WO2017107237A1 - 一种高荧光强度的重组藻胆蛋白串联体的制备方法 - Google Patents

一种高荧光强度的重组藻胆蛋白串联体的制备方法 Download PDF

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WO2017107237A1
WO2017107237A1 PCT/CN2015/099730 CN2015099730W WO2017107237A1 WO 2017107237 A1 WO2017107237 A1 WO 2017107237A1 CN 2015099730 W CN2015099730 W CN 2015099730W WO 2017107237 A1 WO2017107237 A1 WO 2017107237A1
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gene
recombinant
phycobiliprotein
allophycocyanin
fluorescence intensity
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PCT/CN2015/099730
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French (fr)
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陈华新
姜鹏
武静
李富超
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中国科学院海洋研究所
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria

Definitions

  • the invention belongs to the field of recombinant expression preparation of fluorescent proteins in biotechnology, and in particular relates to a method for preparing a recombinant phycobiliprotein cascade which has high fluorescence intensity.
  • the phycobiliprotein is an important pigment protein of algae and has the function of capturing and transmitting light energy.
  • Each molecule of phycobiliprotein contains two structurally similar polypeptide chains ⁇ and ⁇ , and the ⁇ subunit and the ⁇ subunit contain about 160 to 180 amino acid residues, respectively, and the ratio of the two is usually 1:1.
  • the cysteine residue in the subunit is covalently bound to the phycobiliprotein by a thioether bond, and the species of the phycobiliprotein and its interaction with the apoprotein determine the spectroscopic properties of the phycobiliprotein.
  • the phycobiliprotein can emit strong fluorescence, and the most important use of its fluorescent properties is in fluorescent labeling and biomedical research for immunodiagnosis. It can be combined with biotin, avidin or various antibodies to make fluorescent probes. For use in immunofluorescence analysis and detection. In immunofluorescence analysis, phycobiliproteins are required to bind to antibodies as fluorescent markers, ie covalently cross-linking phycobiliprotein molecules with antibody molecules via bifunctional reagents, or phycobiliproteins via crosslinkers with streptavidin After cross-linking, the phycobiliprotein molecule is indirectly coupled to the antibody molecule by the biotin-avidin system (BAS).
  • BAS biotin-avidin system
  • the invention provides a preparation method of a recombinant phycobiliprotein subunit tandem with biotin binding ability. Compared with the recombinant phycobiliprotein subunit monomer, the tandem protein has higher fluorescence intensity, and it has a good application prospect in the immunofluorescence detection of antigen in the fields of biology and biomedicine.
  • the object of the present invention is to provide a process for preparing a recombinant phycobiliprotein cascade having high fluorescence intensity.
  • a method for preparing a recombinant phycobiliprotein cascade with high fluorescence intensity characterized in that the streptavidin gene and the allophycocyanin ⁇ subunit gene are linked by a Linker sequence.
  • the tandem sequence of one or more allophycocyanin ⁇ subunit genes is realized by a linker sequence to form a fusion gene.
  • the fusion gene, the phycobiliprotein lyase gene and the phycoerythrin biosynthesis enzyme gene were co-expressed in E. coli to obtain a recombinant allophycocyanin tandem having a higher fluorescence intensity covalently bound to phycoerythrin.
  • streptavidin gene and the allophycocyanin ⁇ subunit gene are joined by a linker sequence to form a fusion gene, and on this basis, one or more allophycocyanin ⁇ subunit genes are linked by a linker sequence.
  • the recombinant Escherichia coli was subjected to fermentation culture, and the expression of the fusion protein was induced by IPTG, and purified by metal chelate affinity chromatography to obtain a fusion fluorescent protein covalently bound to phycoerythrin.
  • the allophycocyanin ⁇ subunit gene is an allophycocyanin Synechoccus elongatus BP-1 allophycocyanin ⁇ subunit gene, or a homologous gene thereof; the phycoerythrin biosynthesis enzyme gene Ho1 is Synecocystis sp. PCC 6803Ho1
  • pebS is the pebS gene of Prochlorococcus phage P-SSM2
  • cpcS is the Synechoccus elongatus BP-1 cpcS gene, and the linker sequence is:
  • the present invention has the following advantages:
  • the method of genetic engineering uses the linker sequence of the present invention to cascade the allophycocyanin ⁇ subunits, thereby avoiding the mutual interference of the adjacent allophycocyl ⁇ subunits and improving the fluorescence of the recombinant phycobiliprotein. Intensity, has a stronger fluorescent signal in immunofluorescence detection;
  • E. coli is easy to culture, grows fast, and has a short production cycle.
  • the invention utilizes Escherichia coli fermentation to prepare a recombinant phycobiliprotein cascade with high fluorescence intensity, and has the advantages of simple preparation, low cost and the like.
  • Figure 1 is a restriction map of phycobiliprotein monomers and tandem expression vectors constructed in the examples of the present invention.
  • Figure 3 is an absorption spectrum of a purified phycobiliprotein monomer and a tandem sample after purification according to an embodiment of the present invention.
  • Figure 5 is a graph showing the detection of immunofluorescence of AFP by recombinant phycobiliprotein monomer and tandem after purification according to an embodiment of the present invention.
  • the apcA gene was obtained by using Synechococcus elongatus BP-1 genomic DNA as a template, Ho1 using Synechocystis.PCC 6803 genomic DNA as a template, and cpcS using Synechococcus elongatus BP-1 genomic DNA as a template and amplified according to conventional PCR conditions.
  • the SA gene and linker sequences were synthesized by Nanjing Kingsray Biotechnology Co., Ltd. In order to facilitate the fusion PCR reaction, the partial sequence of the SA gene and the apcA gene was added to the 5' and 3' ends, respectively, during the synthesis of the linker sequence.
  • the fusion gene SA-Linker-apcA (abbreviation: SLA) was obtained by fusion PCR with primers SA-F and primer apcAR. After the SLA fragment was recovered, it was digested with restriction endonucleases BamHI and SacI, and the vector pCDFDuet-1 was also digested with the same enzyme. The vector fragment and the fusion gene fragment were separately recovered and then ligated at a molar ratio of 1:5. After ligation overnight at 16 °C, the ligation product was transformed into competent E. coli Top10.
  • the clones were picked from the transformation plates for PCR, and the positive clones were sampled for DNA sequencing, and the plasmid was extracted and identified by restriction endonuclease BamHI and SacI (Fig. 1). The enzyme-clearing and DNA-sequence-tested samples were used for seed conservation, and the successful recombinant plasmid was constructed as: pCDF-SLA.
  • the expression plasmid pCDF-SLA was digested with BglII and Xho, and the cpcS fragment obtained by the above PCR amplification was recovered, and then digested with BglII and SalI.
  • the vector fragment and the cpcS digestion product were separately recovered by a gel recovery kit, and the recovered vector fragment and the cpcS fragment were ligated at a molar ratio of 1:5, and after ligation at 16 ° C overnight, the ligated product was transformed into competent Escherichia coli. Top10.
  • the clone was picked from the transformation plate to make the bacterial solution PCR, and the positive clone was picked for sequencing, and the uncorrected sample was used for seed conservation.
  • the constructed co-expression plasmid was labeled as pCDF-SLA-cpcS.
  • the Ho1 gene was recovered by PCR amplification, and then digested with BglII and SalI.
  • the pRSFDuet-1 vector was digested with BglII and XhoI.
  • the carrier fragment and the Ho1 fragment were separately recovered using a gel recovery kit, and ligated at a molar ratio of 1:5. After ligation overnight at 16 °C, the ligation product was transformed into E. coli Top10.
  • the clone was picked from the transformation plate and the PCR was performed, and the positive clone was sent for sequencing.
  • the uncorrected clones were sequenced and used for seed conservation, and the successfully constructed plasmid was labeled as pRSF-Ho1.
  • the synthetic gene pebS and the plasmid pRSF-Ho1 were digested with NcoI and SalI, and the vector fragment and the gene fragment were separately recovered and ligated at a molar ratio of 1:5. After ligation overnight at 16 °C, the ligation product was transformed into E. coli Top10. The clone was picked from the transformation plate and the PCR was performed, and the positive clone was sent for sequencing. The uncorrected clones were sequenced and used for seed conservation, and the successfully constructed plasmid was labeled as pRSF-Ho1-pebS.
  • the above constructed plasmids pCDF-SLA-cpcS and pRSF-Ho1-pebS were introduced into Escherichia coli BL21 (DE3) by co-transformation, and the transformed Escherichia coli was coated with 100 ⁇ g/mL kanamycin and 100 ⁇ g/mL. On LB plates of mycin, after overnight incubation at 37 °C. Monoclones were picked from the plates and inoculated in 3 mL of LB medium (the medium containing the antibiotic kanamycin and spectinomycin) and cultured overnight at 37 °C.
  • LB medium the medium containing the antibiotic kanamycin and spectinomycin
  • the overnight culture was transferred to 60 mL of LB medium containing the corresponding antibiotic, cultured at 37 ° C, 200 rpm for 4 hours, and added to a final concentration of 1 mM IPTG.
  • the culture was placed at 18 ° C and continued at 160 rpm for 16-20 hours.
  • the cells were collected by centrifugation, and the cells were disrupted by an ultrasonic cell pulverizer.
