CN112521462B

CN112521462B - Horse infectious anemia virus p26-gp90 recombinant protein and preparation method and application thereof

Info

Publication number: CN112521462B
Application number: CN202011474580.XA
Authority: CN
Inventors: 李晓光; 何坚锋; 王哲侃
Original assignee: Hangzhou Ever Genetics Biotech Co ltd
Current assignee: Hangzhou Ever Genetics Biotech Co ltd
Priority date: 2020-12-14
Filing date: 2020-12-14
Publication date: 2023-09-19
Anticipated expiration: 2040-12-14
Also published as: CN112521462A; CN117074671A

Abstract

The application discloses a p26-gp90 recombinant protein of equine infectious anemia virus, belonging to the field of animal virus antibody detection. The recombinant protein comprises an amino acid sequence shown as SEQ ID NO.1 or consists of the amino acid sequence shown as SEQ ID NO. 1. The application further discloses a gene of the recombinant protein, a vector and a host cell containing the gene, a preparation method of the recombinant protein and application of the recombinant protein in detecting an antibody of equine infectious anemia virus. The recombinant protein of the application is used for detecting the equine infectious anemia virus antibody, is convenient and quick, has high sensitivity, has no cross reaction with other pathogens, has strong specificity, and has great clinical significance and wide application prospect.

Description

Horse infectious anemia virus p26-gp90 recombinant protein and preparation method and application thereof

Technical Field

The application belongs to the field of animal virus antibody detection, and particularly relates to a p26-gp90 recombinant protein of equine infectious anemia virus, and a preparation method and application thereof.

Background

Equine infectious anaemia is abbreviated as equine pollakin (EIA, equine Infectious Anemia), infectious diseases of horses, mules, donkeys caused by equine infectious anaemia virus (Equine infectious anemia virus) in the lentivirus subfamily of retrovirus family. Equine infectious anemia virus is a causative agent of infectious disease in equine animals, equine infectious anemia, which is a retrovirus. Equine infectious anaemia is characterized by intermittent fever, wasting, progressive weakness, anaemia, bleeding and edema; symptoms gradually decrease or temporarily disappear during the period of no burn.

The agar gel immunodiffusion assay was developed by the U.S. veterinary microbiologist l. kejin si and nocorks, which has been currently listed in our country as one of the equine transmitted lean specific diagnostic methods. In addition, fluorescent antibody staining techniques and neutralization assays, enzyme-linked immunosorbent assay (ELISA) can also be used as an auxiliary diagnostic assay for equine dysplasia, and in addition, biological assays (vaccinations with healthy foal) can be performed. However, the methods such as immunofluorescence staining technique and enzyme-linked immunosorbent assay (ELISA) require specific instruments and equipment, have corresponding test conditions and skills, and are difficult to popularize in the basic layer.

Disclosure of Invention

In order to solve the technical problems, the application adopts the following technical scheme:

the first aspect of the application provides a p26-gp90 recombinant protein of equine infectious anemia virus, which comprises an amino acid sequence shown as SEQ ID NO. 1.

In the present application, the recombinant protein is also called a fusion protein or a recombinant fusion protein, and is an expression product obtained by recombination of two genes by a DNA recombination technique.

The Capsid protein (CA) p26 protein is the main immunogenic protein of equine infectious anemia virus, which can induce and start the immune system of the organism to generate immune response, and the protein is mostly used as detection antigen at home and abroad at present, but if the kind of detection antigen is too single, there is a risk of missed detection. In order to reduce the risk of omission, the p26 protein and the full-length protein sequence of the other protein gp90 are fused and expressed, so that the diagnostic sensitivity can be remarkably improved. Therefore, the p26 main epitope and the gp90 protein full-length sequence are fused together for expression, so that the diagnostic sensitivity can be improved, and the cross reaction with other pathogens can be reduced.

In some embodiments of the application, preferably, the recombinant protein consists of the amino acid sequence shown in SEQ ID NO. 1.

