48 results about "Allophycocyanin" patented technology

The invention discloses a method for simultaneously separating high-purity phycocyanin and allophycocyanin and application thereof. The method comprises the following steps of: repeatedly freezing and thawing spirulina dry powder, ultrasonically crushing, and centrifuging, wherein supernate is a crude phycobiliprotein extracting solution; then adding ammonia sulfate, and taking out the supernate when the saturation degree is 25-35%; then, adding ammonia sulfate, taking out precipitates when the saturation degree is 60-70%, dialyzing for precipitation to obtain a primarily purified phycobiliprotein extracting solution, and loading the primarily purified phycobiliprotein extracting solution to a hydroxyapatite column; and collecting 0.02-0.03M phosphate buffered solution which contains 0.1-0.2M sodium chloride and has the pH value of 6.5-7.2, and 0.1-0.12M phosphate buffered solution which contains 0.1-0.2M sodium chloride and has the pH value of 6.5-7.2, wherein the 0.02-0.03M phosphate buffered solution is phycocyanin and the 0.1-0.12M phosphate buffered solution is allophycocyanin. The method disclosed by the invention can be used for simultaneously preparing phycocyanin with a purification factor reaching 4.5 and allophycocyanin with a purification factor reaching 3 as well as analogues containing selenium and tellurium, and the purification scale of the method is amplified so as to acquire high-purity protein of a gram level through primary chromatography, so that the purification cost is greatly reduced, and the method can be used for producing the phycocyanin and the allophycocyanin.

The invention relates to a method for preparing high purity phycoerythrin, phycocyanin and allophycocyanin from the algae, belonging to the bioengineering extraction separation technology. The method is as follows: red algae or cyanobacteria is adopted as a raw material, and phosphate buffer is used as an extractant; after algae cells are broken, the raw material of the red algae or the cyanobacteria is salt dissolved, salted out and dialyzed in grade by ammonium sulfate, and crude extract of phycobiliprotein is obtained; the crude extract is in chromatography by a hydroxyapatite column for one time, is gradient eluted by the phosphate buffer, and is frozen and dried, and high purity phycoerythrin, phycocyanin and allophycocyanin are obtained. The purity of the crude extract of the phycobiliprotein prepared by the effective method in the earlier stage reaches the standard of pharmaceutical grade (A620 / A 280 is more than 2.0), thereby simplifying the follow-up purifying procedures, so the high purity phycoerythrin, phycocyanin and allophycocyanin can be respectively obtained by only one time column chromatography. When the method is used to extract high purity phycobiliprotein from the fresh algae and the dry algae, the process is simple, the production cost is low, and the yield of the products is high. Thus, the method is suitable for mass production.

The invention relates to phycocyanin beta subunits fluorescent protein combined with phycoerythrobilin PEB, fusion protein formed by phycocyanin beta subunits fluorescent protein and streptavidin and a mutant thereof, the sequences include sequence 1, sequence 2, sequence 3 and sequence 4; furthermore, the invention discloses a method of the fluorescent protein fused with streptavidin for fluorescent immunological detection directly; phycocyanin beta subunit conserved cysteine residue can be not only combined with phycocyanobilin PCB by a thioether bond, but the fact that the phycocyanin beta subunit conserved cysteine residue can be combined with the phycoerythrobilin PEB by the thioether bond through the genetic engineering can be realized, so as to obtain the novel fluorescent phycocyanin, the spectroscopy of the protein is completely different from that of the phycocyanin beta subunits fluorescent protein combined with PCB, and the protein has high fluorescence efficiency; in addition, as the protein carries His-tag label, purification is not only convenient, but also the dissolubility of the protein can be improved; the protein is combined with the streptavidin to form the fusion protein for fluorescent immunological detection directly, thereby being beneficial to the application of the protein in all kinds of the field.