  • the supernatant was collected, added to Loading Buffer, boiled for 10 minutes, and sampled for SDS-PAGE electrophoresis.
  • the strain with high expression of recombinant protein is selected, and the glycerol is used for the subsequent fermentation and separation and purification of the recombinant protein.
  • the cells were collected by centrifugation, and the cells were suspended in a suspension solution (500 mmol/L NaCl, 15.5 mmol/L Na 2 HPO 4 , 4.5 mmol/L NaH 2 PO 4 , 20 mmol/L imidazole, pH 7.4) in an ice bath. Ultrasonic disruption for 30 min. The disrupted solution was centrifuged at 4 ° C, 8000 rpm for 30 min, and the supernatant was taken.
  • a suspension solution 500 mmol/L NaCl, 15.5 mmol/L Na 2 HPO 4 , 4.5 mmol/L NaH 2 PO 4 , 20 mmol/L imidazole, pH 7.4
  • the nickel column (HiTrap FF, GE) was equilibrated with binding buffer (500 mmol/L NaCl, 15.5 mmol/L Na 2 HPO 4 , 4.5 mmol/L NaH 2 PO 4 , 20 mmol/L imidazole, pH 7.4). After the column, the nickel column was washed with a washing buffer (500 mmol/L NaCl, 15.5 mmol/L Na 2 HPO 4 , 4.5 mmol/L NaH 2 PO 4 , 90 mmol/L imidazole, pH 7.4) to remove the heteroprotein.
  • binding buffer 500 mmol/L NaCl, 15.5 mmol/L Na 2 HPO 4 , 4.5 mmol/L NaH 2 PO 4 , 20 mmol/L imidazole, pH 7.4
  • a washing buffer 500 mmol/L NaCl, 15.5 mmol/L Na 2 HPO 4 , 4.5 mmol/L NaH 2 PO 4 , 90 m
  • elution was carried out with an eluent (500 mmol/L NaCl, 15.5 mmol/L Na 2 HPO 4 , 4.5 mmol/L NaH 2 PO 4 , 500 mmol/L imidazole, pH 7.4).
  • the eluate was passed through a G25 desalting column to remove the imidazole in the protein sample to obtain a recombinant protein solution.
  • the purified protein sample was taken, added to Loading Buffer, boiled for 10 minutes, and sampled for SDS-PAGE electrophoresis.
  • the electrophoresis results are shown in Fig. 2.
  • the band of recombinant phycobiliprotein is located between 35 kDa and 40 kDa, consistent with the theoretical molecular weight of the recombinant phycobiliprotein monomer.
  • a small amount of the purified recombinant phycobiliprotein sample was diluted with 10 mM sodium phosphate buffer (pH 7.4) to 10 -6 mol/L for the measurement of absorption spectrum and fluorescence emission spectrum, respectively.
  • the absorption spectrum was measured by UV1801 ultraviolet-visible spectrophotometer at room temperature. The measurement results are shown in Fig. 3.
  • the maximum absorption peak of the recombinant phycobiliprotein monomer was 550 nm.
  • the fluorescence emission spectrum was measured by F-4500 fluorescence spectrophotometer at room temperature, the fluorescence excitation wavelength was 500 nm, the scanning wavelength was 520-600 nm, the fluorescence emission spectrum was shown in Fig. 4, and the maximum fluorescence emission wavelength was 562 nm.
  • the AFP capture antibody was taken and diluted with a coating buffer (Na 2 CO 3 1.59 g/L, NaHCO 3 2.93 g/L, pH 9.6) to a final concentration of 5 ⁇ g/ml. 100 ⁇ l of the capture antibody solution was taken in the microplate of the fluorescent enzyme plate. The plate was placed at 4 ° C and allowed to stand overnight. The enzyme plate was taken out, the liquid in the well was cleaned, and the tissue was patted dry on the tissue.
  • a coating buffer Na 2 CO 3 1.59 g/L, NaHCO 3 2.93 g/L, pH 9.6
  • PBST KH 2 PO 4 0.27 g/L, Na 2 HPO 4 1.42 g/L, NaCl 8 g/L, KCl 0.2 g/L, Tween-20 0.5 ml/L, pH 7.4
  • 100 ⁇ l of blocking solution (10 g/L, prepared in PBST) was added to each well, placed on a wet box, placed at 37 ° C for 2 h, and washed three times with PBST.
  • AFP protein was serially diluted with PBST solution at concentrations of 51.2 ng/ml, 16.8 ng/ml, 3.2 ng/ml, 0.8 ng/ml, 0.2 ng/ml, 0.05 ng/ml, and 0 ng/ml, respectively.
  • 100 ⁇ l of each AFP protein solution was added to each well, and the mixture was incubated at 37 ° C, shaking at 400 rpm for 1 h, and washed three times with PBST.
  • the biotinylated detection antibody was taken and diluted with PBST to a final concentration of 5 ⁇ g/ml.
  • 100 ⁇ l of each well was added to the reaction well, and the mixture was incubated at 37 ° C, shaking at 400 rpm for 1 h, and washed three times with PBST.
  • the apcA gene was obtained by using Synechococcus elongatus BP-1 genomic DNA as a template, Ho1 using Synechocystis.PCC 6803 genomic DNA as a template, and cpcS using Synechococcus elongatus BP-1 genomic DNA as a template and amplified according to conventional PCR conditions.
  • the SA gene and linker sequences were synthesized by Nanjing Kingsray Biotechnology Co., Ltd. In order to facilitate the fusion PCR reaction, the partial sequence of the SA gene and the apcA gene was added to the 5' and 3' ends, respectively, during the synthesis of the linker sequence.
  • the fusion gene SA-Linker-apcA (abbreviation: SLA) was obtained by fusion PCR with primers SA-F and primer apcAR. After the SLA fragment was recovered, it was digested with restriction endonucleases BamHI and SacI, and the vector pCDFDuet-1 was also digested with the same enzyme. The vector fragment and the fusion gene fragment were separately recovered and then ligated at a molar ratio of 1:5. After ligation overnight at 16 °C, the ligation product was transformed into competent E. coli Top10. The clone was picked from the transformation plate and the PCR was carried out.
  • SLA restriction endonucleases
  • the positive clone was sampled for DNA sequencing, and the plasmid was extracted and identified by restriction endonuclease BamHI and SacI.
  • the enzyme-clearing and DNA-sequence-tested samples were used for seed conservation, and the successful recombinant plasmid was constructed as: pCDF-SLA.
  • the plasmid pCDF-SLA was digested with restriction endonuclease BamHI; the digested vector fragment was recovered by a gel recovery kit, and then treated with alkaline phosphatase CIAP, and the vector fragment was recovered again using a gel recovery kit.
  • the plasmid pCDF-SLA was used as a template, and the primers LAF and LAR were used for PCR amplification to obtain a PCR amplification product containing the Linker sequence and the apcA gene sequence (abbreviated as LA), and the LA was recovered by a gel recovery kit.
  • the restriction endonucleases BamHI and BglII were double-digested, and the digested product was recovered again using a gel recovery kit.
  • the vector fragment obtained by the above recovery and the digested product were ligated at a molar ratio of 1:5, and after ligation at 16 ° C overnight, the ligated product was transformed into competent E. coli Top10.
  • the clone was picked from the transformation plate and the PCR was performed, and the positive clones were picked and inoculated into 3 mL of LB medium. After overnight incubation at 37 ° C, the plasmid was extracted and identified by restriction endonuclease BamHI and SacI double digestion. Based on the restriction map, the recombinant plasmid with the correct insertion direction (Fig. 1) was selected and labeled as pCDF-S(LA) 2 .
  • the expression plasmid pCDF-S(LA) 2 was digested with BglII and Xho, and the cpcS fragment obtained by the above PCR amplification was recovered, and then digested with BglII and SalI.
  • the vector fragment and the cpcS digestion product were separately recovered by a gel recovery kit, and the recovered vector fragment and the cpcS fragment were ligated at a molar ratio of 1:5, and after ligation at 16 ° C overnight, the ligated product was transformed into competent Escherichia coli. Top10.
  • the clone was picked from the transformation plate to make the bacterial solution PCR, and the positive clone was picked for sequencing, and the uncorrected sample was used for seed conservation.
  • the constructed co-expression plasmid was labeled pCDF-S(LA) 2- cpcS.
  • the Ho1 gene was recovered by PCR amplification, and then digested with BglII and SalI.
  • the pRSFDuet-1 vector was digested with BglII and XhoI.
  • the carrier fragment and the Ho1 fragment were separately recovered using a gel recovery kit, and ligated at a molar ratio of 1:5. After ligation overnight at 16 °C, the ligation product was transformed into E. coli Top10.
  • the clone was picked from the transformation plate and the PCR was performed, and the positive clone was sent for sequencing.
  • the uncorrected clones were sequenced and used for seed conservation, and the successfully constructed plasmid was labeled as pRSF-Ho1.
  • the synthetic gene pebS and the plasmid pRSF-Ho1 were digested with NcoI and SalI, and the vector fragment and the gene fragment were separately recovered and ligated at a molar ratio of 1:5. After connecting overnight at 16 ° C, the ligation product is transformed into a large intestine rod Bacteria Top10. The clone was picked from the transformation plate and the PCR was performed, and the positive clone was sent for sequencing. The uncorrected clones were sequenced and used for seed conservation, and the successfully constructed plasmid was labeled as pRSF-Ho1-pebS.
