CN112390862B

CN112390862B - Protein for detecting bluetongue disease and its encoding gene and soluble preparation method

Info

Publication number: CN112390862B
Application number: CN202011162626.4A
Authority: CN
Inventors: 韩雪清; 孔玉方; 王慧煜; 欧先金
Original assignee: Chinese Academy of Inspection and Quarantine CAIQ
Current assignee: Chinese Academy of Inspection and Quarantine CAIQ
Priority date: 2020-10-27
Filing date: 2020-10-27
Publication date: 2022-03-22
Anticipated expiration: 2040-10-27
Also published as: CN112390862A

Abstract

The invention discloses a protein for detecting bluetongue, a coding gene thereof and a soluble preparation method. The invention firstly discloses a protein for detecting bluetongue, and the amino acid sequence of the protein is shown as 2 nd to 219 th position of SEQ ID No. 1. The invention further discloses a coding gene of the protein and a soluble preparation method thereof. The invention starts from the three-dimensional space structure characteristic of VP7 protein, cuts and rearranges the amino acid sequence of VP7 protein to obtain new VP7-N protein for detecting bluetongue, realizes the high-efficiency soluble expression of VP7-N protein in a prokaryotic expression system by introducing the coding gene of VP7-N protein into the prokaryotic expression system, has the characteristics of high expression yield and good solubility, in addition, does not reduce immunogenicity due to prokaryotic expression, has good immunoreaction characteristic, and provides high-quality protein for detecting bluetongue.

Description

Protein for detecting bluetongue, coding gene and soluble preparation method thereof

Technical Field

The invention relates to the technical field of molecular biology. More particularly, it relates to a protein for detecting bluetongue, its coding gene and soluble preparation method.

Background

The bluetongue disease is a non-contact virulent infectious disease caused by animal infection of bluetongue virus (BTV), mainly infects ruminants such as sheep and cattle, the fatality rate reaches 30%, wherein the mortality rate of sheep reaches 80%, and huge economic loss is brought to the livestock industry. The bluetongue virus belongs to RNA virus, encodes 7 structural proteins (abbreviated as VP1-VP7) and 5 non-structural proteins (abbreviated as NS1-NS5), wherein the structural protein VP7 contains an antigenic determinant causing the immune response of host cells of animals, and is an ideal antigenic protein for detecting whether the animals are infected with the bluetongue virus. The VP7 is obtained by recombinant expression and purification, and then is fixed on a solid carrier such as test paper and the like, and generates a detection signal with an animal blood sample to be detected through antigen-antibody immunoreaction, which is a common bluetongue detection method. This first requires recombinant expression of VP7, VP7 is usually in the form of insoluble inclusion bodies in prokaryotic expression systems, which is detrimental to subsequent immune reactions (the prokaryotic expression of VP7 protein of bluetongue virus, schmallenberg virus N protein, and the establishment of indirect ELISA detection methods (master paper).

The major immune antigen protein VP7 of bluetongue virus is usually expressed by inclusion body in a protein prokaryotic expression system, and some attempts have been made to increase the soluble expression of the target protein VP7 by fusion expression, i.e. recombinant expression of "fusion protein-target protein" in series, the solubilizing effect of the fusion protein will improve the soluble expression effect of the target protein VP7, and the commonly used fusion proteins are GST and MBP, etc. (prokaryotic expression of the bluetongue virus VP7 protein and preparation of monoclonal antibody (Shuoshi paper), Liweng, 2007; prokaryotic expression of the bluetongue virus VP7 protein and preparation of monoclonal antibody (Song paper), Song Hongye, 2010).

Although fusion protein expression vectors can improve the solubility of the fusion protein of interest, this soluble state is also usually partially correctly folded, and the rest is mostly heterogeneous, hyperconvergence, affecting the immune response signal; meanwhile, the existence of the fusion protein also brings negative immunoreaction interference, the fusion expression is only partial soluble expression, and the auxiliary fusion protein needs to be cut by using a specific protease in the protein purification process to remove, so that the production cost of the target protein is increased.

