CN105785025A - Kit for detecting tick-borne encephalitis virus infected serum - Google Patents
Kit for detecting tick-borne encephalitis virus infected serum Download PDFInfo
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- CN105785025A CN105785025A CN201410795224.6A CN201410795224A CN105785025A CN 105785025 A CN105785025 A CN 105785025A CN 201410795224 A CN201410795224 A CN 201410795224A CN 105785025 A CN105785025 A CN 105785025A
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Abstract
The invention discloses a kit for detecting tick-borne encephalitis virus infected serum. The kit is a kit 1 or a kit 2. The kit 1 contains tick-borne encephalitis virus antigen; the tick-borne encephalitis virus antigen is a protein represented by 320th-412th amino acids in SEQ ID No.1 in a sequence table; the kit 2 contains a tick-borne encephalitis virus antigen and Ranilla Luciferase fusion protein; and the fusion protein is a protein represented by SEQ ID No.1 in the sequence table. Experiments prove that the kit can be used to detect TBEV infected clinic positive patients' serums and TBEV negative serums.
Description
Technical field
The present invention relates to the test kit detecting tick-brone encephalitis virus infection serum in biomedical sector.
Background technology
Tick-brone encephalitis virus (Tick-borneencephalitisvirus, TBEV), has another name called fores encephalitis virus, belongs to flaviviridae Flavivirus.After TBEV infects, body can cause forest encephalitis, forest encephalitis is a kind of impatient sexually transmitted disease in epidemic disease source to attack central nervous system, with symptoms such as severe headache, periphery sexual relaxation paralysis when acute stage symptom shows as hyperpyrexia, with dysphagia time serious, language system obstacle.After treatment, often leave the sequela such as paralysis, disorder of limb ' s activity, the health of the serious threat people.The Serologic detection that current TBEV infects is but without commercial test kit.
Summary of the invention
The technical problem to be solved is whether the serum how detecting people is infected by tick-brone encephalitis virus.
For solving above-mentioned technical problem, present invention firstly provides luciferase co-immunoprecipitation reagent or test kit 1 that detection tick-brone encephalitis virus infects.
Luciferase co-immunoprecipitation reagent or test kit 1, described reagent or test kit 1 that detection tick-brone encephalitis virus provided by the present invention infects contain the fusion protein being made up of tick-brone encephalitis virus antigen and renilla luciferase；Described tick-brone encephalitis virus antigen name is called EVRP, for following I1) or protein I2):
I1) protein shown in 320-412 amino acids of SEQIDNo.1；
I2) in the aminoacid sequence of the 320-412 position of SEQIDNo.1 through replacement and/or disappearance and/or add that one or several amino acid residue obtains have identical function by I1) derivative protein.
In mentioned reagent or test kit 1, described fusion protein name is called EVRP-Rluc, for following K1) or protein K2):
K1) protein shown in SEQ ID No .1；
K2) in the aminoacid sequence of SEQIDNo.1 through replacement and/or disappearance and/or add that one or several amino acid residue obtains have identical function by K1) derivative protein.
Wherein, SEQIDNo.1 is made up of 412 aminoacid, the 320-412 amino acids of SEQIDNo.1 is the aminoacid sequence of described tick-brone encephalitis virus antigen, the aminoacid sequence that 1-311 amino acids is renilla luciferase of SEQIDNo.1, the aminoacid sequence that 312-319 amino acids is connection peptides of SEQIDNo.1.
In mentioned reagent or test kit 1, described fusion protein is according to following S1) and method S2) prepare:
S1) encoding gene of described fusion protein is imported in eukaryotic cell, obtain reconstitution cell；
S2) albumen expressing described reconstitution cell obtains described fusion protein；
The encoding gene of described fusion protein is following C1) or C2) or C3) shown in nucleic acid molecules:
C1) nucleic acid molecules shown in SEQ ID No .2；
C2) nucleotide sequence and C1) limited has 75% or more than 75% homogeneity, and the cDNA molecule of coding identical function albumen or genomic DNA molecule；
C3) under strict conditions with C1) nucleotide sequence hybridization that limits, and the cDNA molecule of coding identical function albumen or genomic DNA molecule.
Wherein, SEQIDNo.2 is made up of 1236 nucleotide, and encoding amino acid sequence is the protein of SEQIDNo.1.The tick-brone encephalitis virus antigen shown in 320-412 amino acids of the 958-1236 position nucleotide coding SEQIDNo.1 of SEQIDNo.2；The renilla luciferase shown in 1-311 amino acids of the 1-933 position nucleotide coding SEQIDNo.1 of SEQIDNo.2；The connection peptides shown in 312-319 amino acids of the 934-957 position nucleotide coding SEQIDNo.1 of SEQIDNo.2.
Term used herein " homogeneity " refers to the sequence similarity with native sequence nucleic acid." homogeneity " includes having 75% or higher with the nucleotide sequence shown in the SEQIDNo.2 of the present invention, or 85% or higher, or 90% or higher, or the nucleotide sequence of 95% or higher homogeneity.Homogeneity can with the naked eye or computer software be evaluated.Using computer software, the homogeneity between two or more sequences can use percentage ratio (%) to represent, it can be used to the homogeneity evaluating between correlated series.
In mentioned reagent or test kit 1, described stringent condition is in the solution of 2 × SSC, 0.1%SDS, hybridizes and wash film 2 times at 68 DEG C, each 5min, again in the solution of 0.5 × SSC, 0.1%SDS, hybridizes and wash film 2 times at 68 DEG C, each 15min.
Above-mentioned 75% or more than 75% homogeneity, can be the homogeneity of 80%, 85%, 90% or more than 95%.
For solving above-mentioned technical problem, the preparation method that present invention also offers described fusion protein.
The preparation method of described fusion protein provided by the present invention, including following S1) and step S2):
S1) encoding gene of described fusion protein is imported in eukaryotic cell, obtain reconstitution cell；
S2) albumen expressing described reconstitution cell obtains described fusion protein；
The encoding gene of described fusion protein is following C1) or C2) or C3) shown in nucleic acid molecules:
C1) nucleic acid molecules shown in SEQ ID No .2；
C2) nucleotide sequence and C1) limited has 75% or more than 75% homogeneity, and the cDNA molecule of coding identical function albumen or genomic DNA molecule；
C3) under strict conditions with C1) nucleotide sequence hybridization that limits, and the cDNA molecule of coding identical function albumen or genomic DNA molecule.
Term used herein " homogeneity " refers to the sequence similarity with native sequence nucleic acid." homogeneity " includes having 75% or higher with the nucleotide sequence shown in the SEQIDNo.2 of the present invention, or 85% or higher, or 90% or higher, or the nucleotide sequence of 95% or higher homogeneity.Homogeneity can with the naked eye or computer software be evaluated.Using computer software, the homogeneity between two or more sequences can use percentage ratio (%) to represent, it can be used to the homogeneity evaluating between correlated series.
