol mixed solutions (C) under 517nm simultaneously.Calculate the DPPH free radical scavenging activity according to following formula: DPPH clearance rate=[1-(A-C)/B] * 100%.

Tested herein liquid is the phycocyanobilin solution behind the above-mentioned purifying.We respectively with the PCB solution of different concns as being subjected to test solution, shown in the mixed liquor A table composed as follows 1-1, measure the relation between phycocyanobilin antioxidant effect and the concentration.

Table 1-1 phycocyanobilin antioxidant effect is measured system

As shown in Figure 5, phycocyanobilin has stronger removing DPPH free radical ability, and in certain test specification, along with the increase of concentration, the also corresponding increase of its clearance rate has tangible dose-effect relationship, its IC ₅₀Be 160 μ g/L.

Embodiment 10 phycocyanobilins are as the application of natural pigment and antioxidant aspect

Phycocyanobilin behind the purifying has stronger absorption value (as Fig. 5) at 369nm and 680nm place respectively, and it is blue that solution is.Excite through 460nm, maximum emission peak is arranged at the 630nm place.Phycocyanins, C-(comprising phycocyanobilin and apoprotein) is used for aspects such as health care and optical dynamic therapy by the approval of U.S. medicine Surveillance Authority for medicine, so its security has also obtained checking.Therefore, phycocyanobilin can be used as natural pigment and antioxidant, joins in food (as noodles, bread, ice cream etc.) or the beverage (containing the mineral water, sports beverages, nourishing drink of phycocyanobilin etc.), thereby increases the color and luster and the nourishing function of product.

From the foregoing description as can be seen, utilizing genetic engineering technique, is basic characteristics of the present invention by engineering strain fermentative production reorganization phycocyanobilin.Present method does not relate to and takes up an area of algae culture problem wide, that the cycle is long in addition, and the direct production phycocyanobilin does not relate to the breakage problem of phycocyanobilin and apoprotein, easier acquisition high purity, phycocyanobilin cheaply.The reorganization phycocyanobilin that this method is produced has the remarkable antioxygenation that gets, and can be used for preparing purposes such as anti-oxidation medicine, health care or functional food, foodstuff additive, reagent.

Claims (9)

1. a reorganization phycocyanobilin preparation method is characterized in that comprising the steps:

By cloning heme oxidase gene ho1 and ferredoxin oxide-reductase gene pcyA in the algae, above-mentioned ho1 gene and pcyA gene clone to expression vector, are then imported and carry out heterogenous expression in the intestinal bacteria, produce the reorganization phycocyanobilin.

2. the method for claim 1, it is characterized in that also further may further comprise the steps in the described method: intestinal bacteria fragmentation, chloroform extraction and gel permeation chromatography after will transforming obtain described reorganization phycocyanobilin.

3. method as claimed in claim 2 is characterized in that described algae is selected from blue-green algae, red algae, latent algae, dinoflagellate or proto green algae.

4. method as claimed in claim 2 is characterized in that described algae is selected from cytoalgae, synechococcus, Chlamydomonas reinhardtii or thermophilic blue-green algae (Thermosynechococcus elongates).

5. method as claimed in claim 4 is characterized in that described heme oxidase gene ho1 is selected from the ho1 gene that is numbered sll1184, syc2236_c, SYNW0171, CHLREDRAFT_152591 or tll0365 among the Genebank; Described ferredoxin oxide-reductase gene pcyA preferably is numbered the pcyA gene of slr0116, syc0325_d, SYNW1084, CHLREDRAFT_156256 or tll2308 in Genebank.

6. as the arbitrary described method of claim 1-5, it is characterized in that described expression vector is selected from pETDuet-1, pET28a-1, pCDFDuet-1 or pACYCDuet-1.

7. method as claimed in claim 6 is characterized in that described intestinal bacteria are e. coli bl21.

8. the reorganization phycocyanobilin of the arbitrary described method preparation of claim 1-7 is in medicine or Application in Food.

9. application as claimed in claim 8 is characterized in that being used for anti-oxidation medicine or antioxidant food.

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