The present invention relates to a pharmaceutical composition for inhibiting influenza virus infection and reproduction, comprising a effective amount of C-phycocyanin (C-PC), allophycocyanin (APC), spirulina growth factor (SGF) or the mixture thereof. The present invention also provides a method for extracting said pharmaceutical composition, comprising the steps of: (a) adding hypotonic buffer solution to organic blue-green algae powder and mixing thoroughly; (b) incubating the mixture below room temperature overnight; (c) separating and purifying the mixture by a centrifuge; (d) collecting the suspending supernatant and detecting it by a spectrometer to determine ingredients and content; and (e) spray drying the supernatant; characterized in which low-temperature extraction is employed to maintain the bioactivity and nutrients of the pharmaceutical composition.

The present invention relates to a method for inhibiting infection and reproduction of influenza type A WSN virus, which comprises providing an effective amount of a pharmaceutical composition; and contacting said composition with said influenza type A WSN virus, wherein said pharmaceutical composition contains C-phycocyanin (C-PC), allophycocyanin (APC), and spirulina growth factor (SGF). The present invention also provides a method for extracting said pharmaceutical composition, comprising the steps of: (a) adding hypotonic buffer solution to organic blue-green algae powder and mixing thoroughly; (b) incubating the mixture below room temperature overnight; (c) separating and purifying the mixture by a centrifuge; (d) collecting the suspending supernatant and detecting it by a spectrometer to determine ingredients and content; and (e) spray drying the supernatant; characterized in which low-temperature extraction is employed to maintain the bioactivity and nutrients of the pharmaceutical composition.

The invention relates to a method for preparing an allophycocyanin(APC)-marked fluorescent antinuclear antibody, which comprises: separating and purifying spirulina APC by anion exchange chromatography; crosslinking APC and antinuclear antibody derivatives in a liquid phase in a proper molar ratio; and preparing the APC-marked fluorescent antinuclear antibody by purification by high-pressure liquid chromatogram. The fluorescent antinuclear antibody prepared by the method has high crosslinking efficiency, high purity, bright red fluorescence, high stability and high sensibility, and can be used as a common fluorescent probe for indirect immunofluorescent detection of infectious diseases in animals such as chickens and pigs.

The present invention belongs to the field of fluorescent proteins in biotechnology, and particularly relates to a preparation method of a high fluorescence intensity recombinant phycobiliprotein concatermer. According to the preparation method, a streptavidin gene is linked to an allophycocyanin alpha subunit gene through a linker sequence; on the basis, one or a plurality of allophycocyanin alpha subunit genes are connected in series through linker sequences to form a fusion gene; and the fusion gene, a phycobiliprotein lyase gent and a phycoerythrobilin biosynthetic enzyme gene co-express in Escherichia coli to obtain the recombinant allophycocyanin concatermer with characteristics of biotin binding ability and high fluorescence intensity. According to the present invention, tn the immunofluorescence assay, the recombinant phycobiliprotein concatermer can achieve the strong fluorescent signal compared to the recombinant phycobiliprotein monomer; and the prepared recombinant phycobiliprotein concatermer can be adopted as the fluorescent marker for immunofluorescence detection in the field of biology and biomedicine.

The invention belongs to the field of fluorescent protein materials in biotechnology, and specifically relates to a method for preparing an allophycocyanin tripolymer fluorescent protein. The method comprises the following steps: firstly, cloning allophycocyanin alpha sub-gene apoprotein genes and phycobilin biosynthesis enzyme genes hol and pcyA into an expression vector; and secondly, cloning allophycocyanin beta sub-gene apoprotein genes and chromophore lyase genes cpcS and cpcU into another vector, using the two expression vectors to simultaneously transform Escherichia coli, and screening out an engineering bacterium simultaneously expressing the genes, thus obtaining the allophycocyanin tripolymer fluorescent protein. By using the Escherichia coli as the raw material instead of algae to produce the recombinant photo-activated protein, the Escherichia coli is easy to culture and grows fast, thereby greatly shortening the production cycle. Compared with algae, the cell wall of the Escherichia coli can be broken easily, energy can be saved in the purification process, and a high utilization rate can be achieved.

