CN101759795A - Molecular design phycoerythrocyanin beta subunit fluorescent protein combining phycocyanobilin (PCB) and application thereof - Google Patents

Abstract

The invention relates to a molecular design phycoerythrocyanin beta subunit fluorescent protein combining phycocyanobilin (PCB), fusion protein formed by the phycoerythrocyanin 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 phycoerythrocyanin beta subunit can be combined with the phycocyanobilin (PCB), and is prepared into beta subunit of fluorescent phycoerythrocyanin by escherichia coli of genetic engineering. The spectrum property of the phycoerythrocyanin is completely different from that of the natural phycoerythrocyanin, 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 phycoerythrocyanin 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.

Description

The phycoerythrocyanin (pec) beta subunit fluorescent protein and the application thereof in conjunction with phycocyanobilin PCB of molecular designing

Technical field

The present invention the present invention relates to a kind of phycocyanin and phycoerythrin beta subunit fluorescent protein and application thereof in conjunction with phycocyanobilin PCB of molecular designing, belong to chromoprotein material field in the biotechnology, be specifically related to the phycocyanin and phycoerythrin beta subunit fluorescent protein in conjunction with phycocyanobilin PCB of molecular designing, fusion rotein and the mutant thereof that itself and Streptavidin form, and utilize this fusion rotein to detect soluble antigen or detection of antibodies method.

Background technology

Phycobiliprotein (phycobiliprotein) is the function ingredients that blue-green algae and red algae photosynthesis are caught the recovery compound.According to its absorption spectrum and fluorescence spectral characteristic, phycobiliprotein can be divided into phycoerythrin (phycoerythrin, abbreviation CPE), phycoerythrocyanin (pec) (phycoerythrocyanin, abbreviation PEC), Phycocyanins, C-(phycocyanin, be called for short CPC) and become Phycocyanins, C-(allophycocyanin is called for short APC).CPE, PEC, CPC and APC contain alpha and beta subunit, phycobilin in each subunit (phycobilin) is by the sulfydryl covalent attachment of thioether bond and phycobiliprotein apoprotein (apo-phycobiliprotein) cysteine residues, its kind and with the spectral quality of the interaction decision phycobiliprotein of apoprotein.Phycobilin and apoprotein covalent attachment form specific conformation, make CPE mainly absorb the visible light of about 560nm, the fluorescence of the about 580nm of emission; PEC absorbs the visible light of about 570nm, the fluorescence of the about 630nm of emission; CPC absorbs the visible light of about 620nm, the fluorescence of the about 640nm of emission; APC absorbs the visible light of about 650～660nm, launches the fluorescence of about 660～670nm.CPE bonded prothetic group pigment is phycoerythrobilin (phycoerythrobilin is called for short PEB); The alpha subunit bonded prothetic group pigment of PEC is the purple courage element of algae (phycoviolobilin is called for short PVB); The beta subunit bonded prothetic group pigment of PEC is phycocyanobilin (phycocyanobilin is called for short PCB); CPC and APC bonded prothetic group pigment all are phycocyanobilin PCB.

Can from algae, extract and obtain phycoerythrocyanin (pec), and isolate its alpha and beta subunit, 80 cysteine residues of alpha subunit are by the strong covalent attachment phycocyanobilin PCB of thioether, and 82 and 153 cysteine residues of beta subunit are respectively by the strong covalent attachment phycocyanobilin PCB of thioether; Also can utilize intestinal bacteria to produce by genetically engineered.The alpha subunit of PEC can be produced (TooleyAJ, GlazerAN.Biosynthesis of thecyanobacterial light-harvesting polypeptide phycoerythrocyanin holo-alpha subunit in aheterologous host.J Bacteriol.2002 by the bacillus coli gene engineering bacteria; 184 (17): 4666～4671).The beta subunit of PEC also can be produced by the bacillus coli gene engineering bacteria.We find, utilize genetically engineered, can obtain 82 fluorescins in conjunction with phycocyanobilin PCB of phycocyanin and phycoerythrin beta subunit, its spectral quality different with natural phycoerythrocyanin (pec) (as shown in Figure 1 and Figure 2) can also suddenly change, carry out useful mark to its peptide chain amino acid simultaneously.

Streptavidin can be followed the vitamin H specific combination, by Streptavidin-biotin system, can realize the signal amplification, improves detection sensitivity.Streptavidin-biotin system has been widely used in fields such as Biological Detection and medical treatment detection at present.At present mainly be to realize the mark of Streptavidin to target by chemically crosslinked.The present invention passes through genetic engineering technique, Streptavidin gene and phycoerythrocyanin (pec) subunit apoprotein gene splicing rear clone in expression vector, can obtain the fusion rotein of Streptavidin and phycoerythrocyanin (pec) subunit apoprotein at e. coli expression, directly realize being connected of Streptavidin and phycoerythrocyanin (pec) subunit apoprotein; By phycobiliprotein beta lyase energy catalysis phycocyanobilin and phycoerythrocyanin (pec) subunit apoprotein and homologous protein covalent attachment thereof, thereby generation has the phycoerythrocyanin fluorescence protein in conjunction with PCB (its spectrum as shown in Figure 3, Figure 4) of the marked by streptavidin of good photoluminescent property.This albumen can directly apply to fields such as immunofluorescence detection.

Immunofluorescence technique (Immunofluorescence technique) claim fluorescent-antibody technique again, being that the earliest a kind of immunofluorescence technique (Immunofluorescence technique) of development claims fluorescent-antibody technique again in the immuno-labelling technique, is the earliest a kind of of development in the immuno-labelling technique.It is a technology of setting up on the basis of immunology, biological chemistry and microscopy.Just there have been some scholars to attempt antibody molecule is combined with some tracer materials very early, utilize antigen antibody reaction to organize or the location of intracellular antigen material.Coons equals nineteen forty-one to be adopted fluorescein to carry out mark first and succeeds.

Method with fluorescence antibody spike or inspection corresponding antigens claims fluorescent antibody technique; Method with spike of known fluorescent antigen marker or inspection corresponding antibodies claims the fluorescent antigen method.These two kinds of method general name immunofluorescence techniques, because fluorochrome not only can combine with antibody globulin, be used for detecting or locating various antigens, also can with other protein bound, be used for detecting or location antibody, but the fluorescent antigen technology is seldom used in real work, so people's custom is called fluorescent-antibody technique, or is called immunofluorescence technique.More commonly used with the fluorescence antibody method.

The principal feature of this technology is: high specificity, susceptibility height, speed are fast.Main drawback is: the unspecific staining problem solves as yet fully, and technical program also more complicated.Simultaneously, because the background in the general fluorometric assay is than problems such as height, immunofluorence technic is used for quantitative assay certain difficulty.