In the preparation method of above-mentioned fusion protein, described stringent condition is in the solution of 2 × SSC, 0.1%SDS, hybridizes and wash film 2 times at 68 DEG C, each 5min, again in the solution of 0.5 × SSC, 0.1%SDS, hybridizes and wash film 2 times at 68 DEG C, each 15min.
Above-mentioned 75% or more than 75% homogeneity, can be the homogeneity of 80%, 85%, 90% or more than 95%.
In the preparation method of above-mentioned fusion protein, described eukaryotic cell can be COS7 cell.
In the preparation method of above-mentioned fusion protein, the albumen of the described reconstitution cell of described expression obtains described fusion protein and includes cultivating described reconstitution cell and within 4-60 hour, obtain the step of described fusion protein.
In the preparation method of above-mentioned fusion protein, it within described 4-60 hour, can be 12-60 hour.It within described 12-60 hour, can be 12-24 hour.Described 12-24 hour concretely 12 hours, 16 hours, 20 hours or 24 hours.
For solving above-mentioned technical problem, present invention also offers luciferase co-immunoprecipitation reagent or test kit 2 that detection tick-brone encephalitis virus infects.
Reagent provided by the present invention or test kit 2, containing described tick-brone encephalitis virus antigen.
For solving above-mentioned technical problem, present invention also offers following P1) or product P2):
P1) described tick-brone encephalitis virus antigen；
P2) described fusion protein.
For solving above-mentioned technical problem, present invention also offers the biomaterial of following first or second:
The biomaterial that first is relevant to described tick-brone encephalitis virus antigen, for following E1) to E21) in any one:
E1) nucleic acid molecules of described tick-brone encephalitis virus antigen is encoded；
E2) encoding gene of described tick-brone encephalitis virus antigen；
E3) containing E2) expression cassette of described gene；
E4) containing E2) recombinant vector of described gene；
E5) containing E3) recombinant vector of described expression cassette；
E6) containing E2) recombinant microorganism of described gene；
E7) containing E3) recombinant microorganism of described expression cassette；
E8) containing E4) recombinant microorganism of described recombinant vector；
E9) containing E5) recombinant microorganism of described recombinant vector；
E10) containing E2) the transgenic plant cells system of described gene or transgenetic animal cell system；
E11) containing E3) the transgenic plant cells system of described expression cassette or transgenetic animal cell system；
E12) containing E4) the transgenic plant cells system of described recombinant vector or transgenetic animal cell system；
E13) containing E5) the transgenic plant cells system of described recombinant vector or transgenetic animal cell system；
E14) containing E2) Transgenic plant tissue of described gene or transgenic animal tissue；
E15) containing E3) Transgenic plant tissue of described expression cassette or transgenic animal tissue；
E16) containing E4) Transgenic plant tissue of described recombinant vector or transgenic animal tissue；
E17) containing E5) Transgenic plant tissue of described recombinant vector or transgenic animal tissue；
E18) containing E2) the transgenic plant organ of described gene or transgenic animal organ；
E19) containing E3) the transgenic plant organ of described expression cassette or transgenic animal organ；
E20) containing E4) the transgenic plant organ of described recombinant vector or transgenic animal organ；
E21) containing E5) the transgenic plant organ of described recombinant vector or transgenic animal organ；
The biomaterial that second is relevant to described fusion protein, for following G1) to G21) in any one:
G1) nucleic acid molecules of encoding said fusion protein；
G2) encoding gene of described fusion protein；
G3) containing G2) expression cassette of described gene；
G4) containing G2) recombinant vector of described gene；
G5) containing G3) recombinant vector of described expression cassette；
G6) containing G2) recombinant microorganism of described gene；
G7) containing G3) recombinant microorganism of described expression cassette；
G8) containing G4) recombinant microorganism of described recombinant vector；
G9) containing G5) recombinant microorganism of described recombinant vector；
G10) containing G2) the transgenic plant cells system of described gene or transgenetic animal cell system；
G11) containing G3) the transgenic plant cells system of described expression cassette or transgenetic animal cell system；
G12) containing G4) the transgenic plant cells system of described recombinant vector or transgenetic animal cell system；
G13) containing G5) the transgenic plant cells system of described recombinant vector or transgenetic animal cell system；
G14) containing G2) Transgenic plant tissue of described gene or transgenic animal tissue；
G15) containing G3) Transgenic plant tissue of described expression cassette or transgenic animal tissue；
G16) containing G4) Transgenic plant tissue of described recombinant vector or transgenic animal tissue；
G17) containing G5) Transgenic plant tissue of described recombinant vector or transgenic animal tissue；
G18) containing G2) the transgenic plant organ of described gene or transgenic animal organ；
G19) containing G3) the transgenic plant organ of described expression cassette or transgenic animal organ；
G20) containing G4) the transgenic plant organ of described recombinant vector or transgenic animal organ；
G21) containing G5) the transgenic plant organ of described recombinant vector or transgenic animal organ.
Wherein, described nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；Described nucleic acid molecules can also be RNA, such as mRNA or hnRNA etc..
In above-mentioned biomaterial, E1) described nucleic acid molecules concretely following E1a) E1b) or E1c) shown in nucleic acid molecules:
E1a) coded sequence is cDNA molecule or the DNA molecular of the 958-1236 position nucleotide of SEQ ID No .2；
E1b) nucleotide sequence and E1a) limited has 75% or more than 75% homogeneity, and encodes cDNA molecule or the genomic DNA molecule of described tick-brone encephalitis virus antigen；
E1c) under strict conditions with E1a) nucleotide sequence hybridization that limits, and encode cDNA molecule or the genomic DNA molecule of described tick-brone encephalitis virus antigen.
In above-mentioned biomaterial, G1) described nucleic acid molecules concretely following C1) C2) or C3) shown in nucleic acid molecules:
C1) nucleic acid molecules shown in SEQ ID No .2；
C2) nucleotide sequence and C1) limited has 75% or more than 75% homogeneity, and the cDNA molecule of coding identical function albumen or genomic DNA molecule；
C3) under strict conditions with C1) nucleotide sequence hybridization that limits, and the cDNA molecule of coding identical function albumen or genomic DNA molecule.
Those of ordinary skill in the art can adopt known method easily, for instance the method for orthogenesis and point mutation, is that the nucleotide sequence of the protein of SEQIDNo.1 suddenlys change to the encoding amino acid sequence of the present invention.Those are through manually modified, there is the nucleotide of the nucleotide sequence 75% of protein that the aminoacid sequence with the present invention is SEQIDNo.1 or higher homogeneity, as long as aminoacid sequence is the protein of SEQIDNo.1 and has identical function, all it is derived from the nucleotide sequence of the present invention and is equal to the sequence of the present invention.