The invention belongs to the field of rapid food detection and discloses a method for detecting oxidation resistance by utilizing long-wavelength allophycocyanin fluorescence. The method comprises thefollowing steps: with allophycocyanin as a fluorescence indicator, competing free radicals released by AAPH with an antioxidant so as to cause delay of fluorescence decay time of allophycocyanin, with a decay curve without the antioxidant as a blank, calculating an AUC (Area Under the Curve) difference of the two, establishing a standard curve, and performing actual sample detection. The method disclosed by the invention has the advantages that allophycocyanin belongs to a long-wavelength fluorescence indicator, has the excitation wavelength of 653nm and emission wavelength of 668nm and can avoid interference of autofluorescence matters in an actual sample; and allophycocyanin has high pH stability and is non-sensitive to internal and external quenching. According to the advantages, the detection sensitivity is greatly improved.

A Spirulina maxima extract of the present invention and Allophycocyanin (APC), R-phycoerythrin (R-PE), and C-phycocyanin (C-PC), which are components of the Spirulina maxima extract, show an effect of inhibiting cell death and A2E (pyridinium bis-retinoid), oxidation due to blue light, and therefore can be usefully applied as an active ingredient in a composition for preventing and treating retinal disease.

The invention relates to a method for preparing a fluorescent antibody for detecting avian influenza virus and a solid phase immunofluorescence detection kit. According to the invention, an avian influenza virus antibody is marked by allophycocyanin (APC) so as to prepare the avian influenza virus fluorescent antibody; the APC and the antibody are derived by a chemical cross-linking agent SPDP respectively; the derivatives are subjected to liquid phase cross-linking in a proper molar ratio; and then the fluorescent antibody is prepared by high pressure liquid chromatography purification. The solid phase immunofluorescence detection kit comprises the fluorescent antibody, CNBr activated agarose microspheres, the avian influenza virus antibody, cleaning solution and the like. The using method of the kit comprises the following steps of: activating a microsphere carrier and then coating the activated microsphere carrier by using the avian influenza virus antibody; cleaning; combining the microspheres coated with the antibody with a sample (an antigen) to be detected; combining the microspheres coated with the antibody with the fluorescent antibody after cleaning; removing the uncombined fluorescent antibody by cleaning; and observing under a fluorescence microscope and determining the result. The fluorescent antibody prepared by the method is characterized by high cross-linking efficiency, high purity, bright red fluorescence and stable performance. The microsphere solid phase carrier which is adopted by the kit can obviously improve fluorescent detection sensitivity, and is suitable for the quick detection of the avian influenza virus.

The invention belongs to the field of fluorescent protein and recombinant antibodies in biotechnology and particularly relates to a preparation method of fusion fluorescent protein in escherichia coli. The method is characterized in that AFP single-chain antibody genes and allophycocyanin alpha subunit genes are spliced through linker sequence to form chimeric genes, and the chimeric genes, phycobiliprotein lyase genes and phycobilin biosynthetic enzyme genes are expressed in the escherichia coli to obtain fusion fluorescent protein covalently binding phycoerythrin or fusion fluorescent protein covalently binding phycocyanobilin. The method has the advantages that biotechnology is used to produce the fusion protein, of single-chain antibodies and allophycocyanin alpha subunits, in the escherichia coli, the method is environmental friendly and low in cost, and the fusion protein is applicable to fields such as biology and biomedical detection.

The invention discloses fusion protein. The fusion protein is characterized in that the amino acid sequence of the fusion protein is F1 polypeptide, connected protective peptide 1, F2 polypeptide, connected protective peptide 2 and F1 polypeptide, wherein the sequence of the F1 polypeptide is any of SEQID No.1 and SEQID No.2; and the sequence of the F2 polypeptide is any of SEQID No.3 to SEQ ID No.14. An ApcE2 mutant and allophycocyanin subunits BDFP1.1 or BDFP1.2 which are subjected to genetic engineering are merged to obtain the fusion protein, and the problem that the ApcE2 mutant marked fluorescence in lactation animal cells is weak, is solved. In the fluorescence marking process, phytochrome P phi B is added in vitro, so that the brightness of the fusion protein for cell labelling canbe effectively improved.