The phycobiliprotein stable in properties, it is less that its photoluminescent property is subjected to environmental influence, is suitable as fluorescent probe and uses.Phycobiliprotein among the present invention directly has been marked with Streptavidin, utilize indirect detection method, by vitamin H-Streptavidin system, utilize the fluorescence phycobiliprotein of marked by streptavidin to combine, thereby realize detection of antigens with biotin labeled anti-antibody.Vitamin H-Streptavidin has improved the sensitivity that detects, simultaneously because the versatility of anti-antibody makes this detection method can be used for various detection of antigens easily; The fluorescence phycobiliprotein has good photoluminescent property, and the fluorescence emission peak of higher fluorescence efficiency, big wavelength can improve detection sensitivity, reduces the influence of background fluorescence.Present method helps the popularization that the fluorescence phycobiliprotein is used in immunodetection.

Phycobiliprotein is used widely in various fields at present just gradually, particularly in the immunofluorescence context of detection very big exploitation prospect is arranged.Have than high fluorescence efficiency by after transforming, have the phycobiliprotein that can utilize mark, can promote its using value in immunofluorescence detects greatly, this lays a good foundation in the application of medicine and bioengineering field for phycobiliprotein.

Summary of the invention

The object of the present invention is to provide a kind of phycocyanin and phycoerythrin beta subunit fluorescent protein of molecular designing, fusion rotein that itself and Streptavidin form and the aminoacid sequence of mutant thereof in conjunction with phycocyanobilin PCB, and indicate its pigment binding site, provide the fluorescence phycobiliprotein that utilizes marked by streptavidin to detect the immunologic detection method of soluble antigen or antibody simultaneously.The phycocyanin and phycoerythrin beta subunit in conjunction with phycocyanobilin PCB of this class marked by streptavidin have with natural algae in the different structure of phycocyanin and phycoerythrin beta subunit that exists, and its fluorescence spectrum character also has very big difference, simultaneously, what provide that this type of has a marked by streptavidin is used to detect the method for soluble antigen in conjunction with the phycocyanin and phycoerythrin beta subunit of phycocyanobilin PCB: it is to utilize antibody and antigen-specific reaction, Streptavidin can with the principle of vitamin H specific combination, utilize the fluorescence phycobiliprotein of marked by streptavidin to detect soluble antigen or antibody.

In order to address the above problem, the technical solution adopted in the present invention is:

Phycocyanin and phycoerythrin beta subunit fluorescent protein in conjunction with phycocyanobilin PCB, the phycocyanin and phycoerythrin beta subunit coding gene is cloned in expression plasmid, utilize phycocyanin and phycoerythrin beta lyase expression plasmid and phycocyanobilin PCB synthetic enzyme plasmid, can in intestinal bacteria, synthesize phycocyanin and phycoerythrin beta subunit fluorescent protein in conjunction with PCB, and indicated its pigment binding site, such protein sequence N end has the His-tag mark, but the His-tag mark is not limited to the N end.

The sequence of described phycocyanin and phycoerythrin beta subunit fluorescent protein in conjunction with phycocyanobilin PCB is a sequence 1:

MHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADGMLDAFSRVVEQADKKGAYLSNDEINALQAIVADSNKRLDVVNRLTSNASSIVANAYRALVAERPQVFNPGGPCFHHRNQAACIRDLGFILRYVTYSVLAGDTSVMDDRCLNGLRETYQALGTPGDAVASGIKKMKEAALKIANDPNGITKGDCSQLMSELASYFDRAAAAVA

Describedly be meant that in conjunction with phycocyanobilin PCB phycocyanobilin PCB is combined in 201 by thioether bond, be equivalent to 153 of original phycocyanin and phycoerythrin beta subunit.

The phycocyanin and phycoerythrin beta subunit coding gene is cloned in expression plasmid, utilize gene engineering method that it is suddenlyd change, can express and obtain phycocyanin and phycoerythrin beta subunit mutant, to generally can bigger influence not being arranged to property of protein except that suddenling change in conjunction with all sites 130 of pigment, belong to same proteinoid, this albumen is carried out excalation, property of protein is not had considerable influence, also belong to same proteinoid.

The phycocyanin and phycoerythrin beta subunit coding gene is cloned in expression plasmid, utilize gene engineering method that its 130th (being equivalent to the 82nd of original phycocyanin and phycoerythrin beta subunit) cysteine residues sported serine residue, can express obtaining phycocyanin and phycoerythrin beta subunit mutant.

The sequence of the mutant of described phycocyanin and phycoerythrin beta subunit fluorescent protein in conjunction with phycocyanobilin PCB is a sequence 2:

The 201st halfcystine C of sequence 2:(is the binding site of phycocyanobilin PCB, and the 130th Serine S is by the C sudden change)

MHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADGMLDAFSRVVEQADKKGAYLSNDEINALQAIVADSNKRLDVVNRLTSNASSIVANAYRALVAERPQVFNPGGPCFHHRNQAAAIRDLGFILRYVTYSVLAGDTSVMDDRCLNGLRETYQALGTPGDAVASGIKKMKEAALKIANDPNGITKGDCSQLMSELASYFDRAAAAVA

Describedly be meant that in conjunction with phycocyanobilin PCB phycocyanobilin PCB is combined in 201 by thioether bond, be equivalent to 153 of original phycocyanin and phycoerythrin beta subunit.

The phycocyanin and phycoerythrin beta subunit coding gene is cloned in expression plasmid, utilize gene engineering method that it is suddenlyd change, can express the mutant that obtains the phycocyanin and phycoerythrin beta subunit, utilize phycocyanin and phycoerythrin beta lyase expression plasmid and phycocyanobilin PCB synthetic enzyme plasmid, can in intestinal bacteria, synthesize phycocyanin and phycoerythrin beta subunit fluorescent protein, and indicate its pigment binding site in conjunction with PCB.

A kind of phycocyanin and phycoerythrin beta subunit fluorescent protein of marked by streptavidin in conjunction with phycocyanobilin PCB, it is characterized in that: Streptavidin encoding gene and phycocyanin and phycoerythrin beta subunit coding gene are spliced rear clone in expression plasmid, express the fusion rotein that obtains Streptavidin and phycocyanin and phycoerythrin beta subunit.

The phycocyanin and phycoerythrin beta subunit type fluorescence protein sequence in conjunction with phycocyanobilin PCB of described marked by streptavidin is a sequence 3:

The 329th halfcystine C of sequence 3:(is the binding site of phycocyanobilin PCB)

MHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASGSGTDDDDKAMADGMLDAFSRVVEQADKKGAYLSNDEINALQAIVADSNKRLDVVNRLTSNASSIVANAYRALVAERPQVFNPGGPCFHHRNQAACIRDLGFILRYVTYSVLAGDTSVMDDRCLNGLRETYQALGTPGDAVASGIKKMKEAALKIANDPNGITKGDCSQLMSELASYFDRAAAAVA

The phycocyanin and phycoerythrin beta subunit fluorescent protein in conjunction with phycocyanobilin PCB of described a kind of marked by streptavidin, Streptavidin still is not limited to hold at N at the N of PecB end.