Those of ordinary skill in the art can adopt known method easily, for instance the method for orthogenesis and point mutation, is that the nucleotide sequence of the protein shown in 320-412 amino acids of SEQIDNo.1 suddenlys change to the encoding amino acid sequence of the present invention.Those are through manually modified, there is the nucleotide sequence 75% of the protein shown in the 320-412 amino acids that the aminoacid sequence with the present invention is SEQIDNo.1 or the nucleotide of higher homogeneity, as long as aminoacid sequence is the protein shown in 320-412 amino acids of SEQIDNo.1 and has the function of described tick-brone encephalitis virus antigen, all it is derived from the nucleotide sequence of the present invention and is equal to the sequence of the present invention.
Term used herein " homogeneity " refers to the sequence similarity with native sequence nucleic acid." homogeneity " includes having 75% or higher with the nucleotide sequence of the protein of the aminoacid sequence composition shown in the 320-412 position of coding SEQIDNo.1 or SEQIDNo.1 of the present invention, or 85% or higher, or 90% or higher, or the nucleotide sequence of 95% or higher homogeneity.Homogeneity can with the naked eye or computer software be evaluated.Using computer software, the homogeneity between two or more sequences can use percentage ratio (%) to represent, it can be used to the homogeneity evaluating between correlated series.
In above-mentioned biomaterial, described stringent condition is in the solution of 2 × SSC, 0.1%SDS, hybridizes and wash film 2 times at 68 DEG C, each 5min, again in the solution of 0.5 × SSC, 0.1%SDS, hybridizes and wash film 2 times at 68 DEG C, each 15min.
Above-mentioned 75% or more than 75% homogeneity, can be the homogeneity of 80%, 85%, 90% or more than 95%.
In above-mentioned biomaterial, the expression cassette of the encoding gene containing described tick-brone encephalitis virus antigen described in E3), refer to express the DNA of described tick-brone encephalitis virus antigen in host cell, this DNA not only can include starting the promoter that the encoding gene of described tick-brone encephalitis virus antigen is transcribed, and may also include and terminates the terminator that the encoding gene of described tick-brone encephalitis virus antigen is transcribed.Further, described expression cassette may also include enhancer sequence.
Available existing expression vector establishment contains the recombinant vector of the expression cassette of the encoding gene of described tick-brone encephalitis virus antigen.
In above-mentioned biomaterial, the expression cassette of the encoding gene containing described fusion protein described in G3), refer to express the DNA of described fusion protein in host cell, this DNA not only can include starting the promoter that the encoding gene of described fusion protein is transcribed, and may also include and terminates the terminator that the encoding gene of described fusion protein is transcribed.Further, described expression cassette may also include enhancer sequence.
Available existing expression vector establishment contains the recombinant vector of the expression cassette of the encoding gene of described fusion protein.
In above-mentioned biomaterial, described carrier can be plasmid, glutinous grain, phage or viral vector.
In above-mentioned biomaterial, described microorganism can be yeast, antibacterial, algae or fungus, such as escherichia coli.
In above-mentioned biomaterial, described cell line can be COS7 cell line.
In above-mentioned biomaterial, described transgenic plant cells system, described transgenetic animal cell cell line, described Transgenic plant tissue, described transgenic animal tissue, described transgenic plant organ and described transgenic animal organ all do not include propagating materials.
In an embodiment of the invention, EVRP gene is imported in e. coli bl21 by the recombinant vector of the expression cassette containing EVRP gene.The recombinant vector of the described expression cassette containing EVRP gene for by carrier PET32a (+) BamHI and XhoI recognition site between sequence to replace with other sequences of DNA fragmentation shown in the nucleotide of SEQIDNo.2 958-1236 position (keep PET32a (+) constant), obtain recombinant vector, by this recombinant vector called after PET32a-EVRP.
In an embodiment of the invention, the encoding gene of EVRP-Rluc is imported in COS7 cell by the recombinant vector of the expression cassette of the encoding gene containing EVRP-Rluc.The recombinant vector of the expression cassette of the described encoding gene containing EVRP-Rluc for by carrier pcDNA3.1 (+) NheI and XhoI recognition site between sequence to replace with other sequences of DNA fragmentation shown in SEQIDNo.2 (keep pcDNA3.1 (+) constant), obtain recombinant vector, by this recombinant vector called after pcDNA3.1-EVRP-Rluc.
For solving above-mentioned technical problem, present invention also offers luciferase co-immunoprecipitation reagent or test kit 3 that detection tick-brone encephalitis virus infects.
Reagent provided by the present invention or test kit 3, for the reagent containing described biomaterial or test kit.
The reagent of the application or test kit 1 or reagent or test kit 2 or reagent or test kit 3 may also include luciferase other reagent of co-immunoprecipitation system, such as ProteinA/Gplusagarose and/or LIPSBufferA and/or LIPSBufferB and/or the 0.01MPBS solution containing 1% (volumn concentration) hyclone and/or RenillaLuciferaseAssay；
Described LIPSBufferA is made up of solute and water, and wherein each component of solute and concentration thereof are respectively as follows: 20mMTris-HCl (PH7.9), 150mMNaCl, 5mMMgCl2, 0.1% (volumn concentration) TritonX-100；
Described LIPSBufferB is made up of solute and water, and wherein each component of solute and concentration thereof are respectively as follows: 20mMTris-HCl (PH7.9), 150mMNaCl, 5mMMgCl2；
Described ProteinA/Gplusagarose concretely SantaCruz Products, article No. is sc-2003；
Described RenillaLuciferaseAssay reagent concretely Promega Products, article No. is E2810.
The reagent of the application or the Applicable temperature of test kit can be 4 DEG C-37 DEG C.
The luciferase co-immunoprecipitation reagent of above-mentioned detection tick-brone encephalitis virus infection or test kit also include the carrier being described below content: when whether the luciferase co-immunoprecipitation reagent of the detection tick-brone encephalitis virus infection of the application present invention or test kit detection serum are TBEV infection serum, being simultaneously introduced in the reaction system of luciferase co-immunoprecipitation system by test serum, above-mentioned fusion protein EVRP and ProteinA/Gplusagarose, the temperature of immunoprecipitation is 4-37 DEG C.
Described carrier also states that herein below: infect the fluorescence meansigma methods of negative human serum with 3 times of standard deviation sums as judging whether test serum infects the reference value of TBEV using the TBEV of clinical definite, test serum fluorescent value more than or equal to as described in reference value, test serum is that TBEV infects serum, test serum fluorescent value less than as described in reference value, test serum is not for be infected serum by TBEV.
For solving above-mentioned technical problem, present invention also offers following arbitrary application:
I, described tick-brone encephalitis virus antigen application in the luciferase co-immunoprecipitation reagent or test kit of preparation detection tick-brone encephalitis virus infection；
II, described fusion protein application in the luciferase co-immunoprecipitation reagent or test kit of preparation detection tick-brone encephalitis virus infection；
III, described biomaterial application in the luciferase co-immunoprecipitation reagent or test kit of preparation detection tick-brone encephalitis virus infection；
IV, the preparation method of described fusion protein application in the luciferase co-immunoprecipitation reagent or test kit of preparation detection tick-brone encephalitis virus infection.