Therefore, it is required to provide a protein for detecting bluetongue, which can be well soluble expressed in a prokaryotic expression system and has the immune response characteristic.

Disclosure of Invention

The first object of the present invention is to provide a novel protein for detecting bluetongue (abbreviated as VP7-N protein) which can be well soluble expressed in prokaryotic expression system while maintaining its immunoreaction characteristics.

The second purpose of the invention is to provide the coding gene of the protein, a recombinant vector and a recombinant bacterium containing the coding gene, and application of the coding gene and the recombinant bacterium in preparing the protein for detecting the bluetongue.

The third object of the present invention is to provide a soluble preparation method of a protein for detecting bluetongue, which can express the protein in a soluble manner with high efficiency and can obtain the protein with high purity.

To achieve the first object, the invention provides a protein for detecting bluetongue, abbreviated as VP7-N protein, wherein the protein is represented by A1) or A2):

A1) protein with the amino acid sequence shown in SEQ ID No.1, 2 th to 219 th positions;

A2) and (b) a fusion protein obtained by connecting protein tags to the N end or/and the C end of the amino acid sequence shown in the 2 nd to 219 th positions of SEQ ID No. 1.

Among the above proteins, protein-tag (protein-tag) refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate expression, detection, tracking or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

In a specific embodiment of the invention, the amino acid sequence of the fusion protein is shown in SEQ ID No.1, positions 2-227.

The invention further provides a coding gene of the protein for detecting the bluetongue, the nucleotide sequence of the coding gene is shown in SEQ ID NO.2, and the coding gene can obtain soluble protein (namely VP7-N protein) through the expression of a prokaryotic expression system.

To achieve the second object, the present invention provides a recombinant vector comprising a gene encoding the above protein for detecting bluetongue.

Furthermore, the recombinant vector is the VP7-N-28a obtained by connecting the coding gene of the protein for detecting bluetongue between the Nco I and Xho I enzyme cutting sites of pet-28a and keeping other sequences of pet-28a unchanged.

The invention further provides a recombinant bacterium containing the recombinant vector VP7-N-28a, and a host cell of the recombinant bacterium is a competent cell of Escherichia coli BL21(DE 3).

The invention further discloses application of the coding gene, the recombinant vector or the recombinant bacterium in preparation of proteins for detecting bluetongue.

To achieve the third object, the present invention provides a soluble preparation method of a protein for detecting bluetongue, comprising the steps of:

genes encoding proteins for detecting bluetongue were synthesized: the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2;

constructing a recombinant vector containing a coding gene of a protein for detecting bluetongue: the recombinant vector is VP7-N-28a, and the VP7-N-28a is obtained by connecting the coding gene of the protein for detecting bluetongue disease shown in SEQ ID NO.2 between the enzyme cutting sites Nco I and Xho I of pet-28a and keeping other sequences of pet-28a unchanged;

constructing a recombinant vector containing a coding gene of a protein for detecting the bluetongue to obtain a recombinant bacterium: the recombinant strain is BL21(De3)/VP7-N-28a, and the recombinant strain BL21(De3)/VP7-N-28a is obtained by transforming a recombinant vector VP7-N-28a into a competent cell of escherichia coli BL21(De 3);

inducing expression: inoculating the recombinant bacterium BL21(De3)/VP7-N-28a into an LB liquid culture medium, adding IPTG with the final concentration of 0.5mM for induction expression, continuing to culture, and centrifugally collecting thalli to obtain the protein for detecting the bluetongue.

Further, the soluble preparation method further comprises the step of affinity purification.

In the specific embodiment of the invention, the affinity purification is that Tris buffer solution is added into the thalli, and the supernatant is obtained after ultrasonic crushing and centrifugation; and (3) enabling the supernatant to flow through a nickel medium, and washing impurities and eluting to obtain purified protein for detecting the bluetongue.