Describedly be meant that in conjunction with phycocyanobilin PCB phycocyanobilin PCB is combined in 329 by thioether bond, be equivalent to 153 of original phycocyanin and phycoerythrin beta subunit.

A kind of phycocyanin and phycoerythrin beta subunit fluorescent protein mutant of marked by streptavidin in conjunction with phycocyanobilin PCB, Streptavidin encoding gene and phycocyanin and phycoerythrin beta subunit coding gene are spliced rear clone in expression plasmid, utilize gene engineering method that its 258th (being equivalent to the 82nd of original phycocyanin and phycoerythrin beta subunit) cysteine residues sported serine residue, express the mutant of the fusion rotein that obtains Streptavidin and phycocyanin and phycoerythrin beta subunit.

The protein sequence in conjunction with the phycocyanin and phycoerythrin beta subunit fluorescent protein mutant of phycocyanobilin PCB of described marked by streptavidin is a sequence 4:

The 329th halfcystine C of sequence 4:(is the binding site of phycocyanobilin PCB, and the 258th Serine S is by the C sudden change)

MHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASGSGTDDDDKAMADGMLDAFSRVVEQADKKGAYLSNDEINALQAIVADSNKRLDVVNRLTSNASSIVANAYRALVAERPQVFNPGGPCFHHRNQAAAIRDLGFILRYVTYSVLAGDTSVMDDRCLNGLRETYQALGTPGDAVASGIKKMKEAALKIANDPNGITKGDCSQLMSELASYFDRAAAAVA

The phycocyanin and phycoerythrin beta subunit fluorescent protein mutant in conjunction with phycocyanobilin PCB of described marked by streptavidin, be meant that in conjunction with phycocyanobilin PCB phycocyanobilin PCB is combined in 329 by thioether bond, be equivalent to 153 of original phycocyanin and phycoerythrin beta subunit.

In conjunction with the application of the phycocyanin and phycoerythrin beta subunit fluorescent protein of phycocyanobilin PCB, that utilizes marked by streptavidin detects the immunodetection of soluble antigen or antibody in conjunction with the phycocyanin and phycoerythrin beta subunit fluorescent protein of phycocyanobilin PCB.

Described immunologic detection method may further comprise the steps:

(1) can combine with solid phase and can discern antigenic first antibody bag by on the solid phase, confining liquid seals not binding antibody part, and is standby;

(2) the fluorescence phycobiliprotein with determined antigen, the anti-antibody of discerning the mark vitamin H of antigenic second antibody, identification second antibody, mark Streptomycin sulphate avidin adds in the solid phase fully reaction by certain step;

(3) utilize microwell plate fluorescence detector or other corresponding instrument detecting fluorescence, analytical results.

Beneficial effect of the present invention:, be convenient to purify owing to have the His-tag mark in conjunction with the phycocyanin and phycoerythrin beta subunit of PCB, and because this albumen is single subunit constitutes, have more homogeneity than the natural phycoerythrocyanin (pec) of a plurality of subunits of tool; Compare and have and the diverse spectral quality of phycocyanin and phycoerythrin beta subunit that combines PCB, its fluorescence efficiency height, being used for immunodetection has better sensitivity; After forming fusion rotein with Streptavidin, realize the direct mark of Streptavidin, can be directly used in the immunodetection, do not needed to utilize chemical process etc. to carry out marked by streptavidin.

Description of drawings

Fig. 1: 153 cysteine residues are in conjunction with the absorption and the fluorescence spectrum figure of the phycocyanin and phycoerythrin beta subunit fluorescin of PCB, and wherein solid line is an absorption spectrum, and dotted line is a fluorescence spectrum;

Fig. 2: after the 82nd cysteine residues sported L-Ala, 153 cysteine residues were in conjunction with the absorption and the fluorescence spectrum figure of the phycocyanin and phycoerythrin beta subunit fluorescin of PCB, and wherein solid line is an absorption spectrum, and dotted line is a fluorescence spectrum;

Fig. 3: 153 cysteine residues of marked by streptavidin are in conjunction with the absorption and the fluorescence spectrum figure of the phycocyanin and phycoerythrin beta subunit fluorescin of PCB, and wherein solid line is an absorption spectrum, and dotted line is a fluorescence spectrum;

Fig. 4: after the 82nd cysteine residues sports L-Ala, 153 cysteine residues of marked by streptavidin are in conjunction with the absorption and the fluorescence spectrum figure of the phycocyanin and phycoerythrin beta subunit fluorescin of PCB, wherein solid line is an absorption spectrum, and dotted line is a fluorescence spectrum.

The protein amino acid sequence annex:

The 201st halfcystine C of sequence 1:(is the binding site of phycocyanobilin PCB)

MHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADGMLDAFSRVVEQADKKGAYLSNDEINALQAIVADSNKRLDVVNRLTSNASSIVANAYRALVAERPQVFNPGGPCFHHRNQAACIRDLGFILRYVTYSVLAGDTSVMDDRCLNGLRETYQALGTPGDAVASGIKKMKEAALKIANDPNGITKGDCSQLMSELASYFDRAAAAVA

The 201st halfcystine C of sequence 2:(is the binding site of phycocyanobilin PCB, and the 130th Serine S is by the C sudden change)

MHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADGMLDAFSRVVEQADKKGAYLSNDEINALQAIVADSNKRLDVVNRLTSNASSIVANAYRALVAERPQVFNPGGPCFHHRNQAAAIRDLGFILRYVTYSVLAGDTSVMDDRCLNGLRETYQALGTPGDAVASGIKKMKEAALKIANDPNGITKGDCSQLMSELASYFDRAAAAVA

The 329th halfcystine C of sequence 3:(is the binding site of phycocyanobilin PCB)

MHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASGSGTDDDDKAMADGMLDAFSRVVEQADKKGAYLSNDEINALQAIVADSNKRLDVVNRLTSNASSIVANAYRALVAERPQVFNPGGPCFHHRNQAACIRDLGFILRYVTYSVLAGDTSVMDDRCLNGLRETYQALGTPGDAVASGIKKMKEAALKIANDPNGITKGDCSQLMSELASYFDRAAAAVA

The 329th halfcystine C of sequence 4:(is the binding site of phycocyanobilin PCB, and the 258th Serine S is by the C sudden change)

MHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASGSGTDDDDKAMADGMLDAFSRVVEQADKKGAYLSNDEINALQAIVADSNKRLDVVNRLTSNASSIVANAYRALVAERPQVFNPGGPCFHHRNQAAAIRDLGFILRYVTYSVLAGDTSVMDDRCLNGLRETYQALGTPGDAVASGIKKMKEAALKIANDPNGITKGDCSQLMSELASYFDRAAAAVA

Embodiment

The invention will be further described below in conjunction with drawings and Examples, but these embodiment only are used to illustrate the present invention, do not limit the scope of the invention.