  • the above constructed plasmids pCDF-S(LA) 2 -cpcS and pRSF-Ho1-pebS were introduced into E. coli BL21 (DE3) by co-transformation, and the transformed Escherichia coli was coated with 100 ⁇ g/mL kanamycin and 100 ⁇ g/mL spectinomycin on LB plates, after overnight incubation at 37 °C. Monoclones were picked from the plates and inoculated in 3 mL of LB medium (the medium containing the antibiotic kanamycin and spectinomycin) and cultured overnight at 37 °C.
  • LB medium the medium containing the antibiotic kanamycin and spectinomycin
  • the overnight culture was transferred to 60 mL of LB medium containing the corresponding antibiotic, cultured at 37 ° C, 200 rpm for 4 hours, and added to a final concentration of 1 mM IPTG.
  • the culture was placed at 18 ° C and continued at 160 rpm for 16-20 hours.
  • the cells were collected by centrifugation, and the cells were disrupted by an ultrasonic cell pulverizer.
  • the supernatant was collected, added to Loading Buffer, boiled for 10 minutes, and sampled for SDS-PAGE electrophoresis.
  • the strain with high expression of recombinant protein is selected, and the glycerol is used for the subsequent fermentation and separation and purification of the recombinant protein.
  • the cells were collected by centrifugation, and the cells were suspended in a suspension solution (500 mmol/L NaCl, 15.5 mmol/L Na 2 HPO 4 , 4.5 mmol/L NaH 2 PO 4 , 20 mmol/L imidazole, pH 7.4) in an ice bath. Ultrasonic disruption for 30 min. The disrupted solution was centrifuged at 4 ° C, 8000 rpm for 30 min, and the supernatant was taken.
  • a suspension solution 500 mmol/L NaCl, 15.5 mmol/L Na 2 HPO 4 , 4.5 mmol/L NaH 2 PO 4 , 20 mmol/L imidazole, pH 7.4
  • the nickel column (HiTrap FF, GE) was equilibrated with binding buffer (500 mmol/L NaCl, 15.5 mmol/L Na 2 HPO 4 , 4.5 mmol/L NaH 2 PO 4 , 20 mmol/L imidazole, pH 7.4). After the column, the nickel column was washed with washing buffer (500 mmol/L NaCl, 15.5 mmol/L Na 2 HPO 4 , 4.5 mmol/L NaH 2 PO 4 , 90 mmol/L imidazole, pH 7.4) to remove the impurity protein.
  • binding buffer 500 mmol/L NaCl, 15.5 mmol/L Na 2 HPO 4 , 4.5 mmol/L NaH 2 PO 4 , 20 mmol/L imidazole, pH 7.4
  • washing buffer 500 mmol/L NaCl, 15.5 mmol/L Na 2 HPO 4 , 4.5 mmol/L NaH 2 PO 4 , 90 mmol/
  • elution is carried out with an eluent (500 mmol/L NaCl, 15.5 mmol/L Na 2 HPO 4 , 4.5 mmol/L NaH 2 PO 4 , 500 mmol/L imidazole, pH 7.4), and the elution is collected.
  • the solution was passed through a G25 desalting column to remove the imidazole in the protein sample to obtain a recombinant protein solution.
  • the purified protein sample was taken, added to Loading Buffer, boiled for 10 minutes, and sampled for SDS-PAGE electrophoresis.
  • the electrophoresis results are shown in Fig. 2.
  • the band of recombinant phycobiliprotein is between 55 kDa and 70 kDa, consistent with the theoretical molecular weight of the recombinant phycobiliprotein cascade (dimer).
  • a small amount of the purified recombinant phycobiliprotein sample was diluted with 10 mM sodium phosphate buffer (pH 7.4) to 10 -6 mol/L for the measurement of absorption spectrum and fluorescence emission spectrum, respectively.
  • the absorption spectrum was measured by UV1801 ultraviolet-visible spectrophotometer at room temperature.
  • the measurement results are shown in Fig. 3.
  • the maximum absorption peak of the recombinant phycobiliprotein dimer is 550 nm, and the light absorption value is larger than that of the monomer.
  • the fluorescence emission spectrum was measured by F-4500 fluorescence spectrophotometer at room temperature.
  • the fluorescence excitation wavelength was 500 nm
  • the scanning wavelength was 520-600 nm
  • the fluorescence emission spectrum was shown in Fig. 4, and the maximum fluorescence emission wavelength was 562 nm.
  • the fluorescence value was larger than that of the monomer.
  • the AFP capture antibody was taken and diluted with a coating buffer (Na 2 CO 3 1.59 g/L, NaHCO 3 2.93 g/L, pH 9.6) to a final concentration of 5 ⁇ g/ml. 100 ⁇ l of the capture antibody solution was taken in the microplate of the fluorescent enzyme plate. The plate was placed at 4 ° C and allowed to stand overnight. The enzyme plate was taken out, the liquid in the well was cleaned, and the tissue was patted dry on the tissue.
  • a coating buffer Na 2 CO 3 1.59 g/L, NaHCO 3 2.93 g/L, pH 9.6
  • PBST KH 2 PO 4 0.27 g/L, Na 2 HPO 4 1.42 g/L, NaCl 8 g/L, KCl 0.2 g/L, Tween-20 0.5 ml/L, pH 7.4
  • 100 ⁇ l of blocking solution (10 g/L, prepared in PBST) was added to each well, placed on a wet box, placed at 37 ° C for 2 h, and washed three times with PBST.
  • the AFP protein was diluted with PBST solution at concentrations of 51.2 ng/ml, 16.8 ng/ml, 3.2, respectively. Ng/ml, 0.8 ng/ml, 0.2 ng/ml, 0.05 ng/ml, 0 ng/ml. 100 ⁇ l of each AFP protein solution was added to each well, and the mixture was incubated at 37 ° C, shaking at 400 rpm for 1 h, and washed three times with PBST. The biotinylated detection antibody was taken and diluted with PBST to a final concentration of 5 ⁇ g/ml. 100 ⁇ l of each well was added to the reaction well, and the mixture was incubated at 37 ° C, shaking at 400 rpm for 1 h, and washed three times with PBST.
  • a recombinant phycobiliprotein tanosome (dimer) was taken and diluted to 10 ⁇ g/ml with PBST. 100 ⁇ l of the recombinant phycobiliprotein solution was added to each well, and the mixture was incubated at 400 rpm for 1 hour, and then washed 3 times with PBST. 100 ⁇ L of PBST was added to the reaction well, and excited by a fluorescence microplate reader at a wavelength of 530 nm to measure the fluorescence signal at 562 nm. The results of quantitative AFP assay are shown in Figure 5. In the case of the same concentration of AFP, the fluorescent signal with the recombinant phycobiliprotein dimer as the fluorescent probe is higher than that of the monomer.
  • the apcA gene was obtained by using Synechococcus elongatus BP-1 genomic DNA as a template, Ho1 using Synechocystis.PCC 6803 genomic DNA as a template, and cpcS using Synechococcus elongatus BP-1 genomic DNA as a template and amplified according to conventional PCR conditions.
  • the SA gene and linker sequences were synthesized by Nanjing Kingsray Biotechnology Co., Ltd. In order to facilitate the fusion PCR reaction, the partial sequence of the SA gene and the apcA gene was added to the 5' and 3' ends, respectively, during the synthesis of the linker sequence.
  • the fusion gene SA-Linker-apcA (abbreviation: SLA) was obtained by fusion PCR with primers SA-F and primer apcAR. After the SLA fragment was recovered, it was digested with restriction endonucleases BamHI and SacI, and the vector pCDFDuet-1 was also digested with the same enzyme. The vector fragment and the fusion gene fragment were separately recovered and then ligated at a molar ratio of 1:5. After ligation overnight at 16 °C, the ligation product was transformed into competent E. coli Top10. The clone was picked from the transformation plate and the PCR was carried out.
  • SLA restriction endonucleases
  • the positive clone was sampled for DNA sequencing, and the plasmid was extracted and identified by restriction endonuclease BamHI and SacI.
  • the enzyme-clearing and DNA-sequence-tested samples were used for seed conservation, and the successful recombinant plasmid was constructed as: pCDF-SLA.
  • the plasmid pCDF-SLA was digested with restriction endonuclease BamHI; the digested vector fragment was recovered by a gel recovery kit, and then treated with alkaline phosphatase CIAP, and the vector fragment was recovered again using a gel recovery kit.
  • the plasmid pCDF-SLA was used as a template, and the primers LAF and LAR were used for PCR amplification to obtain a PCR amplification product containing the Linker sequence and the apcA gene sequence (abbreviated as LA), and the LA was recovered by a gel recovery kit.
  • the restriction endonucleases BamHI and BglII were double-digested, and the digested product was recovered again using a gel recovery kit.
  • the vector fragment obtained by the above recovery and the fragment cut product of the LA fragment were ligated at a molar ratio of 1:5, and after ligation at 16 ° C overnight, the ligated product was transformed into competent E. coli Top10.