In order to make the fusion protein of the present invention be easy to purification, in sequence table, the amino terminal of the protein shown in sequence 1 or carboxyl terminal label as shown in table 1 can be connected.
Table 1, label sequence
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (is generally 5) | RRRRR |
| Poly-His | 2-10 (is generally 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
It is demonstrated experimentally that utilizing the True Positive Rate that the tick-brone encephalitis virus antigen coalescence protein EVRP-Rluc and luciferase co-immunoprecipitation system detection TBEV of the present invention infect serum is 84%, true negative rate is 100%；Utilizing the tick-brone encephalitis virus antigen EVRP True Positive Rate detecting TBEV infection serum is 60%, and true negative rate is 100%.The tick-brone encephalitis virus antigen coalescence protein EVRP-Rluc and luciferase co-immunoprecipitation system detection TBEV utilizing the present invention infects the True Positive Rate of serum higher than the True Positive Rate utilizing tick-brone encephalitis virus antigen EVRP to detect TBEV infection serum.
The tick-brone encephalitis virus antigen coalescence protein EVRP-Rluc of the present invention can be used to detect TBEV and infects positive human serum and the negative human serum of TBEV infection, tick-brone encephalitis virus antigen coalescence protein EVRP-Rluc can obtain cell transfecting 12 hours the soonest, tick-brone encephalitis virus antigen coalescence protein EVRP-Rluc to prepare required time short.
Accompanying drawing explanation
Fig. 1 is the fluorescent value of recombinant vector pcDNA3.1-EVRP-Rluc rotaring redyeing COS 7 cell different time.
Fig. 2 is the impact on LIPS testing result of the LIPSBufferA volume.Wherein, diluent represents LIPSBufferA.
Fig. 3 is the luciferase co-immunoprecipitation system detection TBEV result infecting serum.Wherein, A represents the TBEV negative infection human serum of clinical definite；B represents that the TBEV of clinical definite infects positive human serum.
Fig. 4 is the polyacrylamide gel electrophoresis result of the reconstitution cell BL21-EVRP tick-brone encephalitis virus antigen expressed.Wherein, 1 is the reconstitution cell BL21-EVRP tick-brone encephalitis virus antigen expressed, and 2 is the reconstitution cell BL21-PET32a albumen expressed, and M is Marker.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention being further described in detail, the embodiment provided is only for illustrating the present invention, rather than in order to limit the scope of the present invention.
Experimental technique in following embodiment, if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all commercially obtain.
PcDNA3.1 in following embodiment (+) for Invitrogen product, article No. is V790-20.
COS7 cell purchased from American DSMZ (ATCC), CRL-1651 in following embodiment.
ProteinA/Gplusagarose in following embodiment is SantaCruz Products, and article No. is sc-2003.
LIPSBufferA in following embodiment is made up of solute and water, and wherein each component of solute and concentration thereof are respectively as follows: 20mMTris-HCl (PH7.9), 150mMNaCl, 5mMMgCl2, 0.1% (volumn concentration) TritonX-100.
LIPSBufferB in following embodiment is made up of solute and water, and wherein each component of solute and concentration thereof are respectively as follows: 20mMTris-HCl (PH7.9), 150mMNaCl, 5mMMgCl2。
The TBEV of the clinical definite in following embodiment infects the clinical diagnosis of positive human serum according to the forest encephalitis symptom (heating, the headache that all occur immunity for patient, and it is attended by the nervous system disease such as disturbance of consciousness), there is Ticks insect stings history, and viral nucleic acid can be detected from blood.The TBEV of clinical definite infects negative human serum and is the physical patient without forest encephalitis symptom or the serum of healthy person.
Embodiment 1, tick-brone encephalitis virus antigen coalescence protein EVRP-Rluc detect TBEV and infect serum
1, the expression of tick-brone encephalitis virus antigen coalescence protein EVRP-Rluc
By pcDNA3.1 (+) sequence between NheI and the XhoI recognition site of carrier replaces with the encoding gene (nucleotide sequence shown in SEQ ID No .2) of the fusion protein of tick-brone encephalitis virus antigen and renilla luciferase, keep pcDNA3.1 (+) other sequences constant, obtain recombinant vector, by this recombinant vector called after, pcDNA3.1-EVRP-Rluc expresses the fusion protein shown in SEQIDNo.1.
Wherein, SEQIDNo.2 encodes the fusion protein of SEQIDNo.1, the aminoacid sequence that 320-412 amino acids is tick-brone encephalitis virus antigen of SEQIDNo.1, the aminoacid sequence that 1-311 amino acids is renilla luciferase of SEQIDNo.1.The coded sequence that 958-1236 position nucleotide is tick-brone encephalitis virus antigen of SEQIDNo.2, namely encodes the protein shown in 320-412 amino acids of SEQIDNo.1；The 1-933 position nucleotide of SEQIDNo.2 is the coded sequence of renilla luciferase, namely encodes the protein shown in 1-311 amino acids of SEQIDNo.1；The connection peptides shown in 312-319 amino acids of the 934-957 position nucleotide coding SEQIDNo.1 of SEQIDNo.2.
By Opti-MEM lipofectamine, pcDNA3.1-EVRP-Rluc is imported in COS7 cell, obtain reconstitution cell, by this reconstitution cell called after COS7-EVRP-Rluc.
After transfection terminates, by COS7-EVRP-Rluc at 37 DEG C, 5%CO2nullWhen，Cultivate in the DMEM culture medium containing 10% hyclone，Cultivating the 4th hour、8 hours、12 hours、16 hours、20 hours、24 hours、36 hours、48 hours、60 hours、Within 72 hours, respectively take 1mL cell culture fluid，And with the 1 × PassiveLysisBuffer of 0.3mL, (1 × PassiveLysisBuffer is made up of 5 × PassiveLysisBuffer (Promega company) and PBS respectively，Wherein，The volume ratio of 5 × PassiveLysisBuffer and PBS is 1:4) cell lysis，Respectively obtain 4 hour cell lysates、8 hour cell lysates、12 hour cell lysates、16 hour cell lysates、20 hour cell lysates、24 hour cell lysates、36 hour cell lysates、48 hour cell lysates、60 hour cell lysates、72 hour cell lysates.
In the 4 hour cell lysates of 20 μ L, add 100 μ L coelenterazine (Promega product, article No. is E2810), after mix homogeneously, obtain 4 hours albumen liquid to be measured.