The present invention relates to a protein, crystal preparation process and its product, in particular, it relates to an allophycocyanin which is extracted from algae under the general condition and can emit strong fluorescence, and can convert it into crystal form. It can be extensively used in the fields of scientific research, medicine, detection and telecommunication, etc.

The invention discloses an allophycocyanin (APC) and Annexin V protein coupling method. The APC and Annexin V protein coupling method comprises the following steps: 1, an Annexin V protein is expressed and purified: specifically, 1.1, a gene sequence is optimized, specifically the gene sequence of the human Annexin V protein is looked up, the human Annexin V gene sequence is optimized, a termination codon sequence is removed, the human Annexin V gene sequence is connected into a prokaryotic expression vector, front and back His tag proteins are retained, and preparation is made for protein purification and subsequent coupled fluorescein purification; and 1.2, the Annexin V protein is subjected to inducible expression, specifically, a recombinant plasmid single colony is picked and inoculated into an LB liquid culture medium containing kanamycin, and culturing is conducted until the concentration OD600 of bacterial liquid is 0.4-0.6, and an inducer is added into the bacterial liquid forinduction culture. When the Annexin V gene sequence is designed, His tags at the two ends of the protein are reserved, the Annexin V protein and an Annexin V-APC conjugate are convenient to purify, only conventional nickel column purification is needed, special purification equipment is not needed, the purification steps are few, the purification time is short, the purification cost is lowered, the good biological activity of the protein is maintained, and the Annexin V protein can be used for cell apoptosis detection.

The invention discloses a preparation method of high-purity spirulina blue. The preparation method is characterized by adopting spirulina powder as a raw material, and performing continuous productionincluding extraction, filtration, salting out, desalination concentration, micro-encapsulation, spray drying and mixing to obtain spirulina blue, which is blue powder. Phycocyanin is dark blue powder, is separated from spirulina, and looks beautiful. The main component of spirulina blue is phycocyanin, which belongs to porphyrin cyanophycin and is one of infrequent chromoproteins in the nature. The phycocyanin has bright color, is protein itself with abundant nutrients, has complete amino acid composition, and is high in amino acid content. The preparation method is short in production periodand doesn't cause secondary pollution. The obtained spirulina blue is high in purity, contains 71.3% of phycocyanin, has the purity A620 / A280 reach 3.6, and can meet the requirement of food and cosmetic industry.

In a method for quantitating an analyte by measuring time resolved transfer of fluorescence energy to or from a label quantitatively associated with the analyte, the present invention provides an improvement comprising measuring the energy transferred from donor compounds having the ability to absorb light energy and then transfer this energy to cross-linked allophycocyanin in a time-resolved manner, where the cross-linked allophycocyanin used according to this invention has not been exposed to strongly chaotropic agents after cross-linking.

The invention relates to a molecular design phycocyanin beta subunit fluorescent protein combining phycoerythrobilin, fusion protein formed by the phycocyanin beta subunit fluorescent protein and streptavidin, and mutant thereof, having the sequences 1, 2, 3 and 4. The invention also discloses a method for directly using the fluorescent protein fused with the streptavidin for fluorescence immunoassay; and due to thioether bond, 153th cysteine residue conserved by phycocyanin beta subunit not only can be combined with phycocyanobilin (PCB), but also can be combined with the phycoerythrobilin (PEB) through genetic engineering, so that a novel fluorescent phycocyanin can be obtained. The spectrum property of the phycocyanin is completely different from that of the phycocyanin beta subunit fluorescent protein combined with the PCB, has higher fluorescence efficiency, and is convenient for purification due to being provided with His-tag label. Furthermore, the dissolubility can be improved, the fusion protein can be formed by the phycocyanin beta subunit fluorescent protein and the streptavidin, the fluorescent protein can be directly used for fluorescence immunoassay, and the invention is beneficial to the application in various fields.