Embodiment 1

Protein amino acid sequence is shown in sequence 1, the phycocyanin and phycoerythrin beta subunit coding gene is cloned in expression plasmid, express obtaining the phycocyanin and phycoerythrin beta subunit, its N end has the His-tag mark, this not only helps it is purified, and also helps to improve its solvability.Phycocyanobilin is combined on the 201st (being equivalent to the 153rd of original phycocyanin and phycoerythrin beta subunit) cysteine residues by thioether is strong.Its spectrum as shown in Figure 1, absorption peak is 595nm, fluorescence emission peak is 625nm.

Embodiment 2

Protein amino acid sequence is shown in sequence 2, the phycocyanin and phycoerythrin beta subunit coding gene is cloned in expression plasmid, utilize gene engineering method that it is suddenlyd change, can express and obtain phycocyanin and phycoerythrin beta subunit mutant, its N end has the His-tag mark, this not only helps it is purified, and also helps to improve its solvability.Phycocyanobilin is combined on the 201st (being equivalent to the 153rd of original phycocyanin and phycoerythrin beta subunit) cysteine residues by thioether is strong.Its spectrum as shown in Figure 2, absorption peak is 595nm, fluorescence emission peak is 625nm.

Embodiment 3

Protein amino acid sequence is shown in sequence 3, Streptavidin encoding gene and phycocyanin and phycoerythrin beta subunit coding gene are spliced rear clone in expression plasmid, the fusion rotein of Streptavidin that expression obtains and phycocyanin and phycoerythrin beta subunit is directly realized the mark of Streptavidin to the phycocyanin and phycoerythrin beta subunit; And the N end has the His-tag mark, and this not only helps it is purified, and also helps to improve its solvability.Phycocyanobilin is combined on the 329th (being equivalent to the 153rd of original phycocyanin and phycoerythrin beta subunit) cysteine residues by thioether is strong.Its spectrum as shown in Figure 3, absorption peak is 595nm, fluorescence emission peak is 625nm.

Embodiment 4

Protein amino acid sequence is shown in sequence 4, Streptavidin encoding gene and phycocyanin and phycoerythrin beta subunit coding gene mutant are spliced rear clone in expression plasmid, utilize gene engineering method that it is suddenlyd change, the fusion rotein mutant of Streptavidin that expression obtains and phycocyanin and phycoerythrin beta subunit mutant is directly realized the mark of Streptavidin to the phycocyanin and phycoerythrin beta subunit; And the N end has the His-tag mark, and this not only helps it is purified, and also helps to improve its solvability.Phycocyanobilin is combined on the 329th (being equivalent to the 153rd of original phycocyanin and phycoerythrin beta subunit) cysteine residues by thioether is strong.Its spectrum as shown in Figure 4, absorption peak is 595nm, fluorescence emission peak is 625nm.

Embodiment 5

(1) mouse monoclonal antibody of carcinomebryonic antigen CEA is cushioned liquid (0.05M, pH9.6 carbonate buffer solution) with bag and is diluted to 2 μ g/mL, get 150 μ L and add black 96 hole enzyme plates, 4 ℃ of bags were by 12 to 20 hours; Remove to wrap antibody,, wash 4 times, dry liquid with lavation buffer solution (the PBS damping fluid that contains the pH7.2 of 0.05%Tween-20) detersive enzyme target by usefulness; Every hole adds sealing damping fluid (the PBS damping fluid that contains the pH7.2 that contains 0.05%Tween-20 of 1% bovine serum albumin) 300 μ L, and 37 ℃ were sealed 30 minutes; The liquid of falling the deblocking with lavation buffer solution (the PBS damping fluid that contains the pH7.2 of 0.05%Tween-20) detersive enzyme target, washs 4 times, dries liquid, and is standby;

(2) get carcinomebryonic antigen CEA, be diluted to 50ng/mL with dilution buffer liquid (the PBS damping fluid that contains the pH7.2 that contains 0.05%Tween-20 of 0.1% bovine serum albumin), getting 150 μ L joins in the ready enzyme plate micropore of step (1), make nine parallel samples, other gets three holes and directly adds dilution buffer liquid as blank, hatched 1.5 hours for 37 ℃, remove liquid, with lavation buffer solution (the PBS damping fluid that contains the pH7.2 of 0.05%Tween-20) detersive enzyme target, wash 4 times, dry liquid, standby;

(3) per three parallel samples are one group, divide following three kinds of steps to finish:

First kind: the rabbit polyclonal antibody of 1. getting carcinomebryonic antigen CEA, be diluted to 2 μ g/mL with dilution buffer liquid (the PBS damping fluid that contains the pH7.2 that contains 0.05%Tween-20 of 0.1% bovine serum albumin), get 150 μ L and join in the ready enzyme plate micropore of step (2), do three parallel samples and blank; Hatched 1.5 hours for 37 ℃, remove liquid,, wash 4 times, dry liquid with lavation buffer solution (the PBS damping fluid that contains the pH7.2 of 0.05%Tween-20) detersive enzyme target, standby; 2. get biotin labeled goat anti-rabbit immunoglobulin antibody, be diluted to 10 μ g/mL with dilution buffer liquid (the PBS damping fluid that contains the pH7.2 that contains 0.05%Tween-20 of 0.1% bovine serum albumin), get 150 μ L and join in the 1. ready enzyme plate micropore of step, do three parallel samples and blank; Hatched 1.5 hours for 37 ℃, remove liquid,, wash 4 times, dry liquid with lavation buffer solution (the PBS damping fluid that contains the pH7.2 of 0.05%Tween-20) detersive enzyme target, standby; 3. get the phycocyanin and phycoerythrin beta subunit fluorescin in conjunction with PCB of labelled streptavidin, 0.05M potassiumphosphate, 0.5M NaCl damping fluid with pH7.2 are diluted to 10 μ g/mL, get 150 μ L and join in the parallel sample and blank hole that 2. step prepare, hatched 0.5 hour for 37 ℃;