The invention has the following beneficial effects:

the invention starts from the three-dimensional space structure characteristic of VP7 protein, cuts and rearranges the amino acid sequence of VP7 protein to obtain new protein (VP 7-N protein for short) for detecting bluetongue, and introduces the coding gene of VP7-N protein into prokaryotic expression system to realize the high-efficiency soluble expression of VP7-N protein in prokaryotic expression system, and has the characteristics of high expression yield and good solubility.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 shows the three-dimensional crystal structure of the VP7 protein; wherein the domains in the circle are defined as N domains; the domains in the box are defined as the C domain.

FIG. 2 shows an electrophoretogram of VP7-N protein induced expression by SDA-PAGE analysis; wherein, 1 is thallus before induction, 2 is protein Marker, and 3 and 4 are thallus after induction; the arrow indicates the VP7-N protein of interest.

FIG. 3 shows an electrophoretogram of the SDA-PAGE analysis of VP7-N protein purification; wherein 1 is the supernatant, 2 is the precipitate, 3 is the protein Marker, 4 is the sample eluted by imidazole at 20mM, 5 is the sample eluted by imidazole at 500mM, and the arrow indicates the target protein VP7-N protein.

FIG. 4 shows the specific detection result of the bluetongue antibody detection test strip; wherein 1-6 are tuberculosis, brucellosis, viral diarrhea, foot and mouth disease, pasteurellosis, and leukemia positive serum; 7-14 are positive bluetongue serum.

Detailed Description

In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. Similar parts in the figures are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.

The VP7 protein from different viruses has slightly different amino acid sequences, and the VP7 protein amino acid sequence shown in SEQ ID NO.3 is designed experimentally, is numbered AAF97690.1 in the www.ncbi.nlm.nih.gov professional website database and contains 349 amino acid residues. The three-dimensional spatial structure of VP7 has been resolved, and the numbers of the corresponding PDB database (protein data base) are 2BTV and 1 BVP. In the three-dimensional structure, 3 VP7 proteins form a 3-mer with a 3-fold axis of symmetry in a head-to-head, foot-to-foot manner. The monomeric VP7 protein consists of 2 independent domains, the structure of which is shown in FIG. 1, with the domains in the circles of FIG. 1 being defined as the N-domain and the domains in the boxes as the C-domain. These 2 domains are not contiguously distributed in the primary amino acid sequence. The N-structural domain is shown as 1-120 th site and 254-349 th site of SEQ ID NO.3, and consists of 216 amino acid residues in total; the C-structural domain is shown in the position 121-253 of SEQ ID NO.3 and consists of 133 amino acid residues in total. Proteins that are closely related in three dimensions but are rather distant in their primary sequence are more common in viruses, but such proteins generally increase the difficulty of recombinant expression.

According to the invention, the amino acid sequence is spliced according to the three-dimensional space structure characteristics, and then the N-structural domain is independently recombined and expressed, so that the soluble expression of the VP7 protein in a prokaryotic system is greatly improved, and the immune antigen reaction of the VP7 protein is not influenced. Specifically, the invention connects the segments shown in the 1 st to 120 th (120 amino acid residues) and the 254 th and 349 th (96 amino acid residues) of SEQ ID NO.3 by a connecting segment (Linker) consisting of a plurality of amino acids to form an independent new protein (shown in the 2 nd to 219 th of SEQ ID NO.1, which is called VP7-N protein for short), and then synthesizes the coding gene of the VP7-N protein to carry out independent recombinant expression in a prokaryotic system.

The invention is further illustrated by the following specific examples.

Example 1 soluble preparation of VP7-N protein

First, obtaining VP7-N protein gene sequence

Passing 6bp nucleotides between a DNA fragment (shown in the 1 st-360 th positions of SEQ ID NO. 4) encoding the amino acid sequence shown in the 1 st-120 th positions of SEQ ID NO.3 and a DNA fragment (shown in the 760 nd 1047 th positions of SEQ ID NO. 4) encoding the amino acid sequence shown in the 254 nd-349 th positions of SEQ ID NO.3 "GGAGGT"(the corresponding amino acid sequence is glycine-glycine") to obtain the gene sequence of VP7-N protein shown in SEQ ID NO.2, and the total length of the sequence is 654 bp.