  • the clone was picked from the transformation plate and the PCR was performed, and the positive clones were picked and inoculated into 3 mL of LB medium. After overnight incubation at 37 ° C, the plasmid was extracted and identified by restriction endonuclease BamHI and SacI double digestion. Based on the restriction map, the recombinant plasmid with the correct insertion direction (Fig. 1) was selected and labeled as pCDF-S(LA) 2 .
  • the plasmid pCDF-S(LA) 2 was digested with restriction endonuclease BamHI; the digested vector fragment was recovered by a gel recovery kit, and then treated with alkaline phosphatase CIAP, and the vector was recovered again using a gel recovery kit. Fragment.
  • the vector fragment obtained by the above recovery and the fragment cut product of the LA fragment were ligated at a molar ratio of 1:5, and after ligation at 16 ° C overnight, the ligated product was transformed into competent E. coli Top10.
  • the clone was picked from the transformation plate and the PCR was performed, and the positive clones were picked and inoculated into 3 mL of LB medium. After overnight incubation at 37 ° C, the plasmid was extracted and identified by restriction endonuclease BamHI and SacI double digestion. Based on the restriction map, the recombinant plasmid with the correct insertion direction (Fig. 1) was selected and labeled as pCDF-S(LA) 3 .
  • the expression plasmid pCDF-S(LA) 3 was digested with BglII and Xho, and the cpcS fragment obtained by the above PCR amplification was recovered, and then digested with BglII and SalI.
  • the vector fragment and the cpcS digestion product were separately recovered by a gel recovery kit, and the recovered vector fragment and the cpcS fragment were ligated at a molar ratio of 1:5, and after ligation at 16 ° C overnight, the ligated product was transformed into competent Escherichia coli. Top10.
  • the clone was picked from the transformation plate to make the bacterial solution PCR, and the positive clone was picked for sequencing, and the uncorrected sample was used for seed conservation.
  • the constructed co-expression plasmid was labeled pCDF-S(LA) 3 -cpcS.
  • the Ho1 gene was recovered by PCR amplification, and then digested with BglII and SalI.
  • the pRSFDuet-1 vector was digested with BglII and XhoI.
  • the carrier fragment and the Ho1 fragment were separately recovered using a gel recovery kit, and ligated at a molar ratio of 1:5. After ligation overnight at 16 °C, the ligation product was transformed into E. coli Top10.
  • the clone was picked from the transformation plate and the PCR was performed, and the positive clone was sent for sequencing.
  • the uncorrected clones were sequenced and used for seed conservation, and the successfully constructed plasmid was labeled as pRSF-Ho1.
  • the synthetic gene pebS and the plasmid pRSF-Ho1 were digested with NcoI and SalI, and the vector fragment and the gene fragment were separately recovered and ligated at a molar ratio of 1:5. After ligation overnight at 16 °C, the ligation product was transformed into E. coli Top10. The clone was picked from the transformation plate and the PCR was performed, and the positive clone was sent for sequencing. The uncorrected clones were sequenced and used for seed conservation, and the successfully constructed plasmid was labeled as pRSF-Ho1-pebS.
  • the above constructed plasmids pCDF-S(LA) 3 -cpcS and pRSF-Ho1-pebS were introduced into Escherichia coli BL21 (DE3) by co-transformation, and the transformed Escherichia coli was coated with 100 ⁇ g/mL kanamycin and 100 ⁇ g/mL spectinomycin on LB plates, after overnight incubation at 37 °C. Monoclones were picked from the plates and inoculated in 3 mL of LB medium (the medium containing the antibiotic kanamycin and spectinomycin) and cultured overnight at 37 °C.
  • LB medium the medium containing the antibiotic kanamycin and spectinomycin
  • the overnight culture was transferred to 60 mL of LB medium containing the corresponding antibiotic, cultured at 37 ° C, 200 rpm for 4 hours, and added to a final concentration of 1 mM IPTG.
  • the culture was placed at 18 ° C and continued at 160 rpm for 16-20 hours.
  • the cells were collected by centrifugation, and the cells were disrupted by an ultrasonic cell pulverizer.
  • the supernatant was collected, added to Loading Buffer, boiled for 10 minutes, and sampled for SDS-PAGE electrophoresis.
  • the strain with high expression of recombinant protein is selected, and the glycerol is used for the subsequent fermentation and separation and purification of the recombinant protein.
  • the cells were collected by centrifugation, and the cells were suspended in a suspension solution (500 mmol/L NaCl, 15.5 mmol/L Na 2 HPO 4 , 4.5 mmol/L NaH 2 PO 4 , 20 mmol/L imidazole, pH 7.4) in an ice bath. Ultrasonic disruption for 30 min. The disrupted solution was centrifuged at 4 ° C, 8000 rpm for 30 min, and the supernatant was taken.
  • a suspension solution 500 mmol/L NaCl, 15.5 mmol/L Na 2 HPO 4 , 4.5 mmol/L NaH 2 PO 4 , 20 mmol/L imidazole, pH 7.4
  • the nickel column (HiTrap FF, GE) was equilibrated with binding buffer (500 mmol/L NaCl, 15.5 mmol/L Na 2 HPO 4 , 4.5 mmol/L NaH 2 PO 4 , 20 mmol/L imidazole, pH 7.4). After the column, the nickel column was washed with washing buffer (500 mmol/L NaCl, 15.5 mmol/L Na 2 HPO 4 , 4.5 mmol/L NaH 2 PO 4 , 90 mmol/L imidazole, pH 7.4) to remove the impurity protein.
  • binding buffer 500 mmol/L NaCl, 15.5 mmol/L Na 2 HPO 4 , 4.5 mmol/L NaH 2 PO 4 , 20 mmol/L imidazole, pH 7.4
  • washing buffer 500 mmol/L NaCl, 15.5 mmol/L Na 2 HPO 4 , 4.5 mmol/L NaH 2 PO 4 , 90 mmol/
  • the purified protein sample was taken, added to Loading Buffer, boiled for 10 minutes, and sampled for SDS-PAGE electrophoresis.
  • the electrophoresis results are shown in Fig. 2.
  • the band of recombinant phycobiliprotein is located between 70 kDa and 100 kDa, with recombinant phycobilisome The theoretical value of the molecular weight of the trisomy is consistent.
  • a small amount of the purified recombinant phycobiliprotein sample was diluted with 10 mM sodium phosphate buffer (pH 7.4) to 10 -6 mol/L for the measurement of absorption spectrum and fluorescence emission spectrum, respectively.
  • the absorption spectrum was measured by UV1801 ultraviolet-visible spectrophotometer at room temperature.
  • the measurement results are shown in Fig. 3.
  • the maximum absorption peak of the recombinant phycobiliprotein monomer is 550 nm, and the light absorption value is higher than that of the monomer and the dimer.
  • the fluorescence emission spectrum was measured by F-4500 fluorescence spectrophotometer at room temperature.
  • the fluorescence excitation wavelength was 500 nm
  • the scanning wavelength was 520-600 nm
  • the fluorescence emission spectrum was shown in Figure 4, and the maximum fluorescence emission wavelength was 562 nm.
  • the fluorescence value was higher than that of monomer and Two bodies.
  • the AFP capture antibody was taken and diluted with a coating buffer (Na 2 CO 3 1.59 g/L, NaHCO 3 2.93 g/L, pH 9.6) to a final concentration of 5 ⁇ g/ml.
  • a coating buffer Na 2 CO 3 1.59 g/L, NaHCO 3 2.93 g/L, pH 9.6
  • 100 ⁇ l of the monoclonal antibody solution was taken in the microplate of the fluorescent plate.
  • the plate was placed at 4 ° C and allowed to stand overnight.
  • the enzyme plate was taken out, the liquid in the well was cleaned, and the tissue was patted dry on the tissue.
  • PBST KH 2 PO 4 0.27 g/L, Na 2 HPO 4 1.42 g/L, NaCl 8 g/L, KCl 0.2 g/L, Tween-20 0.5 ml/L, pH 7.4
  • 100 ⁇ l of blocking solution (10 g/L, prepared in PBST) was added to each well, placed on a wet box, placed at 37 ° C for 2 h, and washed three times with PBST.
  • AFP protein was serially diluted with PBST solution at concentrations of 51.2 ng/ml, 16.8 ng/ml, 3.2 ng/ml, 0.8 ng/ml, 0.2 ng/ml, 0.05 ng/ml, and 0 ng/ml, respectively.
  • 100 ⁇ l of each AFP protein solution was added to each well, and the mixture was incubated at 37 ° C, shaking at 400 rpm for 1 h, and washed three times with PBST.
  • the biotinylated detection antibody was taken and diluted with PBST to a final concentration of 5 ⁇ g/ml.
  • 100 ⁇ l of each well was added to the reaction well, and the mixture was incubated at 37 ° C, shaking at 400 rpm for 1 h, and washed three times with PBST.
  • a recombinant phycobiliprotein tandem (trisomy) was taken and diluted to 10 ⁇ g/ml with PBST. 100 ⁇ l of the recombinant phycobiliprotein solution was added to each well, and the mixture was incubated at 400 rpm for 1 hour, and then washed 3 times with PBST. 100 ⁇ L of PBST was added to the reaction well, and excited by a fluorescence microplate reader at a wavelength of 530 nm to measure the fluorescence signal at 562 nm. The results of AFP quantitative assay are shown in Figure 5. In the case of the same concentration of AFP, the fluorescent signal of the recombinant phycobiliprotein trisomy as the fluorescent probe is higher than that of the monomer and the dimer.