In a second aspect the application provides a gene encoding a recombinant protein according to the first aspect of the application comprising the nucleotide sequence shown in SEQ ID NO. 2.

The gene sequence is used for expressing the recombinant protein in escherichia coli, and the codons are optimized according to the preference of the escherichia coli to the codons. The frequency of usage of synonymous codons varies from species to species, and this codon bias has an impact on the translation process. If an mRNA has many rare codons clustered, this can negatively affect the rate of ribosome movement, greatly reducing protein expression levels. The codon optimization is carried out on the gene sequence, so that the method is suitable for the expression of escherichia coli and can improve the protein expression efficiency.

In a third aspect the application provides an expression vector comprising a gene according to the second aspect of the application.

In some embodiments of the application, the expression vector is pET30a, which is kanamycin resistant, and the expressed fusion protein has a histidine (His) tag.

In a fourth aspect, the application provides a host cell comprising an expression vector according to the third aspect of the application.

Further, the host cell is a eukaryotic host cell or a prokaryotic host cell.

In some embodiments of the application, the host cell is a prokaryotic host cell. Preferably, the host cell is E.coli, more preferably, E.coli is BL21. The method for expressing the bacillus coli has the advantages of short period, low cost, large expression quantity and the like.

In a fifth aspect the present application provides a method of producing a recombinant protein according to the first aspect of the application, comprising the step of inducing protein expression in a host cell according to the fourth aspect of the application.

Further, the host cell is a eukaryotic host cell or a prokaryotic host cell.

In some embodiments of the application, the step of inducing E.coli to perform protein expression is:

s1, culturing the escherichia coli at 37 ℃ by using an LB medium containing 50 mug/mL kanamycin,

s2, when the OD600 of the escherichia coli culture solution is 0.5-0.7, the induction expression is carried out by using IPTG with the final concentration of 1mM, wherein the induction conditions are as follows: 25 ℃, and rotating at 200rpm for 4 hours; by using the induction conditions, the recombinant protein can be expressed more slowly, and the formation of the space conformation can be carried out for a sufficient time, which plays a very important role in the function of the recombinant protein.

S3, centrifuging the culture solution at 4 ℃ and 7000rpm for 10min, and collecting thalli;

s4, crushing thalli by using a Buffer solution Binding Buffer;

s5, ultrasonically crushing thalli, wherein the conditions are as follows: 550w, ultrasonic treatment for 2s and interval for 5s, which are 80-120 times;

s6, centrifuging at 12000rpm for 30min at 4 ℃ to collect supernatant, wherein the recombinant protein is in the supernatant.

Preferably, the induction is performed in step S2 at an E.coli culture OD600 of 0.6.

Preferably, in step S5, the ultrasonic crushing is performed 100 times. The crushing method of the application avoids the situation of recombinant protein loss caused by too severe crushing.

In some embodiments of the application, the method further comprises the step of purifying the recombinant protein. The recombinant protein may be purified by various methods such as ion exchange chromatography, gel filtration chromatography and affinity chromatography. In some embodiments of the application, the method of affinity chromatography is selected, and higher purity can be achieved by one-step purification due to the addition of His tag to the recombinant protein.

In some embodiments of the application, the supernatant comprising the recombinant protein is passed through a Ni column and then eluted with an Elution Buffer, such as an Elution Buffer, to obtain the protein of interest.

Preferably, the formulation of the Elution Buffer solution is: 50mM Tris,0.2M Nacl,0.5M Imidazole,pH8.0.

In a sixth aspect, the application provides the use of a recombinant protein according to the first aspect of the application in the preparation of a kit for detecting equine infectious anemia virus antibodies.

In a seventh aspect, the application provides a kit for detecting antibodies to equine infectious anemia virus, comprising the recombinant protein according to the first aspect of the application.

Further, the kit also comprises mouse IgG and sheep anti-mouse IgG.