The gene sequence of the VP7-N protein shown in SEQ ID NO.2 is obtained by a direct synthesis mode.

Second, construction of recombinant vector

The DNA fragment shown in SEQ ID NO.2 was ligated between the Nco I and Xho I cleavage sites of pet-28a (Novagen, cat. No. 69864-3CN) while leaving the other sequences of pet-28a unchanged, to obtain recombinant vector VP7-N-28 a.

Thirdly, obtaining of recombinant bacteria

Transferring the recombinant vector VP7-N-28a into competent cells of escherichia coli BL21(De3), and screening to obtain a recombinant bacterium BL21(De3)/VP7-N-28 a.

The method comprises the following specific steps:

1. preparation of competent cells of Escherichia coli BL21(De3)

1) A single colony of E.coli BL21(De3) was inoculated into 50ml of LB medium and shake-cultured overnight (250r/min) at 37 ℃ to give an overnight culture.

2) Adding 400ml LB medium to a 2L flask, adding 4ml overnight medium, at 37 deg.C; shaking and culturing to OD600 of 0.4-0.6 to obtain culture solution.

3) And subpackaging the culture solution into 8 pre-cooled sterile polypropylene tubes with the volume of 50ml, placing the tubes on ice for 5-10min, and centrifuging the tubes at the temperature of 4 ℃ for 7min at 1600 g to obtain cell precipitates.

4) The cell pellet was treated with 10ml ice-cold CaCl₂The solution was resuspended and centrifuged at 1100g for 5min at 4 ℃ to obtain cell pellets.

5) The cell pellet was treated with 10ml ice-cold CaCl₂Resuspending the solution, standing on ice for 30min, centrifuging at 1100g for 5min at 4 deg.C, removing supernatant, adding 2ml ice-cold CaCl₂The solution was resuspended in each tube of cells and then dispensed into pre-cooled sterile polypropylene tubes at 250ul per tube and immediately frozen at-70 ℃ to give E.coli BL21(De3) competent cells.

2. The recombinant vector VP7-N-28a was transferred into competent cells of Escherichia coli BL21(De3)

1) 10ng of recombinant vector VP7-N-28a was added to a 15ml sterile round bottom tube and placed on ice.

2) The tube containing the competent cells of E.coli BL21(De3) was held in the hand to melt the inoculum rapidly.

3) Immediately, 100ul of E.coli BL21(De3) competent cells were added to the tube, gently swirled, and placed on ice for 10 min.

4) The tubes were placed in a 42 ℃ water bath for 2min for heat shock, and then 1ml of LB medium was added to each tube and incubated at 37 ℃ for 1h on a roller shaker.

5) And (3) coating the diluted bacteria liquid on a plate containing proper antibiotics, and culturing at 37 ℃ for 12-16 h.

6) And picking positive clones the next day for sequencing verification, wherein the clone bacteria with correct sequencing is recombinant bacteria BL21(De3)/VP7-N-28 a.

Fourth, inducible expression

Inoculating the recombinant bacteria BL21(De3)/VP7-N-28a into an LB liquid culture medium for overnight culture to obtain a seed solution, inoculating the seed solution into the LB liquid culture medium according to the inoculation amount of 1-2% of the volume ratio, culturing for 2-4 hours at 37 ℃ and 200rpm, cooling to 16 ℃ when the optical density (600nm) of the bacterial solution is between 0.6 and 1, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.5mM for induction expression, continuing overnight culture of the bacteria, and centrifugally collecting the bacteria to obtain the induced bacteria. At the same time, the cells before induction were collected as a control and examined by protein denaturing electrophoresis gel (SDS-PAGE).

