CN101759786A - Phycocyanin alpha subunits fluorescent protein combined with phycocyanobilin PCB and application thereof - Google Patents

Abstract

The invention relates to phycocyanin alpha subunits fluorescent protein combined with phycocyanobilin PCB and fusion protein formed by phycocyanin alpha subunits fluorescent protein and streptavidin, the sequences include sequence 1 and sequence 2; furthermore, the invention discloses a method of the fluorescent protein fused with streptavidin for fluorescent immunological detection directly; phycocyanin alpha subunit conserved cysteine residue can be combined with phycocyanobilin PCB by a thioether bond, and the alpha subunit of the fluorescent phycocyanin can be obtained by utilizing escherichia coli through genetic engineering, the spectroscopy of the protein is completely different from that of natural phycocyanin; 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.

Description

Phycocyanins, C-α subunit fluorescent protein and application thereof in conjunction with phycocyanobilin PCB

Technical field

The present invention the present invention relates to a kind of Phycocyanins, C-alpha subunit fluorescent protein and application thereof in conjunction with phycocyanobilin PCB, belong to chromoprotein material field in the biotechnology, be specifically related in conjunction with the Phycocyanins, C-alpha subunit fluorescent protein of phycocyanobilin PCB and the fusion rotein that forms with Streptavidin thereof, and utilize this fusion rotein to detect soluble antigen or detection of antibodies method.

Background technology

Phycobiliprotein (phyeobiliprotein) 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 Phycocyanins, C-, and isolate its alpha and beta subunit, 85 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 CPC can be produced (Tooley AJ by the bacillus coli gene engineering bacteria, Cai YA, GlazerAN.Biosynthesis ofa fluorescent cyanobacterial C-phycocyanin holo-alpha subunit in a heterologous host[J] .Proc NatlAcad Sci USA, 2001,98 (19): 10560～10565).Utilize genetically engineered, can obtain 85 fluorescins of Phycocyanins, C-alpha subunit in conjunction with phycocyanobilin PCB, its spectral quality different with natural Phycocyanins, C-(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 Phycocyanins, C-subunit apoprotein gene splicing rear clone in expression vector, can obtain the fusion rotein of Streptavidin and Phycocyanins, C-subunit apoprotein at e. coli expression, directly realize being connected of Streptavidin and Phycocyanins, C-subunit apoprotein; By phycobiliprotein alpha lyase energy catalysis phycocyanobilin and Phycocyanins, C-subunit apoprotein and homologous protein covalent attachment thereof, thereby generation has the phycocyanin fluorescence protein in conjunction with PCB (its spectrum as shown in Figure 3) 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 the aminoacid sequence of the fusion rotein that a class forms in conjunction with the Phycocyanins, C-alpha subunit of phycocyanobilin PCB and with Streptavidin, 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 Phycocyanins, C-alpha subunit in conjunction with phycocyanobilin PCB of this class marked by streptavidin have with natural algae in the different structure of Phycocyanins, C-alpha 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 Phycocyanins, C-alpha 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:

Phycocyanins, C-alpha subunit coding gene is cloned in expression plasmid, utilize Phycocyanins, C-alpha lyase expression plasmid and phycocyanobilin PCB synthetic enzyme plasmid, can in intestinal bacteria, synthesize Phycocyanins, C-alpha 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.Phycocyanins, C-alpha subunit coding gene is cloned in expression plasmid, utilize gene engineering method that it is suddenlyd change, can express and obtain Phycocyanins, C-alpha subunit mutant, to generally can bigger influence not being arranged to property of protein except that suddenling change in conjunction with all sites 133 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.

Protein sequence in conjunction with the Phycocyanins, C-alpha subunit fluorescent protein of phycocyanobilin PCB is a sequence 1:

The 133rd halfcystine C of sequence 1:(is the binding site of phycocyanobilin PCB)

MHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADGMVKTPITEAIAAADTQGRFLGNTELQSARGRYERAAASLEAARGLTSNAQRLIDGATQAVYQKFPYTTQTPGPQFAADSRGKSKCARDVGHYLRIITYSLVAGGTGPLDEYLIAGLAEINSTFDLSPSWYVEALKHIKANHGLSGQAANEANTYIDYAINALS

Describedly be meant that in conjunction with phycocyanobilin PCB phycocyanobilin PCB is combined in 133 by thioether bond, be equivalent to 85 of original Phycocyanins, C-alpha subunit.