The invention discloses a preparation method of a nostoc sphaeroides grain beverage. The method comprises the following concrete steps of nostoc sphaeroides harvesting and selection, nostoc sphaeroides ion replacement, nostoc sphaeroides enzyme activity passivation, nostoc sphaeroides sterilization treatment, beverage proportioning, filling, pasteurization and packaging. The method has the following beneficial effects that through the ion replacement, the combination with ingredients such as phycocyanobilin, allophycocyanin, phycoerythrobilin, chlorophyll and the like inside nostoc sphaeroidesis performed to form a stable chelate, so that a stable pigment can be formed. Meanwhile, by regulating a PH value and blanching and passivating the enzyme activity, the characteristics of nutritional ingredients of the nostoc sphaeroides is ensured, so that the whole appearance of the whole product is glittering and translucent; the sphere is mellow and full; the mouthfeel is tender and elastic.The functions of thirst quenching, pleasant flavor, rich juice nutrient and the like of the beverage are realized; the effective function, the Q elastic mouthfeel and the glittering and translucent appearance of natural freshwater alga of the nostoc sphaeroides are also realized; in addition, the pleasant flavor in the beverage can be blended into the nostoc sphaeroides.

The invention provides extracting solution for a spirulina maxima and a preparation method thereof. The nutrient content in the extracting solution mainly comprises chlorophyl of the spirulina maxima, phyeoeyani of the spirulina maxima, and polyhexose of the spirulina maxima, and so on, wherein the content of the chlorophyll of the spirulina maxima is higher than 8 percent. The preparation methodmainly comprises the processes of extracting the chlorophyll, extracting the phyeoeyanin, extracting free amino acid, oligopeptides and oligose, and extracting the polyhexose of the spirulina maxima,and so on; particularly, the gummeline embedding process is performed in the process of extracting the chlorophyll of the spirulina maxima. The preparation method extracts sufficiently, reserves the ingredient of the chlorophyll of the spirulina maxima, can be applied to the beverage addition, particularly the beer addition, does not pollute the environment, has convenient operation and is easilyapplied to the industrial production.

A medical composition for suppressing the infection and replication of influenza virus contains C-phycocyanin (C-CP), allophycocyanin (APC), spirulina growth factor (SGF), or their mixture. It is prepared through adding organic blue algae powder to low-tension buffer liquid, stirring, laying aside for 12 hr, separating, purifying taking upper-layer liquid, measuring optical spectrum for determining the contents of active components, and spray drying.

The present invention discloses an Aglae extract for lifting liver functions for a patient, and the Aglae extract is prepared by extracting solution containing Aglae , and the Aglae extract comprises biologically active substances which are selected from one or more of allophycocyanin, sulfur-containing polysacarides, C-phycocyanin and the mixture thereof.

The invention belongs to the field of biotechnology, and specifically relates to a nanograde biocomposite and applications thereof. The nanograde biocomposite is prepared by mixing a nanograde oxidized grapheme-low molecular weight chitosan compound solution and a recombinant allophycocyanin solution with PBS solution, wherein the concentration of the nanograde oxidized grapheme-low molecular weight chitosan compound solution ranges from 0.02 to 10<mu>g / ml, and the concentration of the recombinant allophycocyanin solution ranges from 0.001 to 10mg / ml. The nanograde biocomposite can be uses as biosensors, is high in specificity and sensitivity, and is nanoscaled; and application range of the biocomposite is wide.

A Spirulina maxima extract of the present invention and Allophycocyanin (APC), R-phycoerythrin (R-PE), and C-phycocyanin (C-PC), which are components of the Spirulina maxima extract, show an effect of inhibiting cell death and A2E (pyridinium bis-retinoid), which is oxidized with a blue light, so that the present invention can be usefully applied as an active ingredient in a composition for preventing and treating retinal diseases.