In some embodiments of the application, equine infectious anemia virus antibodies are detected using a double antigen sandwich Jin Biaofa.

In some embodiments of the application, the kit comprises a double antigen sandwich gold-labeled test strip, the test strip having the following reagents:

s1, respectively preparing a recombinant protein colloidal gold complex and a mouse IgG colloidal gold complex;

s2, mixing the recombinant protein colloidal gold complex and the mouse IgG colloidal gold complex to prepare a gold-labeled pad;

s3, drawing on a nitrocellulose membrane by using recombinant protein as a detection line and sheep anti-mouse IgG as a quality control line;

and S4, mounting filter paper, a polyester plate containing a nitrocellulose membrane, a gold mark pad and a sample pad on a bottom plate, wherein a part of the filter paper is overlapped on the polyester plate, a part of the polyester plate is overlapped on the gold mark pad, a part of the gold mark pad is overlapped on the sample pad, a test area and a quality control area are respectively arranged on the polyester plate, a detection line (T line) is arranged in the test area, a quality control line (C line) is arranged in the quality control area, the detection line is close to the gold mark pad, and the quality control line is close to the filter paper, so that the detection strip is prepared.

When the method is used, a biological sample of the equine animal is dripped to the sample pad, the detection result is judged after the equine animal is placed at room temperature for 10min, and the judgment standard is as follows:

(1) two bands appear, one of which is positioned in the quality control area and the other one of which is positioned in the test area, and is positive;

(2) only one strip appears on the quality control line, no strip appears in the test area, and the test area is negative;

(3) the quality control line has no strip, which indicates that the test strip is damaged, and whether the strip appears on the detection line or not, a new test strip should be replaced for retesting.

In some embodiments of the application, a positive equine infectious anemia virus antibody assay indicates that the equine animal biological sample contains equine infectious anemia virus antibodies, meaning that the equine animal has or was infected with equine infectious anemia virus.

In some embodiments of the application, the biological sample is serum or plasma, or any other body fluid that may contain antibodies.

In the present application, the equine animal is selected from the group consisting of horses, mules, donkeys.

The beneficial effects of the application are that

Compared with the prior art, the application has the following beneficial effects:

the p26 and gp90 proteins are main immunogenic antigens of equine infectious anemia viruses and are highly conserved, and can induce and start an immune system of an organism to generate immune response and induce host cells to generate neutralizing antibodies, so that the main antigen epitope of the p26 protein and the full-length sequence of the gp90 protein are fused together for expression, the diagnostic sensitivity can be improved, the cross reaction with other pathogens can be reduced, the specificity is strong, and the method has great clinical significance and wide application prospect.

The colloidal gold labeled immunoassay method adopted by the application is a novel analysis technology, has the characteristics of rapidness, simplicity, convenience, low cost, no pollution and no need of training, is more suitable for on-site detection compared with the traditional method, has the advantages of short color development time, no need of expensive instruments and the like, and has wide market prospect and application value.

Drawings

FIG. 1 shows the results of gel electrophoresis of the purification of the equine infectious anemia virus p26-gp90 fusion protein. 1: loading the crushed cells; 2: flow through; 3:50mM Imidazole elution; 4:0.5M Imidazole elution.

FIG. 2 shows a reagent chart of a test strip according to an embodiment of the present application. 1: a sample pad; 2: a gold mark pad; 3: NC film; 31: a detection line (T line); 32: a quality control line (C line); 4: a filter paper; 5: a bottom plate.

FIG. 3 is a schematic diagram showing the detection result by using the test strip according to one embodiment of the present application. T: detection line, C: and a quality control line.

Fig. 4 shows the overall results of testing clinical horse serum samples using the test strip of the present application.

Detailed Description

In order to make the technical problems, technical schemes and beneficial effects solved by the application more clear, the application is further described in detail below with reference to the embodiments.