According to the method described above, 4 hour cell lysates are replaced with 8 hour cell lysates respectively, 12 hour cell lysates, 16 hour cell lysates, 20 hour cell lysates, 24 hour cell lysates, 36 hour cell lysates, 48 hour cell lysates, 60 hour cell lysates and 72 hour cell lysates, other steps are all constant, respectively obtain 8 hours albumen liquid to be measured, 12 hours albumen liquid to be measured, 16 hours albumen liquid to be measured, 20 hours albumen liquid to be measured, 24 hours albumen liquid to be measured, 36 hours albumen liquid to be measured, 48 hours albumen liquid to be measured, 60 hours albumen liquid to be measured, 72 hours albumen liquid to be measured.
The fluorescence signal in 4 hours albumen liquid to be measured, 8 hours albumen liquid to be measured, 12 hours albumen liquid to be measured, 16 hours albumen liquid to be measured, 20 hours albumen liquid to be measured, 24 hours albumen liquid to be measured, 36 hours albumen liquid to be measured, 48 hours albumen liquid to be measured, 60 hours albumen liquid to be measured, 72 hours albumen liquid to be measured, the expression (Fig. 1) of analyzing proteins is detected respectively with fluorescent signal detector device Glomax20/20Luminometer (Promega company).
Result shows, the fluorescence signal value of the 4th hour after recombinant vector pcDNA3.1-EVRP-Rluc rotaring redyeing COS 7 cell is 8.036 × 106；The fluorescence signal of the 8th hour after recombinant vector pcDNA3.1-EVRP-Rluc rotaring redyeing COS 7 cell is value 2.113 × 107；The fluorescence signal value of the 12nd hour after recombinant vector pcDNA3.1-EVRP-Rluc rotaring redyeing COS 7 cell is 9.246 × 107；The fluorescence signal of the 16th hour after recombinant vector pcDNA3.1-EVRP-Rluc rotaring redyeing COS 7 cell is 1.037 × 108；The fluorescence signal value of the 20th hour after recombinant vector pcDNA3.1-EVRP-Rluc rotaring redyeing COS 7 cell is 9.975 × 107；The fluorescence signal value of the 24th hour after recombinant vector pcDNA3.1-EVRP-Rluc rotaring redyeing COS 7 cell is 1.646 × 108；The fluorescence signal value of the 36th hour after recombinant vector pcDNA3.1-EVRP-Rluc rotaring redyeing COS 7 cell is 1.039 × 108；The fluorescence signal value of the 48th hour after recombinant vector pcDNA3.1-EVRP-Rluc rotaring redyeing COS 7 cell is 1.042 × 108；The fluorescence signal value of the 60th hour after recombinant vector pcDNA3.1-EVRP-Rluc rotaring redyeing COS 7 cell is 8.836 × 107；The fluorescence signal value of the 72nd hour after recombinant vector pcDNA3.1-EVRP-Rluc rotaring redyeing COS 7 cell is 5.721 × 103.Result shows, the expression of the tick-brone encephalitis virus antigen of the 24th hour after recombinant vector pcDNA3.1-EVRP-Rluc rotaring redyeing COS 7 cell and the fusion protein of renilla luciferase is the highest, the higher level that the tick-brone encephalitis virus antigen of the 12-60 hour after recombinant vector pcDNA3.1-EVRP-Rluc rotaring redyeing COS 7 cell and the fusion protein of renilla luciferase all reach.
2, the testing conditions of tick-brone encephalitis virus antigen coalescence protein EVRP-Rluc
Take 24 hour cell lysates in step 1 and determine the volume of the LIPSBufferA of the optimum joining day of ProteinA/Gplusagarose, optimum reaction temperature and optimum when luciferase co-immunoprecipitation system (Luciferaseimmunoprecipitationsystem, LIPS) reacts.
The impact on LIPS of the joining day of 2.1ProteinA/Gplusagarose
(1) in clean 1.7ml centrifuge tube, add the ProteinA/Gplusagarose reagent 100 μ l after mixing, diluent is once abandoned afterwards with diluent (the 0.01MPBS solution containing 1% hyclone) washing, add LIPSBufferA40 μ L mixing to precipitation, obtain ProteinA/Gplusagarose solution；
(2) after taking a blood serum sample LIPSBufferA dilution 10 times, the blood serum sample after being diluted；
(3) LIPS test experience is divided into four groups:
A group: (this fusion protein EVRP-Rluc solution is with the liquid obtained of the fusion protein EVRP-Rluc in LIPSBufferA dissolving step 1, and the fusion protein EVRP-Rluc content in 1 μ l fusion protein EVRP-Rluc solution is 10 for the ProteinA/Gplusagarose solution 40 μ L of step (1) and 1 μ L fusion protein EVRP-Rluc solution-13Mole (fluorescent value is 106)) and the dilution of step (2) after blood serum sample 10 μ L be simultaneously introduced LIPS reaction system, mixing fully, is placed on ice, hatches 1h 4 DEG C of concussions on horizontal shaker；
B group: (this fusion protein EVRP-Rluc solution is with the liquid obtained of the fusion protein EVRP-Rluc in LIPSBufferA dissolving step 1, and the fusion protein EVRP-Rluc content in 1 μ l fusion protein EVRP-Rluc solution is 10 for the ProteinA/Gplusagarose solution 40 μ L of step (1) and 1 μ L fusion protein EVRP-Rluc solution-13Mole (fluorescent value is 106)) mixing, it is placed on ice, horizontal shaker hatches 30min 4 DEG C of concussions, add the mixing of the blood serum sample 10 μ L after the dilution of step (2), be placed on ice, horizontal shaker hatches 30min 4 DEG C of concussions；
C group: the ProteinA/Gplusagarose solution 40 μ L of step (1) mixes with the blood serum sample 10 μ L after the dilution of step (2), it is placed on ice, horizontal shaker hatches 30min 4 DEG C of concussions, (this fusion protein EVRP-Rluc solution is with the liquid obtained of the fusion protein EVRP-Rluc in LIPSBufferA dissolving step 1, and the fusion protein EVRP-Rluc content in 1 μ l fusion protein EVRP-Rluc solution is 10 to add 1 μ L fusion protein EVRP-Rluc solution-13Mole (fluorescent value is 106)) mixing, it is placed on ice, horizontal shaker hatches 30min 4 DEG C of concussions；
D group: (this fusion protein EVRP-Rluc solution is with the liquid obtained of the fusion protein EVRP-Rluc in LIPSBufferA dissolving step 1 to 1 μ L fusion protein EVRP-Rluc solution, and the fusion protein EVRP-Rluc content in 1 μ l fusion protein EVRP-Rluc solution is 10-13Mole (fluorescent value is 106)) and step
(2) the blood serum sample 10 μ L mixing after dilution, it is placed on ice, horizontal shaker hatches 30min 4 DEG C of concussions, add the ProteinA/Gplusagarose solution 40 μ L mixing of step (1), it is placed on ice, horizontal shaker hatches 30min 4 DEG C of concussions；
(4) each group uses LIPSBufferA reagent wash 2 times, after washing, centrifugal 4000rpm, 2min, abandon supernatant, by LIPSBufferB reagent wash 1 time, after washing, centrifugal 4000rpm, 2min, abandon supernatant, after 0.01MPBS reagent wash 1 time, after washing, centrifugal 4000rpm, 2min, abandon supernatant；
(5) add substrate RenillaLuciferaseAssay100 μ l, detect fluorescent value with GLOMAX20/20LUMINOMETER luminometer, record fluorescent value, it is judged that Detection results when different Loading sequence.