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Abstract

提供了一种高荧光强度的重组藻胆蛋白串联体的制备方法：将链霉亲和素基因与别藻蓝蛋白α亚基基因连接，再将一个或多个别藻蓝蛋白α亚基基因串联起来。将得到的融合基因与藻胆蛋白裂合酶基因和藻红胆素生物合成酶基因在大肠杆菌中共表达，获得具有生物素结合能力的、高荧光强度的重组藻胆蛋白串联体，可作为免疫荧光检测的标记物。

Description

一种高荧光强度的重组藻胆蛋白串联体的制备方法 技术领域
本发明属于生物技术中荧光蛋白质重组表达制备领域，具体的说是涉及高荧光强度的重组藻胆蛋白串联体的制备方法。
背景技术
藻胆蛋白是藻类重要的色素蛋白，具有光能捕获和传递的作用。每分子的藻胆蛋白含有两条结构相似的多肽链α和β，α亚基和β亚基分别含有约160～180个氨基酸残基，二者的比例通常为1:1。亚基中的半胱氨酸残基通过硫醚键与藻胆色素共价结合，藻胆色素的种类及其与脱辅基蛋白的相互作用决定了藻胆蛋白的光谱学性质。根据吸收光谱的不同，可将藻胆蛋白分为4大类：即藻红蛋白PE,λmax＝540nm～570nm，藻红蓝蛋白PEC，λmax＝567nm，藻蓝蛋白PC，λmax＝615nm～640nm和别藻蓝蛋白APC，λmax＝650nm～655nm。
藻胆蛋白能发出强烈的荧光，其荧光特性最重要的用途是在免疫诊断用荧光标记、生物医学研究等方面，可将其与生物素、亲和素或各种抗体结合制成荧光探针，用于免疫荧光分析和检测等工作中。在免疫荧光分析中，藻胆蛋白作为荧光标记物需要与抗体结合，即通过双功能试剂将藻胆蛋白分子与抗体分子共价交联，或藻胆蛋白通过交联剂与链霉亲和素交联后，在通过生物素·亲和素系统(BAS)将藻胆蛋白分子间接耦联到抗体分子上。
文献“Functional biosynthesis of an allophycocyan beta subunit in Escherichia coli，Journal of Bioscience and Bioengineering，107(3),246–249,2009”报道了一种利用基因工程方法生产重组别藻蓝蛋白的方法，但是该重组别藻蓝蛋白为单亚基，共价结合的藻蓝胆素，荧光量子效率低，因而荧光强度较低。中国专利“结合藻红胆素的藻蓝蛋白类荧光蛋白质的制备方法，专利授权号：200810025626.2”，提供了一种基因工程方法生产结合藻红胆素的重组别藻蓝蛋白的方法，由于重组蛋白也是单亚基，荧光强度的提升有限。
本发明提供一种具有生物素结合能力的重组藻胆蛋白亚基串联体的制备方法。相对于重组藻胆蛋白亚基单体，串联体蛋白具有更高的荧光强度，在生物学和生物医学等领域中，对抗原的免疫荧光检测中将具有较好的应用前景。
发明内容
本发明目的在于提供高荧光强度的重组藻胆蛋白串联体的制备方法。
为实现上述目的，本发明采用的技术方案为：
一种高荧光强度的重组藻胆蛋白串联体的制备方法，其特征在于：将链霉亲和素基因与别藻蓝蛋白α亚基基因，通过Linker序列连接起来。在此基础上，再通过linker序列实现一个或多个别藻蓝蛋白α亚基基因的串联，形成融合基因。将该融合基因、藻胆蛋白裂合酶基因和藻红胆素生物合成酶基因在大肠杆菌中共表达，获得共价结合藻红胆素的具有更高荧光强度的重组别藻蓝蛋白串联体。
具体是：
1)将链霉亲和素基因和别藻蓝蛋白α亚基基因通过linker序列连接形成融合基因，在此基础上，再通过linker序列连接一个或多个别藻蓝蛋白α亚基基因。
将该融合基因插入到大肠杆菌表达载体pCDFDuet-1的表达框中；将裂合酶基因cpcS插 入到该表达载体的另一个表达框中；
2)将藻红胆素生物合成酶基因Ho1和pebS，分别插入到另一个表达载体pRSFDuet-1的两个表达框中；
3)将两个构建好的表达载体同时转化大肠杆菌，得到重组大肠杆菌；
4)将重组大肠杆菌进行发酵培养，IPTG诱导融合蛋白表达，通过金属螯合亲和层析纯化，得到共价结合藻红胆素的融合荧光蛋白质。
所述别藻蓝蛋白α亚基基因是别藻蓝蛋白Synechoccus elongatus BP-1别藻蓝蛋白α亚基基因，或其同源基因；藻红胆素生物合成酶基因Ho1是Synechocystis sp.PCC 6803Ho1基因，pebS是Prochlorococcus phage P-SSM2的pebS基因，cpcS是指Synechoccus elongatus BP-1cpcS基因，所述的linker序列是：
Figure PCTCN2015099730-appb-000001
与现有技术相比，本发明具有以下优点：
1.通过基因工程的方法，利用本发明中的linker序列将别藻蓝蛋白α亚基串联，避免了相邻的别藻蓝蛋白α亚基功能上相互干扰，提高了重组藻胆蛋白的荧光强度，在免疫荧光检测中具有更强的荧光信号；
2.大肠杆菌易于培养、生长快，生产周期短。本发明利用大肠杆菌发酵制备高荧光强度的重组藻胆蛋白串联体，具有制备简单，成本低等优点。
附图说明
图1是本发明实施例中构建的藻胆蛋白单体和串联体表达载体的酶切图谱。
图2是本发明实施例的经过纯化后重组藻胆蛋白单体和串联体样品的SDS-PAGE电泳。
图3是本发明实施例的经过纯化后重组藻胆蛋白单体和串联体样品的吸收光谱。
图4是本发明实施例的经过纯化后重组藻胆蛋白单体和串联体样品的荧光发射光谱。
图5是本发明实施例的经过纯化后重组藻胆蛋白单体和串联体对AFP免疫荧光检测的检测曲线
具体实施方式
下面通过实施例对本发明的具体实施方式进行阐述，所述的实施方式仅仅用来解释和说明本发明，其并不限制本发明的保护范围。任何本领域技术人员根据公知的知识和现有技术的教导能够想到的等价的变体都包含在本发明的保护范围中。
实施例1
1.基因的克隆
从美国国立生物信息中心(National Centre for Biotechnology Information,NCBI)数据库获取Synechococcus elongatus BP-1apcA，Synechocytissp.PCC 6803Ho1,Prochlorococcus phage P-SSM2pebS,Synechococcus elongatus BP-1cpcS和链霉亲和素的基因SA序列(Accession No.X65082)。由此分别设计扩增apcA，Ho1，cpcS的特异引物(表1)。apcA基因以Synechococcus elongatus BP-1基因组DNA为模板，Ho1以Synechocytissp.PCC 6803基因组DNA为模板，cpcS以Synechococcus elongatus BP-1基因组DNA为模板，按常规PCR条件扩增获得。SA基因和linker序列由南京金斯瑞生物科技有限公司人工合成。为了便于融合PCR反应，linker序列人工合成过程中，在其5’端和3’端分别加上SA基因和apcA基因的部分序列。
2.重组质粒的构建
2.1链霉亲和素基因和别藻蓝蛋白α亚基基因的融合(单体)及其表达载体的构建
以SA、linker和apcA为模板，用引物SA-F和引物apcAR，通过融合PCR扩增，获得融合基因SA-Linker-apcA(简称：SLA)。SLA片段回收后，利用限制性内切酶BamHI和SacI双酶切，同时把载体pCDFDuet-1也利用相同的酶进行双酶切。分别回收载体片段和融合基因片段，然后以1:5的摩尔比进行连接。16℃条件下连接过夜后，将连接产物转化感受态大肠杆菌Top10。从转化平板上挑取克隆做菌液PCR，阳性克隆送样进行DNA测序，同时抽提质粒，用限制性内切酶BamHI和SacI双酶切鉴定(图1)。酶切鉴定以及DNA测序验证无误的样品用于保种，构建成功的重组质粒标记为：pCDF-SLA。
2.2藻胆蛋白裂合酶cpcS共表达载体的构建
将表达质粒pCDF-SLA用BglII和Xho双酶切，将上述PCR扩增得到的cpcS片段回收后，用BglII和SalI双酶切。利用胶回收试剂盒分别回收载体片段和cpcS酶切产物，回收后的载体片段和cpcS片段按照摩尔比1:5的比例进行连接，16℃条件下连接过夜后，将连接产物转化感受态大肠杆菌Top10。从转化平板上挑取克隆做菌液PCR，挑取阳性克隆送样测序，测序无误的样品用于保种。构建好的共表达质粒标记为pCDF-SLA-cpcS。
2.3藻红胆素生物合成酶共表达质粒的构建
将上述PCR扩增得到Ho1基因回收后用BglII和SalI双酶切，pRSFDuet-1载体用BglII和XhoI双酶切。利用胶回收试剂盒，分别回收载体片段和Ho1片段，以1:5摩尔比进行连接。16℃条件下连接过夜后，将连接产物转化大肠杆菌Top10。从转化平板上挑取克隆做菌液PCR，阳性克隆送样测序。测序验证无误的克隆用于保种，构建成功的质粒标记为pRSF-Ho1。