The VP7-N protein is introduced with a vector sequence during induction expression, so that the end of the VP7-N protein is provided with a His6 tag (shown in SEQ ID NO.1 and consisting of 227 amino acid residues), thereby facilitating the rapid affinity purification of the subsequent VP7-N protein.

As shown in FIG. 2, the molecular weight of the protein obtained by induction expression is about 25kda, which is consistent with the theoretical molecular weight, so that the VP7-N protein is well expressed recombinantly.

Affinity purification of VP7-N protein

Suspending the induced bacteria in a Tris buffer solution (20mM Tris, 100mM NacL, pH 8) to obtain a bacteria weight suspension (the mass concentration of the bacteria in the bacteria weight suspension is 10-20%), and performing ultrasonic crushing on the bacteria weight suspension under the ice bath condition to obtain a crushed bacteria body fluid, wherein the ultrasonic crushing condition is as follows: 200w power, 4 seconds on, 4 seconds off, total 20 minutes. Centrifuging the crushed thallus liquid at high speed, 16000g of centrifugal force for 30 minutes to obtain supernatant and sediment; the pellet was resuspended in the same volume of Tris buffer as the supernatant and used as a pellet sample against the supernatant to verify soluble expression of the protein.

The supernatant was passed through a nickel medium (3-5mL) by gravity, and the VP7-N protein containing a His6 tag was specifically bound to the nickel medium, which was then washed with 50mL of tris buffer (containing 20M imidazole) to give a sample (i.e., a sample obtained by washing with 20mM imidazole), and then eluted with 50mL of tris buffer (containing 500M imidazole) to give a sample (i.e., a sample obtained by eluting with 500mM imidazole), and the obtained sample was subjected to protein denaturing electrophoresis gel (SDS-PAGE) assay, whereby a protein of high purity, i.e., VP7-N protein, was obtained in the supernatant in a soluble form, as shown in FIG. 3.

Example 2 immunological reaction of protein VP7-N

Construction of bluetongue antibody test paper strip by using protein VP7-N prepared in example 1

1. Colloidal gold labeling process

10mL of 20nm colloidal gold solution was equilibrated to room temperature in a 15mL centrifuge tube and used at 0.2mol/L K₂CO₃Adjusting pH to 8.2, adding 100 μ g Staphylococcal Protein A (SPA), mixing with a stirrer for 30min, adding 100 μ L2% casein for blocking for 30min, centrifuging at 8000rmp/min at 4 deg.C for 15min, discarding the supernatant to retain concentrated colloidal gold particles, and resuspending the colloidal gold particles with 1mL colloidal gold resuspension (resuspension 20mM TB pH8.5, 1% casein, 2% sucrose). Spraying the gold-labeled pad by using a BIO-DOT three-dimensional gold spraying synoviograph in an amount of 9 mu L/cm, and then placing the gold-labeled pad in a 37 ℃ thermostat for 4h and drying for later use.

2. Sample pad pretreatment

The sample pad is soaked in buffer solution containing 20mM TB (8.5), 0.5% casein, 5% Tween-20 and 0.3% PVP-40 for 30min, and then placed in a constant temperature cabinet at 37 ℃ for 4h to be dried for later use.

3. Test strip assembly

2.5mg/mL protein VP7-N and 1mg/mL anti-SPA rabbit polyclonal antibody are sprayed on an NC membrane by using a BIO-DOT three-dimensional gold spraying synoviograph, are respectively used as a detection line (T) and a quality control line (C), and are placed in a 37 ℃ incubator for 1h for drying. And adhering the sample pad, the NC membrane, the gold label pad and the absorbent paper on a PVC (polyvinyl chloride) base plate, and then cutting the assembled large plate into a bluetongue antibody detection test strip with the width of 4mm by using a BIO-DOT (bidio-DOT) slitting machine for detecting a subsequent sample.