Result shows, the fluorescent value of A group is 4.873 × 105；The fluorescent value of B group is 1.411 × 104；The fluorescent value of C group is 2.69 × 105；The fluorescent value of D group is 1.35 × 105.It is shown that ProteinA/Gplusagarose and fusion protein EVRP-Rluc and patients serum are simultaneously introduced in LIPS reaction system, LIPS reaction can reach optimal result.
The impact on LIPS of 2.2 reaction temperatures
(1) in clean 1.7ml centrifuge tube, add the ProteinA/Gplusagarose reagent 100 μ l after mixing, once abandon diluent afterwards with diluent (the 0.01MPBS solution containing 1% hyclone) washing, add LIPSBufferA40 μ l mixing to precipitation；
(2) after taking a blood serum sample LIPSBufferA dilution 10 times, blood serum sample after being diluted, (this fusion protein EVRP-Rluc solution is with the liquid obtained of the fusion protein EVRP-Rluc in LIPSBufferA dissolving step 1, and the fusion protein EVRP-Rluc content in 1 μ l fusion protein EVRP-Rluc solution is 10 to add 1 μ l fusion protein EVRP-Rluc solution in the blood serum sample after 10 μ l dilutions-13Mole (fluorescent value is 106))；
(3) step (1) is abundant with liquid mixing in step (2), and the mixing liquid obtained carries out following two groups of experiments:
A group: be placed on ice, hatches 1h 4 DEG C of concussions on horizontal shaker；
B group: be placed under 37 DEG C of constant temperatures, 1h is hatched in concussion；
(4) each group all uses LIPSBufferA reagent wash 2 times, after washing, centrifugal 4000rpm, 2min, abandon supernatant, by LIPSBufferB reagent wash 1 time, after washing, centrifugal 4000rpm, 2min, abandon supernatant, after 0.01MPBS reagent wash 1 time, after washing, centrifugal 4000rpm, 2min, abandon supernatant；
(5) add substrate RenillaLuciferaseAssay100 μ l, detect fluorescent value with GLOMAX20/20LUMINOMETER luminometer, record fluorescent value, it is judged that the Detection results under condition of different temperatures.
Result shows, the fluorescent value of A group is 6.06 × 105；The fluorescent value of B group is 5.92 × 105.It is shown that when reaction temperature is 4 DEG C and 37 DEG C, LIPS reaction result does not have significant difference, and luciferase co-immunoprecipitation system (Luciferaseimmunoprecipitationsystem, LIPS) can carry out at 4 DEG C or 37 DEG C
The impact on LIPS of the volume of 2.4LIPSBufferA
(1) after taking a blood serum sample LIPSBufferA dilution 10 times, blood serum sample after being diluted, (this fusion protein EVRP-Rluc solution is with the liquid obtained of the fusion protein EVRP-Rluc in LIPSBufferA dissolving step 1, and the fusion protein EVRP-Rluc content in 1 μ l fusion protein EVRP-Rluc solution is 10 to add 1 μ l fusion protein EVRP-Rluc solution in the blood serum sample after 10 μ l dilutions-13Mole (fluorescent value is 106))；
(2) in clean 1.7ml centrifuge tube add mixing after ProteinA/Gplusagarose reagent 100 μ l, then with the 0.01MPBS solution washing containing 1% (volumn concentration) hyclone once after abandoning supernatant；LIPSBufferA10 μ l (i.e. A group), 20 μ l (i.e. B group), 40 μ l (i.e. C group), 60 μ l (i.e. D group) or 80 μ l (i.e. E group) mixing is added in precipitation；
(3) step (1) is fully mixed with the liquid in step (2), be placed on ice, horizontal shaker hatches 1h 4 DEG C of concussions；
(4) each group uses LIPSBufferA reagent wash 2 times, after washing, centrifugal 4000rpm, 2min, abandon supernatant, by LIPSBufferB reagent wash 1 time, after washing, centrifugal 4000rpm, 2min, abandon supernatant, after 0.01MPBS reagent wash 1 time, after washing, centrifugal 4000rpm, 2min, abandon supernatant；
(5) add substrate RenillaLuciferaseAssay100 μ l, detect fluorescent value with GLOMAX20/20LUMINOMETER luminometer, record fluorescent value, it is judged that the Detection results (Fig. 2) under different LIPSBufferA volume conditions.
It is shown that when the volume of LIPSBufferA is 40 μ l, LIPS reaction can reach optimal result.
3, tick-brone encephalitis virus antigen coalescence protein EVRP-Rluc detects TBEV infection serum
Infecting negative human serum sample with the TBEV of the TBEV infection positive human serum's sample and 21 parts of clinical definites detecting 50 parts of clinical definites respectively of the fusion protein EVRP-Rluc in step 1, comprise the following steps that, reaction carries out at 4 DEG C:
1) in clean 1.7ml centrifuge tube add mixing after ProteinA/Gplusagarose reagent 100 μ l, then with the 0.01MPBS solution washing containing 1% (volumn concentration) hyclone once after abandoning supernatant；
2) to step 1) precipitation in add LIPSBufferA40 μ l mixing；
3) after taking a blood serum sample LIPSBufferA dilution 10 times, blood serum sample after being diluted, (this fusion protein EVRP-Rluc solution is with the liquid obtained of the fusion protein EVRP-Rluc in LIPSBufferA dissolving step 1, and the fusion protein EVRP-Rluc content in 1 μ l fusion protein EVRP-Rluc solution is 10 to add 1 μ l fusion protein EVRP-Rluc solution in the blood serum sample after 10 μ l dilutions-13Mole (fluorescent value is 106))；
4) by step 2) with step 3) in liquid fully mix, be placed on ice, horizontal shaker hatch 1h 4 DEG C of concussions；
5) LIPSBufferA reagent wash is used 2 times, after washing, centrifugal 4000rpm, 2min, remove supernatant, by LIPSBufferB reagent wash 1 time, after washing, centrifugal 4000rpm, 2min, remove supernatant, after 0.01MPBS reagent wash 1 time, after washing, centrifugal 4000rpm, 2min, remove supernatant；
6) to step 5) precipitation in add substrate RenillaLuciferaseAssay reagent (Promega product, article No. is E2810) 100 μ L, fluorescent value, record fluorescent value (table 2 and Fig. 3) is detected with GLOMAX20/20LUMINOMETER luminometer.
Table 2, each serum sample LIPS reaction fluorescent value and testing result
Note: "+" represent that serum sample is judged as TBEV and infects serum；"-" represents that serum sample is judged as and is not infected serum by TBEV；" " indicates without these data.