人工合成的基因pebS和质粒pRSF-Ho1均用NcoI和SalI酶切，分别回收载体片段和基因片段，以1:5摩尔比进行连接。16℃条件下连接过夜后，将连接产物转化大肠杆菌Top10。从转化平板上挑取克隆做菌液PCR，阳性克隆送样测序。测序验证无误的克隆用于保种，构建成功的质粒标记为pRSF-Ho1-pebS。
3.重组藻胆蛋白单体表达菌株的构建
将上述构建好的质粒pCDF-SLA-cpcS和pRSF-Ho1-pebS通过共转化方式导入大肠杆菌BL21(DE3)，转化后的大肠杆菌涂布在含有100μg/mL卡那霉素和100μg/mL壮观霉素的LB平板上，37℃过夜培养后。从平板上挑取单克隆，分别接种于3mL LB培养基(培养基中含有抗生素卡那霉素和壮观霉素)中，37℃培养过夜。将过夜培养物，转接到含有相应抗生素的60mL LB培养基中，37℃，200rpm条件下培养4小时，加入终浓度为1mM IPTG诱导，培养物置于18℃，160rpm继续培养16-20小时。离心收集菌体，利用超声波细胞粉碎机破碎细胞，收集上清液，加入Loading Buffer后煮沸处理10分钟，取样进行SDS-PAGE电泳。选择重组蛋白表达量高的菌株，保甘油种用于后续发酵与重组蛋白的分离纯化。
4.重组蛋白的发酵与分离纯化
吸取甘油种200μL，接种于含有卡那霉素和壮观霉素的15mL LB培养基中，37℃过夜培养后。将过夜培养物接种于含有卡那霉素和壮观霉素的300mL LB培养基中。37℃，200rpm条件下培养4小时，加入终浓度为1mM IPTG，18℃条件下诱导20小时。
离心收集菌体，将菌体悬浮于破碎缓液(500mmol/L NaCl，15.5mmol/L Na2HPO4，4.5mmol/L NaH2PO4，20mmol/L咪唑，pH 7.4)中，冰浴中超声破碎30min。破碎液于4℃，8000rpm条件下离心30min，取上清液。用结合缓冲液(500mmol/L NaCl，15.5mmol/L Na2HPO4，4.5mmol/L NaH2PO4，20mmol/L咪唑，pH 7.4)平衡镍柱(HiTrap FF， GE)，上清液过柱后，再用洗涤缓冲液洗涤(500mmol/L NaCl，15.5mmol/L Na2HPO4，4.5mmol/L NaH2PO4，90mmol/L咪唑，pH 7.4)镍柱，除去杂蛋白。待A280值不再变化时，用洗脱液(500mmol/L NaCl，15.5mmol/L Na2HPO4，4.5mmol/L NaH2PO4，500mmol/L咪唑，pH 7.4)进行洗脱，收集洗脱液，再过G25脱盐柱，除去蛋白样品中的咪唑，得到重组蛋白溶液。
取纯化后的蛋白样品，加入Loading Buffer后煮沸处理10分钟，取样进行SDS-PAGE电泳，电泳结果如图2。重组藻胆蛋白的条带位于35kDa和40kDa之间，与重组藻胆蛋白单体的分子量理论值相符。
5.重组蛋白的光谱学性质
取少量纯化后的重组藻胆蛋白样品，用50mM磷酸钠缓冲液(pH7.4)稀释至10-6mol/L，分别用于吸收光谱和荧光发射光谱的测定。吸收光谱采用UV1801紫外可见光分光光度计室温条件下测定，测量结果见图3，重组藻胆蛋白单体的最大吸收峰为550nm。荧光发射光谱采用F-4500荧光分光光度计室温下测定，荧光激发波长为500nm，扫描波长为520-600nm，荧光发射光谱见图4，最大荧光发射波长为562nm。
6.重组藻胆蛋白(单体)对AFP免疫荧光检测
取AFP捕获抗体，用包被缓冲液(Na2CO31.59g/L,NaHCO32.93g/L,pH 9.6)稀释至终浓度为5μg/ml。取100μl捕获抗体溶液于荧光酶标板微孔中。将酶标板置于4℃静置，过夜吸附。取出酶标板，甩净孔内液体，在面巾纸上拍干。向孔内加入200μl PBST(KH2PO40.27g/L,Na2HPO41.42g/L,NaCl 8g/L,KCl 0.2g/L,Tween-20 0.5ml/L,pH 7.4)，400rpm振荡洗涤3min，甩净拍干，重复2次。向反应孔中各加入100μl封闭液(10g/L，PBST配制)，置于湿盒上，37℃放置2h，PBST洗涤3次。
取AFP蛋白用PBST溶液进行梯度稀释，浓度分别为51.2ng/ml、16.8ng/ml、3.2ng/ml、0.8ng/ml、0.2ng/ml、0.05ng/ml、0ng/ml。每孔各加AFP蛋白溶液100μl，37℃，400rpm振荡孵育1h，PBST洗涤3次。取生物素化的检测抗体，用PBST稀释至终浓度为5μg/ml。向反应孔中各加入100μl，37℃，400rpm振荡孵育1h，PBST洗涤3次。
取重组藻胆蛋白(单体)样品，用PBST稀释至10μg/ml。向反应孔中各加入100μl重组藻胆蛋白溶液，400rpm振荡孵育1h后，用PBST洗涤3次。向反应孔中加入100μL PBST，用荧光酶标仪以530nm波长光激发，测量562nm处的荧光信号。AFP定量测定结果见图5。
表1 PCR扩增中使用的特异引物
Figure PCTCN2015099730-appb-000002
实施例2
1.基因的克隆
从美国国立生物信息中心(National Centre for Biotechnology Information,NCBI)数据库获取Synechococcus elongatus BP-1apcA，Synechocytissp.PCC 6803Ho1,Prochlorococcus phage P-SSM2pebS,Synechococcus elongatus BP-1cpcS和链霉亲和素的基因SA序列(Accession No.X65082)。由此分别设计扩增apcA，Ho1，cpcS的特异引物(表1)。apcA基因以Synechococcus elongatus BP-1基因组DNA为模板，Ho1以Synechocytissp.PCC 6803基因组DNA为模板，cpcS以Synechococcus elongatus BP-1基因组DNA为模板，按常规PCR条件扩增获得。SA基因和linker序列由南京金斯瑞生物科技有限公司人工合成。为了便于融合PCR反应，linker序列人工合成过程中，在其5’端和3’端分别加上SA基因和apcA基因的部分序列。
2.重组质粒的构建
2.1别藻蓝蛋白α亚基串联体(二体)的融合及其表达载体的构建
以SA、linker和apcA为模板，用引物SA-F和引物apcAR，通过融合PCR扩增，获得融合基因SA-Linker-apcA(简称：SLA)。SLA片段回收后，利用限制性内切酶BamHI和SacI双酶切，同时把载体pCDFDuet-1也利用相同的酶进行双酶切。分别回收载体片段和融合基因片段，然后以1:5的摩尔比进行连接。16℃条件下连接过夜后，将连接产物转化感受态大肠杆菌Top10。从转化平板上挑取克隆做菌液PCR，阳性克隆送样进行DNA测序，同时抽提质粒，用限制性内切酶BamHI和SacI双酶切鉴定。酶切鉴定以及DNA测序验证无误的样品用于保种，构建成功的重组质粒标记为：pCDF-SLA。
用限制性内切酶BamHI单酶切质粒pCDF-SLA；酶切后的载体片段利用胶回收试剂盒回收后，再用碱性磷酸酶CIAP处理，再次利用胶回收试剂盒回收载体片段。以质粒pCDF-SLA为模板，用引物LAF和LAR做PCR扩增，得到包含有Linker序列和apcA基因序列(简写为：LA)的PCR扩增产物，利用胶回收试剂盒将LA回收后，用限制性内切酶BamHI和BglII双酶切，再次利用胶回收试剂盒回收酶切产物。
将上述回收得到的载体片段和酶切产物按照1:5的摩尔比进行连接，16℃条件下连接过夜后，将连接产物转化感受态大肠杆菌Top10。从转化平板上挑取克隆做菌液PCR，挑取阳性克隆接种于3mL LB培养基中。37℃培养过夜后，抽提质粒，用限制性内切酶BamHI和SacI双酶切鉴定。根据酶切图谱，挑选出插入方向正确的重组质粒(图1)，并标记为pCDF-S(LA)2。
2.2藻胆蛋白裂合酶cpcS共表达载体的构建
将表达质粒pCDF-S(LA)2用BglII和Xho双酶切，将上述PCR扩增得到的cpcS片段回收后，用BglII和SalI双酶切。利用胶回收试剂盒分别回收载体片段和cpcS酶切产物，回收后的载体片段和cpcS片段按照摩尔比1:5的比例进行连接，16℃条件下连接过夜后，将连接产物转化感受态大肠杆菌Top10。从转化平板上挑取克隆做菌液PCR，挑取阳性克隆送样测序，测序无误的样品用于保种。构建好的共表达质粒标记为pCDF-S(LA)2-cpcS。