Examples

The following examples are presented herein to demonstrate preferred embodiments of the present application. It will be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the practice of the application, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the application.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, the disclosure of which is incorporated herein by reference as is commonly understood by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the application described herein. Such equivalents are intended to be encompassed by the claims.

The experimental methods in the following examples are conventional methods unless otherwise specified. The instruments used in the following examples are laboratory conventional instruments unless otherwise specified; the test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.

EXAMPLE 1 construction of the Gene expression vector for the equine infectious anemia Virus p26-gp90 fusion protein

The equine infectious anemia virus p26 Gene was designed based on the NCBI Gene bank: ABE03841 protein sequence. The surface protein (Envelope polyprotein) gp90 Gene was designed based on the NCBI Gene bank: AAC24024 protein sequence.

The amino acid sequence of the p26-gp90 recombinant protein fusion is as follows (SEQ ID NO. 1):

EFIDGAGNRNFRPLTPRGYTTWVNTIQQHNLLNEASVNLFGILSVDCTSEEMNAFLDVVPGQAGQKQVLLDALDKIAEDWDNRHPLPNAPLVAPPQGPIPMTARFIRGLGVPRERQMEPAFDQFRQTYRQWIIEAMTEGIKVMTGKPKAQNIRQGPKEPYPEFVDRLLSQIESEGHSTEITRFLTDTLTIQNANEECRNAMRHLRPEDSLEEKMYACRDFGSTKLSKNSMAESKEARDQEMNLKEESKEEKRRNDWWKIGMFLLCLAGTTGGILWWYEGLPQQHYIGLVAIGGRLNGSGQSNAIECWGSFPGCRPFQNYFSYETNRSMHMNNNTATLLEAYHREITFIYKSSCTDSDHCQEYQCKKVDLINSSSNSVRVVENETTTEYWGFKWLECNQTENLKTILVPENEMVNINDSDTWIPKGCNETWARVKRCPIDILYGIHPIRLCVQPPFFLVQEKGIANNSRISNCGPTIFLGVLEDNKGVIRGNSTICKVNITEIKRKDYTGIYQVPIFYTCNFTNITSCNNESIISVIMYDTNQVQYLLCNNNNSNNYNCVVQSFGVIGQAHLELPRLNKRIRNQSFNQYNCSINNKTELETWKLVKTSGITPLPIS

since the frequency of usage of synonymous codons is different for different species, this codon bias has an impact on the translation process. If an mRNA has many rare codons clustered, this can negatively affect the rate of ribosome movement, greatly reducing protein expression levels.

In the application, escherichia coli is used as an expression system, in order to obtain higher expression efficiency and higher expression quantity, codon optimization is carried out when exogenous protein expression is carried out, and the exogenous protein expression is reversely translated into a nucleotide sequence, so that the obtained nucleotide sequence is as follows (SEQ ID NO. 2):