Second, specificity verification of bluetongue antibody detection test paper strip

The specificity of the blue tongue antibody detection test strip is verified by using positive serum of tuberculosis, brucellosis, viral diarrhea, foot-and-mouth disease, pasteurellosis, leukemia and blue tongue disease stored in animal inspection and quarantine institute of Chinese institute. Diluting positive serum of tuberculosis, brucellosis, viral diarrhea, foot and mouth disease, pasteurellosis, leukemia and bluetongue with sample diluent (20mM TB pH8.5, 1% Tween-20, 0.3% casein) at a ratio of 1:4, namely adding 60 muL of the sample diluent into 20 muL of the serum sample, respectively dripping the sample diluent on sample pads of different bluetongue antibody detection test strips, and observing the result after 5-10 min.

The results are shown in fig. 4, and show that the bluetongue antibody detection test strip has no cross reaction with positive serum of tuberculosis, brucellosis, viral diarrhea, foot-and-mouth disease, pasteurellosis and leukemia, can detect the bluetongue, and shows that the bluetongue antibody detection test strip has better specificity.

It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.

SEQUENCE LISTING

<110> scientific research institute of Chinese inspection and quarantine

<120> protein for detecting bluetongue, and coding gene and soluble preparation method thereof

<130> JLP20I0989

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 227

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Met Asp Thr Ile Ala Ala Arg Ala Leu Thr Val Met Arg Ala Cys

1 5 10 15

Ala Thr Leu Gln Glu Ala Arg Ile Val Leu Glu Ala Asn Val Met Glu

20 25 30

Ile Leu Gly Ile Ala Ile Asn Arg Tyr Asn Gly Leu Thr Leu Arg Gly

35 40 45

Val Thr Met Arg Pro Thr Ser Leu Ala Gln Arg Asn Glu Met Phe Phe

50 55 60

Met Cys Leu Asp Met Met Leu Ser Ala Ala Gly Ile Asn Val Gly Pro

65 70 75 80

Ile Ser Pro Asp Tyr Thr Gln His Met Ala Thr Ile Gly Val Leu Ala

85 90 95

Thr Pro Glu Ile Pro Phe Thr Thr Glu Ala Ala Asn Glu Ile Ala Arg

100 105 110

Val Thr Gly Glu Thr Ser Thr Trp Gly Gly Gly Asp Lys Thr Leu Asn

115 120 125

Gln Tyr Pro Ala Leu Thr Ala Glu Ile Phe Asn Val Tyr Ser Phe Arg

130 135 140

Asp His Thr Trp His Gly Leu Arg Thr Ala Ile Leu Asn Arg Thr Thr

145 150 155 160

Leu Pro Asn Met Leu Pro Pro Ile Phe Pro Pro Asn Asp Arg Asp Ser

165 170 175

Ile Leu Thr Ile Leu Leu Leu Ser Thr Leu Ala Asp Val Tyr Ser Val

180 185 190

Leu Arg Pro Glu Phe Ala Ile His Gly Val Asn Pro Met Pro Gly Pro

195 200 205

Leu Thr Arg Ala Ile Ala Arg Ala Ala Tyr Ala Leu Glu His His His

210 215 220

His His His

225

<210> 2

<211> 654

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggacacca tcgctgctcg tgctctgacc gttatgcgtg cttgcgctac cctgcaggaa 60

gctcgtatcg ttctggaagc taacgttatg gaaatcctgg gtatcgctat caaccgttac 120

aacggtctga ccctgcgtgg tgttaccatg cgtccgacct ctctggctca gcgtaacgaa 180

atgttcttca tgtgcctgga catgatgctg tctgctgctg gtatcaacgt tggtccgatc 240

tctccggact acacccagca catggctacc atcggtgttc tggctacccc ggaaatcccg 300

ttcaccaccg aagctgctaa cgaaatcgct cgtgttaccg gtgaaacctc tacctggggt 360

ggaggtgaca aaaccctgaa ccagtacccg gctctgaccg ctgaaatctt caacgtttac 420

tctttccgtg accacacctg gcacggtctg cgtaccgcta tcctgaaccg taccaccctg 480

ccgaacatgc tgccgccgat cttcccgccg aacgaccgtg actctatcct gaccatcctg 540

ctgctgtcta ccctggctga cgtttactct gttctgcgtc cggaattcgc tatccacggt 600

gttaacccga tgccgggtcc gctgacccgt gctatcgctc gtgctgctta cgct 654

<210> 3

<211> 349

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Asp Thr Ile Ala Ala Arg Ala Leu Thr Val Met Arg Ala Cys Ala