2.3藻红胆素生物合成酶共表达质粒的构建
将上述PCR扩增得到Ho1基因回收后用BglII和SalI双酶切，pRSFDuet-1载体用BglII和XhoI双酶切。利用胶回收试剂盒，分别回收载体片段和Ho1片段，以1:5摩尔比进行连接。16℃条件下连接过夜后，将连接产物转化大肠杆菌Top10。从转化平板上挑取克隆做菌液PCR，阳性克隆送样测序。测序验证无误的克隆用于保种，构建成功的质粒标记为pRSF-Ho1。
人工合成的基因pebS和质粒pRSF-Ho1均用NcoI和SalI酶切，分别回收载体片段和基因片段，以1:5摩尔比进行连接。16℃条件下连接过夜后，将连接产物转化大肠杆 菌Top10。从转化平板上挑取克隆做菌液PCR，阳性克隆送样测序。测序验证无误的克隆用于保种，构建成功的质粒标记为pRSF-Ho1-pebS。
3.重组藻胆蛋白单体表达菌株的构建
将上述构建好的质粒pCDF-S(LA)2-cpcS和pRSF-Ho1-pebS通过共转化方式导入大肠杆菌BL21(DE3)，转化后的大肠杆菌涂布在含有100μg/mL卡那霉素和100μg/mL壮观霉素的LB平板上，37℃过夜培养后。从平板上挑取单克隆，分别接种于3mL LB培养基(培养基中含有抗生素卡那霉素和壮观霉素)中，37℃培养过夜。将过夜培养物，转接到含有相应抗生素的60mL LB培养基中，37℃，200rpm条件下培养4小时，加入终浓度为1mM IPTG诱导，培养物置于18℃，160rpm继续培养16-20小时。离心收集菌体，利用超声波细胞粉碎机破碎细胞，收集上清液，加入Loading Buffer后煮沸处理10分钟，取样进行SDS-PAGE电泳。选择重组蛋白表达量高的菌株，保甘油种用于后续发酵与重组蛋白的分离纯化。
4.重组蛋白的发酵与分离纯化
吸取甘油种200μL，接种于含有卡那霉素和壮观霉素的15mL LB培养基中，37℃过夜培养后。将过夜培养物接种于含有卡那霉素和壮观霉素的300mL LB培养基中。37℃，200rpm条件下培养4小时，加入终浓度为1mM IPTG，18℃条件下诱导20小时。
离心收集菌体，将菌体悬浮于破碎缓液(500mmol/L NaCl，15.5mmol/L Na2HPO4，4.5mmol/L NaH2PO4，20mmol/L咪唑，pH 7.4)中，冰浴中超声破碎30min。破碎液于4℃，8000rpm条件下离心30min，取上清液。用结合缓冲液(500mmol/L NaCl，15.5mmol/L Na2HPO4，4.5mmol/L NaH2PO4，20mmol/L咪唑，pH 7.4)平衡镍柱(HiTrap FF，GE)，上清液过柱后，再用洗涤缓冲液洗涤(500mmol/L NaCl，15.5mmol/L Na2HPO4，4.5mmol/L NaH2PO4，90mmol/L咪唑，pH 7.4)镍柱，除去杂蛋白，待A280值不再变化时，用洗脱液(500mmol/L NaCl，15.5mmol/L Na2HPO4，4.5mmol/L NaH2PO4，500mmol/L咪唑，pH 7.4)进行洗脱，收集洗脱液，再过G25脱盐柱，除去蛋白样品中的咪唑，得到重组蛋白溶液。
取纯化后的蛋白样品，加入Loading Buffer后煮沸处理10分钟，取样进行SDS-PAGE电泳，电泳结果如图2。重组藻胆蛋白的条带在55kDa和70kDa之间，与重组藻胆蛋白串联体(二体)的分子量理论值相符。
5.重组蛋白的光谱学性质
取少量纯化后的重组藻胆蛋白样品，用50mM磷酸钠缓冲液(pH7.4)稀释至10-6mol/L，分别用于吸收光谱和荧光发射光谱的测定。吸收光谱采用UV1801紫外可见光分光光度计室温条件下测定，测量结果见图3，重组藻胆蛋白二体的最大吸收峰为550nm，其光吸收值大于单体。荧光发射光谱采用F-4500荧光分光光度计室温下测定，荧光激发波长为500nm，扫描波长为520-600nm，荧光发射光谱见图4，最大荧光发射波长为562nm，其荧光值大于单体。
6.重组藻胆蛋白串联体(二体)对AFP免疫荧光检测
取AFP捕获抗体，用包被缓冲液(Na2CO31.59g/L,NaHCO32.93g/L,pH 9.6)稀释至终浓度为5μg/ml。取100μl捕获抗体溶液于荧光酶标板微孔中。将酶标板置于4℃静置，过夜吸附。取出酶标板，甩净孔内液体，在面巾纸上拍干。向孔内加入200μl PBST(KH2PO40.27g/L,Na2HPO41.42g/L,NaCl 8g/L,KCl 0.2g/L,Tween-20 0.5ml/L,pH 7.4)，400rpm振荡洗涤3min，甩净拍干，重复2次。向反应孔中各加入100μl封闭液(10g/L，PBST配制)，置于湿盒上，37℃放置2h，PBST洗涤3次。
取AFP蛋白用PBST溶液进行梯度稀释，浓度分别为51.2ng/ml、16.8ng/ml、3.2 ng/ml、0.8ng/ml、0.2ng/ml、0.05ng/ml、0ng/ml。每孔各加AFP蛋白溶液100μl，37℃，400rpm振荡孵育1h，PBST洗涤3次。取生物素化的检测抗体，用PBST稀释至终浓度为5μg/ml。向反应孔中各加入100μl，37℃，400rpm振荡孵育1h，PBST洗涤3次。
取重组藻胆蛋白串联体(二体)，用PBST稀释至10μg/ml。向反应孔中各加入100μl重组藻胆蛋白溶液，400rpm振荡孵育1h后，用PBST洗涤3次。向反应孔中加入100μL PBST，用荧光酶标仪以530nm波长光激发，测量562nm处的荧光信号。AFP定量测定结果见图5，在AFP浓度相同的情况下，以重组藻胆蛋白二体为荧光探针的荧光信号高于单体。
实施例3
1.基因的克隆
从美国国立生物信息中心(National Centre for Biotechnology Information,NCBI)数据库获取Synechococcus elongatus BP-1apcA，Synechocytissp.PCC 6803Ho1,Prochlorococcus phage P-SSM2pebS,Synechococcus elongatus BP-1cpcS和链霉亲和素的基因SA序列(Accession No.X65082)。由此分别设计扩增apcA，Ho1，cpcS的特异引物(表1)。apcA基因以Synechococcus elongatus BP-1基因组DNA为模板，Ho1以Synechocytissp.PCC 6803基因组DNA为模板，cpcS以Synechococcus elongatus BP-1基因组DNA为模板，按常规PCR条件扩增获得。SA基因和linker序列由南京金斯瑞生物科技有限公司人工合成。为了便于融合PCR反应，linker序列人工合成过程中，在其5’端和3’端分别加上SA基因和apcA基因的部分序列。
2.重组质粒的构建
2.1别藻蓝蛋白α亚基串联体(三体)的融合及其表达载体的构建
以SA、linker和apcA为模板，用引物SA-F和引物apcAR，通过融合PCR扩增，获得融合基因SA-Linker-apcA(简称：SLA)。SLA片段回收后，利用限制性内切酶BamHI和SacI双酶切，同时把载体pCDFDuet-1也利用相同的酶进行双酶切。分别回收载体片段和融合基因片段，然后以1:5的摩尔比进行连接。16℃条件下连接过夜后，将连接产物转化感受态大肠杆菌Top10。从转化平板上挑取克隆做菌液PCR，阳性克隆送样进行DNA测序，同时抽提质粒，用限制性内切酶BamHI和SacI双酶切鉴定。酶切鉴定以及DNA测序验证无误的样品用于保种，构建成功的重组质粒标记为：pCDF-SLA。
用限制性内切酶BamHI单酶切质粒pCDF-SLA；酶切后的载体片段利用胶回收试剂盒回收后，再用碱性磷酸酶CIAP处理，再次利用胶回收试剂盒回收载体片段。以质粒pCDF-SLA为模板，用引物LAF和LAR做PCR扩增，得到包含有Linker序列和apcA基因序列(简写为：LA)的PCR扩增产物，利用胶回收试剂盒将LA回收后，用限制性内切酶BamHI和BglII双酶切，再次利用胶回收试剂盒回收酶切产物。
将上述回收得到的载体片段和LA片段酶切产物按照1:5的摩尔比进行连接，16℃条件下连接过夜后，将连接产物转化感受态大肠杆菌Top10。从转化平板上挑取克隆做菌液PCR，挑取阳性克隆接种于3mL LB培养基中。37℃培养过夜后，抽提质粒，用限制性内切酶BamHI和SacI双酶切鉴定。根据酶切图谱，挑选出插入方向正确的重组质粒(图1)，并标记为pCDF-S(LA)2。