When the fluorescent value of LIPS reaction is more than or equal to 21 parts of negative serum specimen fluorescence meansigma methodss and 3 times of standard deviation sums (namely 1.74 × 105) time, serum sample is judged as TBEV and infects serum；When the fluorescent value of LIPS reaction is less than negative serum meansigma methods and 3 times of standard deviation sums (namely 1.74 × 105) time, serum sample is judged as and is not infected serum by TBEV.Statistics luciferase co-immunoprecipitation system detection TBEV infects True Positive Rate and the true negative rate of serum.The TBEV that True Positive Rate is clinical definite infects the percentage rate that positive human serum adopts luciferase co-immunoprecipitation system detection results to be TBEV infection positive serum；The TBEV that true negative rate is clinical definite infects the percentage rate that negative human serum adopts luciferase co-immunoprecipitation system detection results to be TBEV infection negative serum.
Result shows, tick-brone encephalitis virus antigen coalescence protein EVRP-Rluc infects from the TBEV of these 50 parts of clinical definites and detects 42 parts of TBEV positive human serum infected positive human serum's sample, and it is 84% that luciferase co-immunoprecipitation system detection TBEV infects the True Positive Rate of serum；Tick-brone encephalitis virus antigen coalescence protein EVRP-Rluc infects from the TBEV of these 21 parts of clinical definites and detects 21 parts of TBEV clinical patients with negative serum infected negative human serum sample, and it is 100% that luciferase co-immunoprecipitation system detection TBEV infects the true negative rate of serum.
Experiments show that, the tick-brone encephalitis virus antigen coalescence protein EVRP-Rluc of the present invention can be used to detect the TBEV positive human serum infected and negative human serum.
Comparative example 1, tick-brone encephalitis virus antigen EVRP detect TBEV and infect human serum
1, the expression of tick-brone encephalitis virus antigen EVRP
By PET32a (+) sequence between BamHI and the XhoI recognition site of carrier replaces with the encoding gene (nucleotide sequence shown in the 958-1236 position nucleotide of SEQ ID No .2) of tick-brone encephalitis virus antigen, keep PET32a (+) other sequences constant, obtain recombinant vector, by this recombinant vector called after PET32a-EVRP.PET32a-EVRP is imported in e. coli bl21, obtain reconstitution cell, by this reconstitution cell called after BL21-EVRP.By PET32a (+) import in e. coli bl21, obtain reconstitution cell, by this reconstitution cell called after BL21-PET32a.
IPTG induces reconstitution cell BL21-EVRP to express tick-brone encephalitis virus antigen EVRP, and is purified with HIS protein purification test kit, obtains tick-brone encephalitis virus antigen EVRP (Fig. 4).
IPTG induces reconstitution cell BL21-PET32a expressing protein, and IPTG induces the BL21-PET32a total protein expressed as comparison (Fig. 4).
2, tick-brone encephalitis virus Detection of antigen TBEV infects serum
Detecting TBEV infection positive human serum's sample of 50 parts of clinical definites and the negative human serum sample of TBEV infection of 25 parts of clinical definites with the tick-brone encephalitis virus antigen in step 1 respectively, comprise the following steps that, reaction carries out at 37 DEG C:
1) by the tick-brone encephalitis virus antigen in step 1 with being coated liquid (pH=9.6, the carbonate buffer solution of 0.05M) dilution, obtain tick-brone encephalitis virus antigenic solution, in tick-brone encephalitis virus antigenic solution, the concentration of tick-brone encephalitis virus antigen is 1.5 μ g/ml, tick-brone encephalitis virus antigenic solution is added in ELISA microwell plate, 100 μ l/ holes, 4 DEG C are coated overnight；
2), after being coated, wash 1 time with PBST.Add ELISA confining liquid (0.01MPBS containing 3%BSA), 200 μ l/ holes, 300rpm, close 1h at 37 DEG C；
3) PBST rinses 3 times, it is separately added into the PBS containing 1%FBS respectively according to the blood serum sample of 1:100 and 1:1000 dilution, 100 μ l/ holes (every hole one dilute serum sample), diluent is the PBS containing 1%FBS, and 1h is hatched in 37 DEG C of 320rpm concussions；
4) PBST rinses 3 times, adds with the PBS containing 1%FBS according to the 1:1000 HRP-Goat anti human IgG50 μ l/ hole diluted, 37 DEG C, hatch 30min under 320rpm；
5) PBST washs 4 times, adds substrate TMB (Tian Gen Reagent Company product), the room temperature reaction 15min of horseradish peroxidase, adds ELISA stop buffer (2MH2SO4Aqueous solution) terminate reaction；
6) microplate reader detection OD450nmLight absorption value, record result (table 3).
Table 3, each serum sample ELISA reaction OD450nmLight absorption value and testing result
Note: "+" represent that serum sample is judged as TBEV and infects serum；"-" represents that serum sample is judged as and is not infected serum by TBEV；" " indicates without these data.
OD when ELISA reaction450nmMore than or equal to 0.846, (this numerical value is the 25 parts of negative serum specimen OD detected450nmMeansigma methods and 3 times of standard deviation sums) time, serum sample is judged as TBEV and infects serum；OD when ELISA reaction450nmDuring less than 0.846, serum sample is judged as and is not infected serum by TBEV.Statistics tick-brone encephalitis virus Detection of antigen TBEV infects True Positive Rate and the true negative rate of human serum.The TBEV that True Positive Rate is clinical definite infects the percentage rate that positive human serum adopts tick-brone encephalitis virus Detection of antigen result to be TBEV infection positive serum；The TBEV that true negative rate is clinical definite infects the percentage rate that negative human serum adopts tick-brone encephalitis virus Detection of antigen result to be TBEV infection negative serum.
It is shown that tick-brone encephalitis virus antigen EVRP infects from the TBEV of these 50 parts of clinical definites detects 30 parts of TBEV positive human serum infected positive human serum's sample, it is 60% that detection TBEV infects the True Positive Rate of serum；Tick-brone encephalitis virus antigen EVRP infects from the TBEV of these 25 parts of clinical definites and detects 25 parts of TBEV clinical patients with negative serum infected negative human serum sample, and it is 100% that detection TBEV infects the true negative rate of serum.
Experiments show that, all do not use the tick-brone encephalitis virus antigen coalescence protein EVRP-Rluc of the present invention to detect the efficiency of TBEV infection positive human serum and the efficiency height of the negative human serum of TBEV infection by the efficiency of the tick-brone encephalitis virus antigen EVRP efficiency and the negative human serum of TBEV infection detecting TBEV infection positive human serum.
The tick-brone encephalitis virus antigen coalescence protein EVRP-Rluc of the present invention can be used to detect TBEV and infects positive human serum and the negative human serum of TBEV infection, tick-brone encephalitis virus antigen coalescence protein EVRP-Rluc can obtain cell transfecting 12 hours the soonest, tick-brone encephalitis virus antigen coalescence protein EVRP-Rluc to prepare required time short.