Second kind: the rabbit polyclonal antibody of 1. getting carcinomebryonic antigen CEA, be diluted to 2 μ g/mL with dilution buffer liquid (the PBS damping fluid that contains the pH7.2 that contains 0.05%Tween-20 of 0.1% bovine serum albumin), get 150 μ L and join in the ready enzyme plate micropore of step (2), do three parallel samples and blank; Hatched 1.5 hours for 37 ℃, remove liquid,, wash 4 times, dry liquid with lavation buffer solution (the PBS damping fluid that contains the pH7.2 of 0.05%Tween-20) detersive enzyme target, standby; 2. the phycocyanin and phycoerythrin beta subunit fluorescin in conjunction with PCB of getting biotin labeled goat anti-rabbit immunoglobulin antibody and labelled streptavidin mixes and adds in the dilution buffer liquid (the PBS damping fluid that contains the pH7.2 that contains 0.05%Tween-20 of 0.1% bovine serum albumin), the final concentration of two components all is respectively 10 μ g/mL, get above-mentioned solution 150 μ L and join in the parallel sample and blank hole that 1. step prepare, hatched 1.5 hours for 37 ℃;

The third: the phycocyanin and phycoerythrin beta subunit fluorescent protein in conjunction with phycocyanobilin PCB of getting the rabbit polyclonal antibody of carcinomebryonic antigen CEA, biotin labeled goat anti-rabbit immunoglobulin antibody and labelled streptavidin mixes and adds in the dilution buffer liquid (the PBS damping fluid that contains the pH7.2 that contains 0.05%Tween-20 of 0.1% bovine serum albumin), the rabbit polyclonal antibody final concentration of carcinomebryonic antigen CEA is 2 μ g/mL, and the final concentration of two other component all is respectively 10 μ g/mL; Get above-mentioned solution 150 μ L and join in the parallel sample and blank hole that 1. step prepare, hatched 1.5 hours for 37 ℃;

(4) step (3) gained enzyme plate is removed liquid, 0.05M potassiumphosphate, 0.5M NaCl damping fluid detersive enzyme target with pH7.2, wash 4 times, after drying liquid, every hole adds 0.05M potassiumphosphate, the 0.5M NaCl damping fluid of 150 μ L pH7.2, places the microwell plate fluorescence detector to detect fluorescent value.

Embodiment 6

(1) mouse monoclonal antibody of carcinomebryonic antigen CEA is cushioned liquid (0.05M, pH9.6 carbonate buffer solution) with bag and is diluted to 2 μ g/mL, get 150 μ L and add black 96 hole enzyme plates, two groups of parallel samples respectively 4 ℃ and 37 ℃ of bags by 12 to 20 hours; Remove to wrap antibody,, wash 4 times, dry liquid with lavation buffer solution (the PBS damping fluid that contains the pH7.2 of 0.05%Tween-20) detersive enzyme target by usefulness; Every hole adds sealing damping fluid (the PBS damping fluid that contains the pH7.2 that contains 0.05%Tween-20 of 1% bovine serum albumin) 300 μ L, and 37 ℃ were sealed 30 minutes; The liquid of falling the deblocking with lavation buffer solution (the PBS damping fluid that contains the pH7.2 of 0.05%Tween-20) detersive enzyme target, washs 4 times, dries liquid, and is standby;

(2) get carcinomebryonic antigen CEA, be diluted to 50ng/mL with dilution buffer liquid (the PBS damping fluid that contains the pH7.2 that contains 0.05%Tween-20 of 0.1% bovine serum albumin), getting 150 μ L joins in the ready enzyme plate micropore of step (1), make six parallel samples, other gets two holes and directly adds dilution buffer liquid as blank, hatched 1.5 hours for 37 ℃, remove liquid, with lavation buffer solution (the PBS damping fluid that contains the pH7.2 of 0.05%Tween-20) detersive enzyme target, wash 4 times, dry liquid, standby;

(3) each is organized parallel sample and finishes according to the following steps respectively:

1. get the rabbit polyclonal antibody of carcinomebryonic antigen CEA, be diluted to 2 μ g/mL with dilution buffer liquid (the PBS damping fluid that contains the pH7.2 that contains 0.05%Tween-20 of 0.1% bovine serum albumin), get 150 μ L and join in the ready enzyme plate micropore of step (2), do three parallel samples and blank; Hatched 1.5 hours for 37 ℃, remove liquid,, wash 4 times, dry liquid with lavation buffer solution (the PBS damping fluid that contains the pH7.2 of 0.05%Tween-20) detersive enzyme target, standby;

2. the phycocyanin and phycoerythrin beta subunit fluorescin in conjunction with PCB of getting biotin labeled goat anti-rabbit immunoglobulin antibody and labelled streptavidin mixes and adds in the dilution buffer liquid (the PBS damping fluid that contains the pH7.2 that contains 0.05%Tween-20 of 0.1% bovine serum albumin), the final concentration of two components all is respectively 10 μ g/mL, get above-mentioned solution 150 μ L and join in the parallel sample and blank hole that 1. step prepare, hatched 1.5 hours for 37 ℃;

(4) step (3) gained enzyme plate is removed liquid, 0.05M potassiumphosphate, 0.5M NaCl damping fluid detersive enzyme target with pH7.2, wash 4 times, after drying liquid, every hole adds 0.05M potassiumphosphate, the 0.5M NaCl damping fluid of 150 μ L pH7.2, places the microwell plate fluorescence detector to detect fluorescent value.

Embodiment 7

(1) mouse monoclonal antibody of carcinomebryonic antigen CEA is cushioned liquid (0.05M, pH9.6 carbonate buffer solution) with bag and is diluted to 2 μ g/mL, get 150 μ L and add black 96 hole enzyme plates, 4 ℃ of bags were by 12 to 20 hours; Remove to wrap antibody,, wash 4 times, dry liquid with lavation buffer solution (the PBS damping fluid that contains the pH7.2 of 0.05%Tween-20) detersive enzyme target by usefulness; Every hole adds sealing damping fluid (the PBS damping fluid that contains the pH7.2 that contains 0.05%Tween-20 of 1% bovine serum albumin) 300 μ L, and 37 ℃ were sealed 30 minutes; The liquid of falling the deblocking with lavation buffer solution (the PBS damping fluid that contains the pH7.2 of 0.05%Tween-20) detersive enzyme target, washs 4 times, dries liquid, and is standby;

(2) get carcinomebryonic antigen CEA, be diluted to 50ng/mL with dilution buffer liquid (the PBS damping fluid that contains the pH7.2 that contains 0.05%Tween-20 of 0.1% bovine serum albumin), getting 150 μ L joins in the ready enzyme plate micropore of step (1), make 27 parallel samples, other gets nine holes and directly adds dilution buffer liquid as blank, hatched 1.5 hours for 37 ℃, remove liquid, with lavation buffer solution (the PBS damping fluid that contains the pH7.2 of 0.05%Tween-20) detersive enzyme target, wash 4 times, dry liquid, standby;