用限制性内切酶BamHI单酶切质粒pCDF-S(LA)2；酶切后的载体片段利用胶回收试剂盒回收后，再用碱性磷酸酶CIAP处理，再次利用胶回收试剂盒回收载体片段。
将上述回收得到的载体片段和LA片段酶切产物按照1:5的摩尔比进行连接，16℃条件下连接过夜后，将连接产物转化感受态大肠杆菌Top10。从转化平板上挑取克隆做菌液PCR，挑取阳性克隆接种于3mL LB培养基中。37℃培养过夜后，抽提质粒，用限 制性内切酶BamHI和SacI双酶切鉴定。根据酶切图谱，挑选出插入方向正确的重组质粒(图1)，并标记为pCDF-S(LA)3。
2.2藻胆蛋白裂合酶cpcS共表达载体的构建
将表达质粒pCDF-S(LA)3用BglII和Xho双酶切，将上述PCR扩增得到的cpcS片段回收后，用BglII和SalI双酶切。利用胶回收试剂盒分别回收载体片段和cpcS酶切产物，回收后的载体片段和cpcS片段按照摩尔比1:5的比例进行连接，16℃条件下连接过夜后，将连接产物转化感受态大肠杆菌Top10。从转化平板上挑取克隆做菌液PCR，挑取阳性克隆送样测序，测序无误的样品用于保种。构建好的共表达质粒标记为pCDF-S(LA)3-cpcS。
2.3藻红胆素生物合成酶共表达质粒的构建
将上述PCR扩增得到Ho1基因回收后用BglII和SalI双酶切，pRSFDuet-1载体用BglII和XhoI双酶切。利用胶回收试剂盒，分别回收载体片段和Ho1片段，以1:5摩尔比进行连接。16℃条件下连接过夜后，将连接产物转化大肠杆菌Top10。从转化平板上挑取克隆做菌液PCR，阳性克隆送样测序。测序验证无误的克隆用于保种，构建成功的质粒标记为pRSF-Ho1。
人工合成的基因pebS和质粒pRSF-Ho1均用NcoI和SalI酶切，分别回收载体片段和基因片段，以1:5摩尔比进行连接。16℃条件下连接过夜后，将连接产物转化大肠杆菌Top10。从转化平板上挑取克隆做菌液PCR，阳性克隆送样测序。测序验证无误的克隆用于保种，构建成功的质粒标记为pRSF-Ho1-pebS。
3.重组藻胆蛋白单体表达菌株的构建
将上述构建好的质粒pCDF-S(LA)3-cpcS和pRSF-Ho1-pebS通过共转化方式导入大肠杆菌BL21(DE3)，转化后的大肠杆菌涂布在含有100μg/mL卡那霉素和100μg/mL壮观霉素的LB平板上，37℃过夜培养后。从平板上挑取单克隆，分别接种于3mL LB培养基(培养基中含有抗生素卡那霉素和壮观霉素)中，37℃培养过夜。将过夜培养物，转接到含有相应抗生素的60mL LB培养基中，37℃，200rpm条件下培养4小时，加入终浓度为1mM IPTG诱导，培养物置于18℃，160rpm继续培养16-20小时。离心收集菌体，利用超声波细胞粉碎机破碎细胞，收集上清液，加入Loading Buffer后煮沸处理10分钟，取样进行SDS-PAGE电泳。选择重组蛋白表达量高的菌株，保甘油种用于后续发酵与重组蛋白的分离纯化。
4.重组蛋白的发酵与分离纯化
吸取甘油种200μL，接种于含有卡那霉素和壮观霉素的15mL LB培养基中，37℃过夜培养后。将过夜培养物接种于含有卡那霉素和壮观霉素的300mL LB培养基中。37℃，200rpm条件下培养4小时，加入终浓度为1mM IPTG，18℃条件下诱导20小时。
离心收集菌体，将菌体悬浮于破碎缓液(500mmol/L NaCl，15.5mmol/L Na2HPO4，4.5mmol/L NaH2PO4，20mmol/L咪唑，pH 7.4)中，冰浴中超声破碎30min。破碎液于4℃，8000rpm条件下离心30min，取上清液。用结合缓冲液(500mmol/L NaCl，15.5mmol/L Na2HPO4，4.5mmol/L NaH2PO4，20mmol/L咪唑，pH 7.4)平衡镍柱(HiTrap FF，GE)，上清液过柱后，再用洗涤缓冲液洗涤(500mmol/L NaCl，15.5mmol/L Na2HPO4，4.5mmol/L NaH2PO4，90mmol/L咪唑，pH 7.4)镍柱，除去杂蛋白，待A280值不再变化时，用洗脱液(500mmol/L NaCl，15.5mmol/L Na2HPO4，4.5mmol/L NaH2PO4，500mmol/L咪唑，pH 7.4)进行洗脱，收集流出液，再过G25脱盐柱，除去蛋白样品中的咪唑，得到重组蛋白溶液。
取纯化后的蛋白样品，加入Loading Buffer后煮沸处理10分钟，取样进行SDS-PAGE电泳，电泳结果如图2。重组藻胆蛋白的条带位于70kDa和100kDa之间，与重组藻胆 蛋白三体的分子量理论值相符。
5.重组蛋白的光谱学性质
取少量纯化后的重组藻胆蛋白样品，用50mM磷酸钠缓冲液(pH7.4)稀释至10-6mol/L，分别用于吸收光谱和荧光发射光谱的测定。吸收光谱采用UV1801紫外可见光分光光度计室温条件下测定，测量结果见图3，重组藻胆蛋白单体的最大吸收峰为550nm，其光吸收值高于单体和二体。荧光发射光谱采用F-4500荧光分光光度计室温下测定，荧光激发波长为500nm，扫描波长为520-600nm，荧光发射光谱见图4，最大荧光发射波长为562nm，其荧光值高于单体和二体。
6.重组藻胆蛋白串联体(三体)对AFP免疫荧光检测
取AFP捕获抗体，用包被缓冲液(Na2CO31.59g/L,NaHCO32.93g/L,pH 9.6)稀释至终浓度为5μg/ml。取100μl单抗溶液于荧光酶标板微孔中。将酶标板置于4℃静置，过夜吸附。取出酶标板，甩净孔内液体，在面巾纸上拍干。向孔内加入200μl PBST(KH2PO40.27g/L,Na2HPO41.42g/L,NaCl 8g/L,KCl 0.2g/L,Tween-20 0.5ml/L,pH 7.4)，400rpm振荡洗涤3min，甩净拍干，重复2次。向反应孔中各加入100μl封闭液(10g/L，PBST配制)，置于湿盒上，37℃放置2h，PBST洗涤3次。
取AFP蛋白用PBST溶液进行梯度稀释，浓度分别为51.2ng/ml、16.8ng/ml、3.2ng/ml、0.8ng/ml、0.2ng/ml、0.05ng/ml、0ng/ml。每孔各加AFP蛋白溶液100μl，37℃，400rpm振荡孵育1h，PBST洗涤3次。取生物素化的检测抗体，用PBST稀释至终浓度为5μg/ml。向反应孔中各加入100μl，37℃，400rpm振荡孵育1h，PBST洗涤3次。
取重组藻胆蛋白串联体(三体)，用PBST稀释至10μg/ml。向反应孔中各加入100μl重组藻胆蛋白溶液，400rpm振荡孵育1h后，用PBST洗涤3次。向反应孔中加入100μL PBST，用荧光酶标仪以530nm波长光激发，测量562nm处的荧光信号。AFP定量测定结果见图5，在AFP浓度相同的情况下，以重组藻胆蛋白三体为荧光探针的荧光信号高于单体和二体。

Claims (3)

  1. 一种高荧光强度的重组藻胆蛋白串联体的制备方法，其特征在于：将链霉亲和素基因与别藻蓝蛋白α亚基基因，通过Linker序列连接起来；在此基础上，再通过Linker序列实现一个或多个别藻蓝蛋白α亚基基因的串联，形成融合基因；将该融合基因、藻胆蛋白裂合酶基因和藻红胆素生物合成酶基因在大肠杆菌中共表达，获得共价结合藻红胆素的具有更高荧光强度的重组别藻蓝蛋白串联体。
  2. 按权利要求1所述的高荧光强度的重组藻胆蛋白串联体的制备方法，其特征在于：
    1)将链霉亲和素基因和别藻蓝蛋白α亚基基因通过linker序列连接起来，在此基础上，再通过linker序列串联一个或多个别藻蓝蛋白α亚基基因，将该融合基因插入到大肠杆菌表达载体pCDFDuet-1的表达框中；将裂合酶基因cpcS插入到pCDFDuet-1的另一个表达框中；
    2)将藻红胆素生物合成酶基因Ho1和pebS，分别插入到另一个表达载体pRSFDuet-1的两个表达框中；
    3)将上述步骤1)和步骤2)构建的表达载体同时转化大肠杆菌，得到重组大肠杆菌；
    4)将重组大肠杆菌进行发酵培养，IPTG诱导融合蛋白表达，通过金属螯合亲和层析纯化，得到共价结合藻红胆素的重组藻胆蛋白单体和串联体。
  3. 按权利要求1，2所述的高荧光强度的重组藻胆蛋白串联体的制备方法，其特征在于：
    linker序列是：
    GGATCCGCCGGAGCGGAAGCAAAAGGAGCGGAAGCAAAAGGAGCGGAAGCAAAAGGAGCGGAAGCAAAAGCGGAATTC。
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