Streptavidin encoding gene and Phycocyanins, C-alpha subunit coding gene are spliced rear clone in expression plasmid, utilize Phycocyanins, C-alpha lyase expression plasmid and phycocyanobilin PCB synthetic enzyme plasmid, can in intestinal bacteria, synthesize Phycocyanins, C-alpha subunit fluorescent protein, and indicate its pigment binding site in conjunction with PCB.

Above-mentioned aminoacid sequence is a sequence 2:

The 261st halfcystine C of sequence 2:(is the binding site of phycocyanobilin PCB)

MHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASGSGTDDDDKAMADGMVKTPITEAIAAADTQGRFLGNTELQSARGRYERAAASLEAARGLTSNAQRLIDGATQAVYQKFPYTTQTPGPQFAADSRGKSKCARDVGHYLRIITYSLVAGGTGPLDEYLIAGLAEINSTFDLSPSWYVEALKHIKANHGLSGQAANEANTYIDYAINALS

A kind of Phycocyanins, C-alpha subunit fluorescent protein in conjunction with phycocyanobilin PCB of marked by streptavidin, Streptavidin still is not limited to hold at N at the N of CpcA end.

Marked by streptavidin be meant that in conjunction with phycocyanobilin PCB phycocyanobilin PCB is combined in 261 by thioether bond, be equivalent to 85 of original Phycocyanins, C-alpha subunit.

Application in conjunction with the Phycocyanins, C-alpha subunit fluorescent protein of phycocyanobilin PCB:

Utilize the fluorescence phycobiliprotein detection soluble antigen of marked by streptavidin or the immunodetection of antibody.

The fluorescence phycobiliprotein of described marked by streptavidin is the Phycocyanins, C-alpha subunit proteinoid in conjunction with phycocyanobilin PCB of marked by streptavidin.

The fluorescence phycobiliprotein detection soluble antigen of marked by streptavidin or the method for antibody utilized provided by the invention, its step is as follows:

(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 Phycocyanins, C-alpha subunit of PCB, and because this albumen is single subunit constitutes, have more homogeneity than the natural Phycocyanins, C-of a plurality of subunits of tool; Compare and have and the diverse spectral quality of Phycocyanins, C-alpha 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: the absorption of natural Phycocyanins, C-and fluorescence spectrum figure, wherein solid line is an absorption spectrum, dotted line is a fluorescence spectrum;

Fig. 2: 85 cysteine residues are in conjunction with the absorption and the fluorescence spectrum figure of the Phycocyanins, C-alpha subunit fluorescin of PCB, and wherein solid line is an absorption spectrum, and dotted line is a fluorescence spectrum;

Fig. 3: 85 cysteine residues of marked by streptavidin are in conjunction with the absorption and the fluorescence spectrum figure of the Phycocyanins, C-alpha subunit fluorescin of PCB, and wherein solid line is an absorption spectrum, and dotted line is a fluorescence spectrum.

The protein amino acid sequence annex:

The 133rd halfcystine C of sequence 1:(is the binding site of phycocyanobilin PCB)

MHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADGMVKTPITEAIAAADTQGRFLGNTELQSARGRYERAAASLEAARGLTSNAQRLIDGATQAVYQKFPYTTQTPGPQFAADSRGKSKCARDVGHYLRIITYSLVAGGTGPLDEYLIAGLAEINSTFDLSPSWYVEALKHIKANHGLSGQAANEANTYIDYAINALS

The 261st halfcystine C of sequence 2:(is the binding site of phycocyanobilin PCB)

MHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASGSGTDDDDKAMADGMVKTPITEAIAAADTQGRFLGNTELQSARGRYERAAASLEAARGLTSNAQRLIDGATQAVYQKFPYTTQTPGPQFAADSRGKSKCARDVGHYLRIITYSLVAGGTGPLDEYLIAGLAEINSTFDLSPSWYVEALKHIKANHGLSGQAANEANTYIDYAINALS

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, Phycocyanins, C-alpha subunit coding gene is cloned in expression plasmid, express obtaining Phycocyanins, C-alpha 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 133rd (being equivalent to the 85th of original Phycocyanins, C-alpha subunit) cysteine residues by thioether is strong.Its spectrum as shown in Figure 2, absorption peak is 619nm, fluorescence emission peak is 641nm.