1 5 10 15

Thr Leu Gln Glu Ala Arg Ile Val Leu Glu Ala Asn Val Met Glu Ile

20 25 30

Leu Gly Ile Ala Ile Asn Arg Tyr Asn Gly Leu Thr Leu Arg Gly Val

35 40 45

Thr Met Arg Pro Thr Ser Leu Ala Gln Arg Asn Glu Met Phe Phe Met

50 55 60

Cys Leu Asp Met Met Leu Ser Ala Ala Gly Ile Asn Val Gly Pro Ile

65 70 75 80

Ser Pro Asp Tyr Thr Gln His Met Ala Thr Ile Gly Val Leu Ala Thr

85 90 95

Pro Glu Ile Pro Phe Thr Thr Glu Ala Ala Asn Glu Ile Ala Arg Val

100 105 110

Thr Gly Glu Thr Ser Thr Trp Gly Pro Ala Arg Gln Pro Tyr Gly Phe

115 120 125

Phe Leu Glu Thr Glu Glu Val Tyr Gln Pro Gly Arg Trp Phe Met Arg

130 135 140

Ala Ala Gln Val Val Thr Pro Val Val Cys Gly Pro Asp Met Ile Gln

145 150 155 160

Val Ser Leu Asn Ala Gly Ala Arg Gly Asp Val Gln Gln Ile Phe Gln

165 170 175

Gly Arg Asn Asp Pro Met Met Ile Tyr Leu Val Trp Arg Arg Ile Glu

180 185 190

Asn Phe Ser Met Pro Gln Gly Asn Ser Gln Arg Thr Leu Ala Gly Val

195 200 205

Thr Val Ser Val Gly Gly Val Asp Met Arg Ala Gly Arg Ile Ile Ala

210 215 220

Trp Asp Gly Gln Ala Val Leu Gln Ile His Asn Pro Thr Gln Gln Asn

225 230 235 240

Ala Met Val Gln Ile Gln Val Val Phe Tyr Ile Ser Met Asp Lys Thr

245 250 255

Leu Asn Gln Tyr Pro Ala Leu Thr Ala Glu Ile Phe Asn Val Tyr Ser

260 265 270

Phe Arg Asp His Thr Trp His Gly Leu Arg Thr Ala Ile Leu Asn Arg

275 280 285

Thr Thr Leu Pro Asn Met Leu Pro Pro Ile Phe Pro Pro Asn Asp Arg

290 295 300

Asp Ser Ile Leu Thr Ile Leu Leu Leu Ser Thr Leu Ala Asp Val Tyr

305 310 315 320

Ser Val Leu Arg Pro Glu Phe Ala Ile His Gly Val Asn Pro Met Pro

325 330 335

Gly Pro Leu Thr Arg Ala Ile Ala Arg Ala Ala Tyr Ala

340 345

<210> 4

<211> 1050

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atggacacca tcgctgctcg tgctctgacc gttatgcgtg cttgcgctac cctgcaggaa 60