(3) each is organized parallel sample and finishes according to the following steps respectively:

1. get the rabbit polyclonal antibody of carcinomebryonic antigen CEA, be diluted to 2 μ g/mL with dilution buffer liquid (the PBS damping fluid that contains the pH7.2 that contains 0.05%Tween-20 of 0.1% bovine serum albumin), get 150 μ L and join in the ready enzyme plate micropore of step (2), do three parallel samples and blank; Hatched 1.5 hours for 37 ℃, remove liquid,, wash 4 times, dry liquid with lavation buffer solution (the PBS damping fluid that contains the pH7.2 of 0.05%Tween-20) detersive enzyme target, standby;

2. the phycocyanin and phycoerythrin beta subunit fluorescent protein in conjunction with phycocyanobilin PCB of getting biotin labeled goat anti-rabbit immunoglobulin antibody and labelled streptavidin mixes and adds in the dilution buffer liquid (the PBS damping fluid that contains the pH7.2 that contains 0.05%Tween-20 of 0.1% bovine serum albumin), the final concentration of two components is respectively: 5 μ g/mL, 5 μ g/mL, 5 μ g/mL, 10 μ g/mL, 5 μ g/mL, 20 μ g/mL, 10 μ g/mL, 5 μ g/mL, 10 μ g/mL, 10 μ g/mL, 10 μ g/mL, 20 μ g/mL, 20 μ g/mL, 5 μ g/mL, 20 μ g/mL, 10 μ g/mL, 20 μ g/mL, 20 μ g/mL, getting above-mentioned solution 150 μ L joins respectively in the parallel sample and blank hole that 1. step prepare, the corresponding one group of parallel sample of each concentration was hatched 1.5 hours for 37 ℃;

(4) step (3) gained enzyme plate is removed liquid, 0.05M potassiumphosphate, 0.5M NaCl damping fluid detersive enzyme target with pH7.2, wash 4 times, after drying liquid, every hole adds 0.05M potassiumphosphate, the 0.5M NaCl damping fluid of 150 μ L pH7.2, places the microwell plate fluorescence detector to detect fluorescent value.

Embodiment 8

(1) mouse monoclonal antibody of carcinomebryonic antigen CEA is cushioned liquid (0.05M, pH9.6 carbonate buffer solution) with bag and is diluted to 2 μ g/mL, get 150 μ L and add black 96 hole enzyme plates, 4 ℃ of bags were by 12 to 20 hours; Remove to wrap antibody,, wash 4 times, dry liquid with lavation buffer solution (the PBS damping fluid that contains the pH7.2 of 0.05%Tween-20) detersive enzyme target by usefulness; Every hole adds sealing damping fluid (the PBS damping fluid that contains the pH7.2 that contains 0.05%Tween-20 of 1% bovine serum albumin) 300 μ L, and 37 ℃ were sealed 30 minutes; The liquid of falling the deblocking with lavation buffer solution (the PBS damping fluid that contains the pH7.2 of 0.05%Tween-20) detersive enzyme target, washs 4 times, dries liquid, and is standby;

(2) get carcinomebryonic antigen CEA, be diluted to 50ng/mL with dilution buffer liquid (the PBS damping fluid that contains the pH7.2 that contains 0.05%Tween-20 of 0.1% bovine serum albumin), getting 150 μ L joins in the ready enzyme plate micropore of step (1), make six parallel samples, other gets two holes and directly adds dilution buffer liquid as blank, hatched 1.5 hours for 37 ℃, remove liquid, with lavation buffer solution (the PBS damping fluid that contains the pH7.2 of 0.05%Tween-20) detersive enzyme target, wash 4 times, dry liquid, standby;

(3) each is organized parallel sample and finishes according to the following steps respectively:

1. get the rabbit polyclonal antibody of carcinomebryonic antigen CEA, be diluted to 2 μ g/mL with dilution buffer liquid (the PBS damping fluid that contains the pH7.2 that contains 0.05%Tween-20 of 0.1% bovine serum albumin), get 150 μ L and join in the ready enzyme plate micropore of step (2), do three parallel samples and blank; Hatched 1.5 hours for 37 ℃, remove liquid,, wash 4 times, dry liquid with lavation buffer solution (the PBS damping fluid that contains the pH7.2 of 0.05%Tween-20) detersive enzyme target, standby;

2. get and mixing of biotin labeled goat anti-rabbit immunoglobulin antibody and labelled streptavidin add dilution buffer liquid and (contain the PBS damping fluid of the pH7.2 that contains 0.05%Tween-20 of 0.1% bovine serum albumin or the 0.05M potassiumphosphate of pH7.2 in conjunction with the phycocyanin and phycoerythrin beta subunit fluorescent protein of phycocyanobilin PCB, 0.5M the NaCl damping fluid), the final concentration of two components is respectively: biotin labeled goat anti-rabbit immunoglobulin antibody 20 μ g/mL, the phycocyanin and phycoerythrin beta subunit fluorescin 10 μ g/mL in conjunction with PCB of labelled streptavidin, getting above-mentioned solution 150 μ L joins respectively in the parallel sample and blank hole that 1. step prepare, the corresponding one group of parallel sample of each solution was hatched 1.5 hours for 37 ℃;

(4) step (3) gained enzyme plate is removed liquid, 0.05M potassiumphosphate, 0.5M NaCl damping fluid detersive enzyme target with pH7.2, wash 4 times, after drying liquid, every hole adds 0.05M potassiumphosphate, the 0.5M NaCl damping fluid of 150 μ L pH7.2, places the microwell plate fluorescence detector to detect fluorescent value.

Embodiment 9

(1) mouse monoclonal antibody of carcinomebryonic antigen CEA is cushioned liquid (0.05M, pH9.6 carbonate buffer solution) with bag and is diluted to 2 μ g/mL, get 150 μ L and add black 96 hole enzyme plates, 4 ℃ of bags were by 12 to 20 hours; Remove to wrap antibody,, wash 4 times, dry liquid with lavation buffer solution (the PBS damping fluid that contains the pH7.2 of 0.05%Tween-20) detersive enzyme target by usefulness; Every hole adds sealing damping fluid (the PBS damping fluid that contains the pH7.2 that contains 0.05%Tween-20 of 1% bovine serum albumin) 300 μ L, and 37 ℃ were sealed 30 minutes; The liquid of falling the deblocking with lavation buffer solution (the PBS damping fluid that contains the pH7.2 of 0.05%Tween-20) detersive enzyme target, washs 4 times, dries liquid, and is standby;