Embodiment 2

Protein amino acid sequence is shown in sequence 2, Streptavidin encoding gene and Phycocyanins, C-alpha subunit coding gene are spliced rear clone in expression plasmid, the fusion rotein of Streptavidin that expression obtains and Phycocyanins, C-alpha subunit is directly realized the mark of Streptavidin to Phycocyanins, C-alpha 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 261st (being equivalent to the 85th of original Phycocyanins, C-alpha subunit) cysteine residues by thioether is strong.Its spectrum as shown in Figure 3, absorption peak is 619nm, fluorescence emission peak is 641nm.

Embodiment 3

(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 Phycocyanins, C-alpha 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 Phycocyanins, C-alpha 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 Phycocyanins, C-alpha subunit fluorescin in conjunction with 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 4

(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 Phycocyanins, C-alpha 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 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 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 Phycocyanins, C-alpha 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 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 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, 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 Phycocyanins, C-alpha subunit fluorescin of 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 Phycocyanins, C-alpha 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 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 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 Phycocyanins, C-alpha 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 is respectively: biotin labeled goat anti-rabbit immunoglobulin antibody 20 μ g/mL, the Phycocyanins, C-alpha 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 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), 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 Phycocyanins, C-alpha 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 is respectively: biotin labeled goat anti-rabbit immunoglobulin antibody 20 μ g/mL, the Phycocyanins, C-alpha 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 Phycocyanins, C-alpha protein subunit also can be implemented the present invention equally.Simultaneously, except 85 halfcystines,,, also can implement the present invention equally if Phycocyanins, C-alpha protein subunit photoluminescent property is not had considerable influence to all the other sudden changes that Phycocyanins, C-alpha subunit aminoacid sequence carries out; N end or C end to Phycocyanins, C-alpha subunit aminoacid sequence carry out excalation, also can not produce considerable influence to Phycocyanins, C-alpha 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 Phycocyanins, C-alpha subunit in conjunction with PCB 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 an 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 (11)

1. the Phycocyanins, C-alpha subunit fluorescent protein in conjunction with phycocyanobilin PCB is characterized in that: Phycocyanins, C-alpha subunit coding gene is cloned in expression plasmid, express obtaining Phycocyanins, C-alpha subunit.

2. the Phycocyanins, C-alpha subunit fluorescent protein in conjunction with phycocyanobilin PCB according to claim 1, it is characterized in that: described proteinic sequence is a sequence 1.

3. the Phycocyanins, C-alpha 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 Phycocyanins, C-alpha 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 133 by thioether bond, be equivalent to 85 of original Phycocyanins, C-alpha subunit.

5. Phycocyanins, C-alpha subunit fluorescent protein mutant in conjunction with phycocyanobilin PCB, it is characterized in that: Phycocyanins, C-alpha subunit coding gene is cloned in expression plasmid, utilize gene engineering method that it is suddenlyd change, can express and obtain Phycocyanins, C-alpha subunit mutant, to generally can bigger influence not being arranged to property of protein except that suddenling change in conjunction with all sites 133 of pigment, 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 Phycocyanins, C-alpha subunit fluorescent protein in conjunction with phycocyanobilin PCB of a marked by streptavidin, it is characterized in that: Streptavidin encoding gene and Phycocyanins, C-alpha subunit coding gene are spliced rear clone in expression plasmid, express the fusion rotein that obtains Streptavidin and Phycocyanins, C-alpha subunit.

7. the Phycocyanins, C-alpha subunit fluorescent protein in conjunction with phycocyanobilin PCB of marked by streptavidin according to claim 6, it is characterized in that: described proteinic sequence is a sequence 2.

8. the Phycocyanins, C-alpha subunit fluorescent protein in conjunction with phycocyanobilin PCB of a kind of marked by streptavidin according to claim 6 is characterized in that: Streptavidin is at the N of CpcA end, but is not limited to hold at N.

9. according to the Phycocyanins, C-alpha subunit fluorescent protein in conjunction with phycocyanobilin PCB of claim 6,7 or 8 described marked by streptavidin, it is characterized in that: describedly be meant that in conjunction with phycocyanobilin PCB phycocyanobilin PCB is combined in 261 by thioether bond, be equivalent to 85 of original Phycocyanins, C-alpha subunit.

10. in conjunction with the application of the Phycocyanins, C-alpha subunit fluorescent protein of phycocyanobilin PCB, it is characterized in that: that utilizes marked by streptavidin detects the immunodetection of soluble antigen or antibody in conjunction with the Phycocyanins, C-alpha subunit fluorescent protein of phycocyanobilin PCB.

11. the application of the Phycocyanins, C-alpha subunit fluorescent protein in conjunction with phycocyanobilin PCB according to claim 10, it is characterized in that: described immunodetection 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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