gctcgtatcg ttctggaagc taacgttatg gaaatcctgg gtatcgctat caaccgttac 120

aacggtctga ccctgcgtgg tgttaccatg cgtccgacct ctctggctca gcgtaacgaa 180

atgttcttca tgtgcctgga catgatgctg tctgctgctg gtatcaacgt tggtccgatc 240

tctccggact acacccagca catggctacc atcggtgttc tggctacccc ggaaatcccg 300

ttcaccaccg aagctgctaa cgaaatcgct cgtgttaccg gtgaaacctc tacctggggt 360

ccggctcgtc agccgtacgg tttcttcctg gaaaccgaag aagtttacca gccgggtcgt 420

tggttcatgc gtgctgctca ggttgttacc ccggttgttt gcggtccgga catgatccag 480

gtttctctga acgctggtgc tcgtggtgac gttcagcaga tcttccaggg tcgtaacgac 540

ccgatgatga tctacctggt ttggcgtcgt atcgaaaact tctctatgcc gcagggtaac 600

tctcagcgta ccctggctgg tgttaccgtt tctgttggtg gtgttgacat gcgtgctggt 660

cgtatcatcg cttgggacgg tcaggctgtt ctgcagatcc acaacccgac ccagcagaac 720

gctatggttc agatccaggt tgttttctac atctctatgg acaaaaccct gaaccagtac 780

ccggctctga ccgctgaaat cttcaacgtt tactctttcc gtgaccacac ctggcacggt 840

ctgcgtaccg ctatcctgaa ccgtaccacc ctgccgaaca tgctgccgcc gatcttcccg 900

ccgaacgacc gtgactctat cctgaccatc ctgctgctgt ctaccctggc tgacgtttac 960

tctgttctgc gtccggaatt cgctatccac ggtgttaacc cgatgccggg tccgctgacc 1020

cgtgctatcg ctcgtgctgc ttacgcttga 1050

Claims

1. a protein for detecting bluetongue, is characterized in that, described protein is shown in A1) or A2):

A1) The amino acid sequence is the protein shown in positions 2-219 of SEQ ID No.1;

A2) A fusion protein obtained by linking a protein tag to the N-terminal or/and C-terminal of the amino acid sequence shown in positions 2-219 of SEQ ID No. 1.

2. A protein encoding gene for detecting bluetongue disease, wherein the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

3. A recombinant vector comprising the encoding gene of claim 2.

4. recombinant vector according to claim 3, is characterized in that, described recombinant vector is that the coding gene described in claim 2 is connected between Nco I and Xho I restriction enzyme site of pet-28a, and keeps The recombinant vector VP7-N-28a obtained with the other sequences of pet-28a unchanged.

5 . The recombinant bacteria containing the recombinant vector of claim 3 or 4 , wherein the host cells of the recombinant bacteria are Escherichia coli BL21 (DE3) competent cells. 6 .

6. The application of the encoding gene according to claim 2, or the recombinant vector according to claim 3 or 4, or the recombinant bacteria according to claim 5 in preparing a protein for detecting bluetongue disease.

7. A soluble preparation method for detecting the protein of bluetongue disease, characterized in that, the soluble preparation method comprises the steps:

Synthesize the gene encoding the protein for detecting bluetongue: the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2;

Construct a recombinant vector comprising the encoding gene of the protein used for detecting bluetongue disease: the recombinant vector is the recombinant vector VP7-N-28a, and the recombinant vector VP7-N-28a is a combination of the SEQ ID NO. The coding gene for the protein shown for detecting bluetongue disease is connected between the Nco I and Xho I restriction sites of pet-28a, and other sequences of pet-28a are kept unchanged;

The recombinant bacteria obtained by constructing the recombinant vector containing the encoding gene of the protein used for detecting bluetongue disease: the recombinant bacteria are recombinant bacteria BL21(De3)/VP7-N-28a, and the recombinant bacteria BL21(De3)/VP7 -N-28a is obtained by transforming the recombinant vector VP7-N-28a into E. coli BL21 (De3) competent cells;

Induced expression: Inoculate the recombinant bacteria BL21(De3)/VP7-N-28a into LB liquid medium, add IPTG with a final concentration of 0.5mM to induce expression, continue to culture, and collect the bacteria by centrifugation, which can be used to detect blue Tongue protein.

8. The soluble preparation method according to claim 7, characterized in that, the method further comprises the step of further affinity purification.