(2) get carcinomebryonic antigen CEA, be diluted to 50ng/mL with dilution buffer liquid (the PBS damping fluid that contains the pH7.2 that contains 0.05%Tween-20 of 0.1% bovine serum albumin), getting 150 μ L joins in the ready enzyme plate micropore of step (1), make nine parallel samples, other gets three holes and directly adds dilution buffer liquid as blank, hatched 1.5 hours for 37 ℃, remove liquid, with lavation buffer solution (the PBS damping fluid that contains the pH7.2 of 0.05%Tween-20) detersive enzyme target, wash 4 times, dry liquid, standby;

(3) each is organized parallel sample and finishes according to the following steps respectively:

1. get the rabbit polyclonal antibody of carcinomebryonic antigen CEA, be diluted to 2 μ g/mL with dilution buffer liquid (the PBS damping fluid that contains the pH7.2 that contains 0.05%Tween-20 of 0.1% bovine serum albumin), get 150 μ L and join in the ready enzyme plate micropore of step (2), do three parallel samples and blank; Hatched 1.5 hours for 37 ℃, remove liquid,, wash 4 times, dry liquid with lavation buffer solution (the PBS damping fluid that contains the pH7.2 of 0.05%Tween-20) detersive enzyme target, standby;

2. the phycocyanin and phycoerythrin beta subunit fluorescent protein in conjunction with phycocyanobilin PCB of getting biotin labeled goat anti-rabbit immunoglobulin antibody and labelled streptavidin mixes and adds in the dilution buffer liquid (the PBS damping fluid that contains the pH7.2 that contains 0.05%Tween-20 of 0.1% bovine serum albumin), the final concentration of two components is respectively: biotin labeled goat anti-rabbit immunoglobulin antibody 20 μ g/mL, the phycocyanin and phycoerythrin beta subunit fluorescin 10 μ g/mL in conjunction with PCB of labelled streptavidin, get above-mentioned solution 150 μ L and join respectively in the parallel sample and blank hole that 1. step prepare, three groups of parallel samples respectively 37 ℃ hatched 0.5 hour, 1 hour, 1.5 hour;

(4) step (3) gained enzyme plate is removed liquid, 0.05M potassiumphosphate, 0.5M NaCl damping fluid detersive enzyme target with pH7.2, wash 4 times, after drying liquid, every hole adds 0.05M potassiumphosphate, the 0.5M NaCl damping fluid of 150 μ L pH7.2, places the microwell plate fluorescence detector to detect fluorescent value.

Embodiment 10

(1) mouse monoclonal antibody of carcinomebryonic antigen CEA is cushioned liquid (0.05M, pH9.6 carbonate buffer solution) with bag and is diluted to 2 μ g/mL, get 150 μ L and add black 96 hole enzyme plates, 4 ℃ of bags were by 12 to 20 hours; Remove to wrap antibody,, wash 4 times, dry liquid with lavation buffer solution (the PBS damping fluid that contains the pH7.2 of 0.05%Tween-20) detersive enzyme target by usefulness; Every hole adds sealing damping fluid (the PBS damping fluid that contains the pH7.2 that contains 0.05%Tween-20 of 1% bovine serum albumin) 300 μ L, and 37 ℃ were sealed 30 minutes; The liquid of falling the deblocking with lavation buffer solution (the PBS damping fluid that contains the pH7.2 of 0.05%Tween-20) detersive enzyme target, washs 4 times, dries liquid, and is standby;

(2) get carcinomebryonic antigen CEA, be diluted to 50ng/mL with dilution buffer liquid (the PBS damping fluid that contains the pH7.2 that contains 0.05%Tween-20 of 0.1% bovine serum albumin), 25ng/mL, 12.5ng/mL, 6.25ng/mL, 3.125ng/mL, 1.562ng/mL, 0.781ng/mL, 0.390ng/mL, getting 150 μ L respectively joins in the ready enzyme plate micropore of step (1), each concentration is made two parallel samples, other gets two holes and directly adds dilution buffer liquid as blank, hatched 1.5 hours for 37 ℃, remove liquid, with lavation buffer solution (the PBS damping fluid that contains the pH7.2 of 0.05%Tween-20) detersive enzyme target, wash 4 times, dry liquid, standby;

(3) finish according to the following steps:

1. get the rabbit polyclonal antibody of carcinomebryonic antigen CEA, be diluted to 2 μ g/mL with dilution buffer liquid (the PBS damping fluid that contains the pH7.2 that contains 0.05%Tween-20 of 0.1% bovine serum albumin), get 150 μ L and join in the ready enzyme plate micropore of step (2), do three parallel samples and blank; Hatched 1.5 hours for 37 ℃, remove liquid,, wash 4 times, dry liquid with lavation buffer solution (the PBS damping fluid that contains the pH7.2 of 0.05%Tween-20) detersive enzyme target, standby;

2. the phycocyanin and phycoerythrin beta subunit fluorescent protein in conjunction with phycocyanobilin PCB of getting biotin labeled goat anti-rabbit immunoglobulin antibody and labelled streptavidin mixes and adds in the dilution buffer liquid (the PBS damping fluid that contains the pH7.2 that contains 0.05%Tween-20 of 0.1% bovine serum albumin), the final concentration of two components is respectively: biotin labeled goat anti-rabbit immunoglobulin antibody 20 μ g/mL, the phycocyanin and phycoerythrin beta subunit fluorescin 10 μ g/mL in conjunction with PCB of labelled streptavidin, get above-mentioned solution 150 μ L and join respectively in the parallel sample and blank hole that 1. step prepare, hatched 1.5 hours for 37 ℃;

(4) step (3) gained enzyme plate is removed liquid, 0.05M potassiumphosphate, 0.5M NaCl damping fluid detersive enzyme target with pH7.2, wash 4 times, after drying liquid, every hole adds 0.05M potassiumphosphate, the 0.5M NaCl damping fluid of 150 μ L pH7.2, places the microwell plate fluorescence detector to detect fluorescent value.

In the above-mentioned albumen, the residing position of His-tag mark and marked by streptavidin does not limit and is positioned at albumen n end, is positioned at other position only otherwise the activity that influences the phycocyanin and phycoerythrin beta protein subunit also can be implemented the present invention equally.Simultaneously, except 82 and 153 halfcystines,,, also can implement the present invention equally if phycocyanin and phycoerythrin beta protein subunit photoluminescent property is not had considerable influence to all the other sudden changes that phycocyanin and phycoerythrin beta subunit aminoacid sequence carries out; N end or C end to phycocyanin and phycoerythrin beta subunit aminoacid sequence carry out excalation, also can not produce considerable influence to phycocyanin and phycoerythrin beta protein subunit photoluminescent property, also can implement the present invention equally.