Claims (10)
1. detecting luciferase co-immunoprecipitation reagent or test kit that tick-brone encephalitis virus infects, described reagent or test kit contain the fusion protein being made up of tick-brone encephalitis virus antigen and renilla luciferase；Described tick-brone encephalitis virus antigen is following I1) or protein I2):
I1) protein shown in 320-412 amino acids of SEQIDNo.1；
I2) in the aminoacid sequence of the 320-412 position of SEQIDNo.1 through replacement and/or disappearance and/or add that one or several amino acid residue obtains have identical function by I1) derivative protein.
2. reagent according to claim 1 or test kit, it is characterised in that: described fusion protein is following K1) or protein K2):
K1) protein shown in SEQ ID No .1；
K2) in the aminoacid sequence of SEQIDNo.1 through replacement and/or disappearance and/or add that one or several amino acid residue obtains have identical function by K1) derivative protein.
3. reagent according to claim 1 and 2 or test kit, it is characterised in that: described fusion protein is prepared in accordance with the method for claim 4.
4. the preparation method of arbitrary described fusion protein in claim 1-3, including following S1) and step S2):
S1) encoding gene of described fusion protein arbitrary in claim 1-3 is imported in eukaryotic cell, obtain reconstitution cell；
S2) albumen expressing described reconstitution cell obtains described fusion protein；
The encoding gene of described fusion protein is following C1) or C2) or C3) shown in nucleic acid molecules:
C1) nucleic acid molecules shown in SEQ ID No .2；
C2) nucleotide sequence and C1) limited has 75% or more than 75% homogeneity, and the cDNA molecule of coding identical function albumen or genomic DNA molecule；
C3) under strict conditions with C1) nucleotide sequence hybridization that limits, and the cDNA molecule of coding identical function albumen or genomic DNA molecule.
5. method according to claim 4, it is characterised in that: the albumen of the described reconstitution cell of described expression obtains described fusion protein and includes cultivating described reconstitution cell and within 4-60 hour, obtain the step of described fusion protein.
6. detecting luciferase co-immunoprecipitation reagent or test kit that tick-brone encephalitis virus infects, described reagent or test kit contain the tick-brone encephalitis virus antigen described in claim 1.
7. following P1) or product P2):
P1) the tick-brone encephalitis virus antigen described in claim 1；
P2) fusion protein described in claim 1 or 2 or 3 or 4 or 5.
8. the biomaterial of following first or second:
The biomaterial that first is relevant to the tick-brone encephalitis virus antigen described in claim 1, for following E1) to E21) in any one:
E1) nucleic acid molecules of coding tick-brone encephalitis virus antigen described in claim 1；
E2) encoding gene of the tick-brone encephalitis virus antigen described in claim 1；
E3) containing E2) expression cassette of described gene；
E4) containing E2) recombinant vector of described gene；
E5) containing E3) recombinant vector of described expression cassette；
E6) containing E2) recombinant microorganism of described gene；
E7) containing E3) recombinant microorganism of described expression cassette；
E8) containing E4) recombinant microorganism of described recombinant vector；
E9) containing E5) recombinant microorganism of described recombinant vector；
E10) containing E2) the transgenic plant cells system of described gene or transgenetic animal cell system；
E11) containing E3) the transgenic plant cells system of described expression cassette or transgenetic animal cell system；
E12) containing E4) the transgenic plant cells system of described recombinant vector or transgenetic animal cell system；
E13) containing E5) the transgenic plant cells system of described recombinant vector or transgenetic animal cell system；
E14) containing E2) Transgenic plant tissue of described gene or transgenic animal tissue；
E15) containing E3) Transgenic plant tissue of described expression cassette or transgenic animal tissue；
E16) containing E4) Transgenic plant tissue of described recombinant vector or transgenic animal tissue；
E17) containing E5) Transgenic plant tissue of described recombinant vector or transgenic animal tissue；
E18) containing E2) the transgenic plant organ of described gene or transgenic animal organ；
E19) containing E3) the transgenic plant organ of described expression cassette or transgenic animal organ；
E20) containing E4) the transgenic plant organ of described recombinant vector or transgenic animal organ；
E21) containing E5) the transgenic plant organ of described recombinant vector or transgenic animal organ；
The biomaterial that second described fusion protein arbitrary to claim 1-5 is relevant, for following G1) to G21) in any one:
G1) nucleic acid molecules of arbitrary described fusion protein in coding claim 1-5；
G2) encoding gene of arbitrary described fusion protein in claim 1-5；
G3) containing G2) expression cassette of described gene；
G4) containing G2) recombinant vector of described gene；
G5) containing G3) recombinant vector of described expression cassette；
G6) containing G2) recombinant microorganism of described gene；
G7) containing G3) recombinant microorganism of described expression cassette；
G8) containing G4) recombinant microorganism of described recombinant vector；
G9) containing G5) recombinant microorganism of described recombinant vector；
G10) containing G2) the transgenic plant cells system of described gene or transgenetic animal cell system；
G11) containing G3) the transgenic plant cells system of described expression cassette or transgenetic animal cell system；
G12) containing G4) the transgenic plant cells system of described recombinant vector or transgenetic animal cell system；
G13) containing G5) the transgenic plant cells system of described recombinant vector or transgenetic animal cell system；
G14) containing G2) Transgenic plant tissue of described gene or transgenic animal tissue；
G15) containing G3) Transgenic plant tissue of described expression cassette or transgenic animal tissue；
G16) containing G4) Transgenic plant tissue of described recombinant vector or transgenic animal tissue；
G17) containing G5) Transgenic plant tissue of described recombinant vector or transgenic animal tissue；
G18) containing G2) the transgenic plant organ of described gene or transgenic animal organ；
G19) containing G3) the transgenic plant organ of described expression cassette or transgenic animal organ；
G20) containing G4) the transgenic plant organ of described recombinant vector or transgenic animal organ；
G21) containing G5) the transgenic plant organ of described recombinant vector or transgenic animal organ.
9. detect luciferase co-immunoprecipitation reagent or test kit that tick-brone encephalitis virus infects, containing the biomaterial described in claim 8.
10. following arbitrary application:
I, the application in the luciferase co-immunoprecipitation reagent or test kit of preparation detection tick-brone encephalitis virus infection of the tick-brone encephalitis virus antigen described in claim 1；
II, arbitrary described fusion protein application in the luciferase co-immunoprecipitation reagent or test kit of preparation detection tick-brone encephalitis virus infection in claim 1-5；
III, the application in the luciferase co-immunoprecipitation reagent or test kit of preparation detection tick-brone encephalitis virus infection of the biomaterial described in claim 8；
IV, the application in the luciferase co-immunoprecipitation reagent or test kit of preparation detection tick-brone encephalitis virus infection of the method described in claim 4 or 5.
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