Aiming at the technical problems in the prior art, the invention provides a rat peripheral blood Pig-a gene mutation test flow cytometry detection method, which comprises the following steps of 1, collecting target tissue cells,2, dyeing a surface antigen,3, dyeing with a nucleic acid dye application solution,and 4, after dyeing is finished, detecting the sample by using a double-laser flow cytometer to obtain data. The detection method distinguishes red blood cells by using forward angular scatter (FSC) and sideangular scatter (SSC), Syto13 labels reticulocyte, CD59-allophycocyanin (APC) labels mutant cells, and the rat peripheral blood Pig-a gene mutation test flow cytometry detection method is simpler, more convenient and economical.

The invention relates to allophycocyanin subunits fluorescent protein combined with phycocyanobilin PCB and fusion protein formed by allophycocyanin subunits fluorescent protein and streptavidin, the sequences are from sequence 1 to sequence 10; furthermore, the invention discloses a method of the fluorescent protein fused with streptavidin for fluorescent immunological detection directly; allophycocyanin subunit conserved cysteine residue is combined with the phycocyanobilin PCB by a thioether bond, and the subunit of the fluorescent allophycocyanin can be obtained by utilizing escherichia coli through genetic engineering, the spectroscopy of the protein carries His-tag label, therefore, purification is not only convenient, but also the dissolubility of the protein can be improved; the protein is combined with the streptavidin to form the fusion protein for fluorescent immunological detection directly, thereby being beneficial to the application of the protein in all kinds of the field.

The invention discloses an artificial binding protein capable of specifically recognizing allophycocyanin. The amino acid sequence is as follows: HMVDNKFNKEIVNAITEIHHLPNLNLEQRWAFIFSLFDDPSQSANLLAEAKKLNDAQAPKSGGGGSGGGGIGVDNKFNKEWQNAHNEIIWLPNLNWEQKWAFINSLYDDPSQSANLLAEAKKLNDAQAPKAAASDYKDDDDKLEHHHHHH. The protein has the characteristics of strong specificity, high affinity, strong thermal stability, strong acid-base tolerance and the like, and can be applied to the fields of detection, purification and the like of allophycocyanin. In addition, the invention also relates to a DNA fragment for encoding the artificial binding protein and an acquisition method of the artificial binding protein. According to the invention, a protein A dimer with high stability and high solubility is selected as a skeleton protein, and on the basis of the skeleton protein, the protein with antigen binding activity is finally obtained through the processes of genetic engineering modification, mutant library construction, affinity screening and the like.

The invention relates to allophycocyanin subunits fluorescent protein combined with phycoerythrobilin PEB and fusion protein formed by allophycocyanin subunits fluorescent protein and streptavidin, the sequences are from sequence 1 to sequence 10; furthermore, the invention discloses a method of the fluorescent protein fused with streptavidin for fluorescent immunological detection directly; allophycocyanin subunit conserved cysteine residue is combined with the phycoerythrobilin PEB by a thioether bond, and the subunit of the fluorescent allophycocyanin can be obtained by utilizing escherichia coli through genetic engineering, the spectroscopy of the protein carries His-tag label, therefore, purification is not only convenient, but also the dissolubility of the protein can be improved; the protein is combined with the streptavidin to form the fusion protein for fluorescent immunological detection directly, thereby being beneficial to the application of the protein in all kinds of the field.

The invention belongs to the field of producing protein through microbiological fermentation, and in particular relates to a microbiological preparation method of inulin source recombinant allophycocyanin. The method comprises the following steps: with inulin as raw material, degrading the raw material as a seed solution, culturing Escherichia coli P2 through a shaking table or a fermentation tank under the effect of a fermentation medium, and then inductively expressing through isopropyl-beta-D-thiopyran galactoside (IPTG) to obtain the inulin source recombinant allophycocyanin. The recombinant allophycocyanin can be applied to the fields of food, healthcare and medical and functional materials. The preparation method of the inulin source recombinant allophycocyanin is simple, and an application path with low cost and high yield is found for the bio-utilization of the inulin; compared with a method of using glucose as a carbon source, the microbiological preparation method disclosed by the invention has the advantage that the raw material cost of producing the allophycocyanin in fermentation production is greatly reduced.