Above-mentioned detection method is applicable to utilizes various different antibodies or antigen, different solid phase carrier, fluoroscopic examination instrument to detect soluble antigen or antibody.But because the antibody type that may relate to and various, the phycobiliprotein kind is also more, the phycocyanin and phycoerythrin beta subunit in conjunction with PCB (82 cysteine mutation are Serine) that the present invention only chooses carcinomebryonic antigen and a kind of mouse resource monoclonal antibody thereof and rabbit source polyclonal antibody, marked by streptavidin is example, be solid phase carrier with 96 orifice plates, utilize the microwell plate fluorescence detector, the present invention is illustrated.Those skilled in the art can be according to above-mentioned disclosed content, and material other materials and instrument are implemented the present invention.

Claims (17)

1. phycocyanin and phycoerythrin beta subunit fluorescent protein in conjunction with phycocyanobilin PCB is characterized in that: phycocyanobilin PCB is combined in 201 of reorganization phycocyanin and phycoerythrin beta subunits by thioether bond, is equivalent to 153 of original phycocyanin and phycoerythrin beta subunit.

2. the phycocyanin and phycoerythrin beta subunit fluorescent protein in conjunction with phycocyanobilin PCB according to claim 1 is characterized in that: described proteinic sequence is a sequence 1.

3. the phycocyanin and phycoerythrin beta subunit fluorescent protein in conjunction with phycocyanobilin PCB according to claim 1 and 2 is characterized in that: described protein sequence N end has the His-tag mark, but the His-tag mark is not limited to the N end.

4. according to claim 1,2 or 3 described phycocyanin and phycoerythrin beta subunit fluorescent proteins in conjunction with phycocyanobilin PCB, it is characterized in that: describedly be meant that in conjunction with phycocyanobilin PCB phycocyanobilin PCB is combined in 201 by thioether bond, be equivalent to 153 of original phycocyanin and phycoerythrin beta subunit.

5. phycocyanin and phycoerythrin beta subunit fluorescent protein mutant in conjunction with phycocyanobilin PCB, it is characterized in that: the phycocyanin and phycoerythrin beta subunit coding gene is cloned in expression plasmid, utilize gene engineering method that it is suddenlyd change, expression obtains phycocyanin and phycoerythrin beta subunit mutant, to except that in conjunction with pigment 201 all sites suddenly change and generally can bigger influence not arranged property of protein, belong to same proteinoid, this albumen is carried out excalation, property of protein is not had considerable influence, belong to same proteinoid yet.

6. the phycocyanin and phycoerythrin beta subunit fluorescent protein mutant in conjunction with phycocyanobilin PCB according to claim 5, it is characterized in that: the phycocyanin and phycoerythrin beta subunit coding gene is cloned in expression plasmid, utilize gene engineering method that its 130th (being equivalent to the 82nd of original phycocyanin and phycoerythrin beta subunit) cysteine residues sported serine residue, can express obtaining phycocyanin and phycoerythrin beta subunit mutant.

7. the mutant of the phycocyanin and phycoerythrin beta subunit fluorescent protein in conjunction with phycocyanobilin PCB according to claim 6, it is characterized in that: the sequence of described mutant is a sequence 2.

8. according to claim 5,6 or 7 described phycocyanin and phycoerythrin beta subunit fluorescent protein mutant in conjunction with phycocyanobilin PCB, it is characterized in that: describedly be meant that in conjunction with phycocyanobilin PCB phycocyanobilin PCB is combined in 201 by thioether bond, be equivalent to 153 of original phycocyanin and phycoerythrin beta subunit.

9. the phycocyanin and phycoerythrin beta subunit fluorescent protein in conjunction with phycocyanobilin PCB of a marked by streptavidin, it is characterized in that: Streptavidin encoding gene and phycocyanin and phycoerythrin beta subunit coding gene are spliced rear clone in expression plasmid, express the fusion rotein that obtains Streptavidin and phycocyanin and phycoerythrin beta subunit.

10. the phycocyanin and phycoerythrin beta subunit fluorescent protein in conjunction with phycocyanobilin PCB of marked by streptavidin according to claim 9, it is characterized in that: described proteinic sequence is a sequence 3.

11. the phycocyanin and phycoerythrin beta subunit fluorescent protein in conjunction with phycocyanobilin PCB according to claim 9 or 10 described marked by streptavidin is characterized in that: Streptavidin still is not limited to hold at N at the N of PecB end.

12. phycocyanin and phycoerythrin beta subunit fluorescent protein in conjunction with phycocyanobilin PCB according to claim 9,10,11 described marked by streptavidin, it is characterized in that: describedly be meant that in conjunction with phycocyanobilin PCB phycocyanobilin PCB is combined in 329 by thioether bond, be equivalent to 153 of original phycocyanin and phycoerythrin beta subunit.

13. the phycocyanin and phycoerythrin beta subunit fluorescent protein mutant in conjunction with phycocyanobilin PCB of a marked by streptavidin, it is characterized in that: Streptavidin encoding gene and phycocyanin and phycoerythrin beta subunit coding gene are spliced rear clone in expression plasmid, utilize gene engineering method that its 258th (being equivalent to the 82nd of original phycocyanin and phycoerythrin beta subunit) cysteine residues sported serine residue, express the mutant of the fusion rotein that obtains Streptavidin and phycocyanin and phycoerythrin beta subunit.

14. the phycocyanin and phycoerythrin beta subunit fluorescent protein mutant in conjunction with phycocyanobilin PCB of marked by streptavidin according to claim 13, it is characterized in that: described proteinic sequence is a sequence 4.

15. phycocyanin and phycoerythrin beta subunit fluorescent protein mutant in conjunction with phycocyanobilin PCB according to claim 13 or 14 described marked by streptavidin, it is characterized in that: describedly be meant that in conjunction with phycocyanobilin PCB phycocyanobilin PCB is combined in 329 by thioether bond, be equivalent to 153 of original phycocyanin and phycoerythrin beta subunit.

16. the application in conjunction with the phycocyanin and phycoerythrin beta subunit fluorescent protein of phycocyanobilin PCB is characterized in that: that utilizes marked by streptavidin detects the immunodetection of soluble antigen or antibody in conjunction with the phycocyanin and phycoerythrin beta subunit fluorescent protein of phycocyanobilin PCB.

17. the application of the phycocyanin and phycoerythrin beta subunit fluorescent protein in conjunction with phycocyanobilin PCB according to claim 16, it is characterized in that: described immunologic detection method may further comprise the steps:

(1) can combine with solid phase and can discern antigenic first antibody bag by on the solid phase, confining liquid seals not binding antibody part, and is standby;

(2) the fluorescence phycobiliprotein with determined antigen, the anti-antibody of discerning the mark vitamin H of antigenic second antibody, identification second antibody, mark Streptomycin sulphate avidin adds in the solid phase fully reaction by certain step;

(3) utilize microwell plate fluorescence detector or other corresponding instrument detecting fluorescence, analytical results.

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