GAATTTATTGATGGCGCGGGCAACCGCAACTTTCGCCCGCTGACCCCGCGCGGCTATACCACCTGGGTGAACACCATTCAGCAGCATAACCTGCTGAACGAAGCGAGCGTGAACCTGTTTGGCATTCTGAGCGTGGATTGCACCAGCGAAGAAATGAACGCGTTTCTGGATGTGGTGCCGGGCCAGGCGGGCCAGAAACAGGTGCTGCTGGATGCGCTGGATAAAATTGCGGAAGATTGGGATAACCGCCATCCGCTGCCGAACGCGCCGCTGGTGGCGCCGCCGCAGGGCCCGATTCCGATGACCGCGCGCTTTATTCGCGGCCTGGGCGTGCCGCGCGAACGCCAGATGGAACCGGCGTTTGATCAGTTTCGCCAGACCTATCGCCAGTGGATTATTGAAGCGATGACCGAAGGCATTAAAGTGATGACCGGCAAACCGAAAGCGCAGAACATTCGCCAGGGCCCGAAAGAACCGTATCCGGAATTTGTGGATCGCCTGCTGAGCCAGATTGAAAGCGAAGGCCATAGCACCGAAATTACCCGCTTTCTGACCGATACCCTGACCATTCAGAACGCGAACGAAGAATGCCGCAACGCGATGCGCCATCTGCGCCCGGAAGATAGCCTGGAAGAAAAAATGTATGCGTGCCGCGATTTTGGCAGCACCAAACTGAGCAAAAACAGCATGGCGGAAAGCAAAGAAGCGCGCGATCAGGAAATGAACCTGAAAGAAGAAAGCAAAGAAGAAAAACGCCGCAACGATTGGTGGAAAATTGGCATGTTTCTGCTGTGCCTGGCGGGCACCACCGGCGGCATTCTGTGGTGGTATGAAGGCCTGCCGCAGCAGCATTATATTGGCCTGGTGGCGATTGGCGGCCGCCTGAACGGCAGCGGCCAGAGCAACGCGATTGAATGCTGGGGCAGCTTTCCGGGCTGCCGCCCGTTTCAGAACTATTTTAGCTATGAAACCAACCGCAGCATGCATATGAACAACAACACCGCGACCCTGCTGGAAGCGTATCATCGCGAAATTACCTTTATTTATAAAAGCAGCTGCACCGATAGCGATCATTGCCAGGAATATCAGTGCAAAAAAGTGGATCTGATTAACAGCAGCAGCAACAGCGTGCGCGTGGTGGAAAACGAAACCACCACCGAATATTGGGGCTTTAAATGGCTGGAATGCAACCAGACCGAAAACCTGAAAACCATTCTGGTGCCGGAAAACGAAATGGTGAACATTAACGATAGCGATACCTGGATTCCGAAAGGCTGCAACGAAACCTGGGCGCGCGTGAAACGCTGCCCGATTGATATTCTGTATGGCATTCATCCGATTCGCCTGTGCGTGCAGCCGCCGTTTTTTCTGGTGCAGGAAAAAGGCATTGCGAACAACAGCCGCATTAGCAACTGCGGCCCGACCATTTTTCTGGGCGTGCTGGAAGATAACAAAGGCGTGATTCGCGGCAACAGCACCATTTGCAAAGTGAACATTACCGAAATTAAACGCAAAGATTATACCGGCATTTATCAGGTGCCGATTTTTTATACCTGCAACTTTACCAACATTACCAGCTGCAACAACGAAAGCATTATTAGCGTGATTATGTATGATACCAACCAGGTGCAGTATCTGCTGTGCAACAACAACAACAGCAACAACTATAACTGCGTGGTGCAGAGCTTTGGCGTGATTGGCCAGGCGCATCTGGAACTGCCGCGCCTGAACAAACGCATTCGCAACCAGAGCTTTAACCAGTATAACTGCAGCATTAACAACAAAACCGAACTGGAAACCTGGAAACTGGTGAAAACCAGCGGCATTACCCCGCTGCCGATTAGC

recombinant gene sequences were synthesized by the division of bioengineering (Shanghai) and ligated with pET30a plasmid to form recombinant expression vectors.

EXAMPLE 2 expression of the equine infectious anemia Virus p26-gp90 fusion protein

The equine infectious anemia virus p26-gp90 fusion gene plasmid is transformed into escherichia coli BL21, coated on an LB plate containing 50 mug/mL kanamycin (Shanghai, cat# K0408), cultured overnight at 37 ℃, a monoclonal colony is selected, the monoclonal colony is cultured to an OD600 of about 0.6 by 300mL LB culture medium containing the same concentration of kanamycin at 37 ℃, and induced expression is carried out by using IPTG (Shanghai, cat# IB 0168) with a final concentration of 1 mM: 25 ℃, rotating at 200rpm for 4 hours. After induction, the culture medium was centrifuged at 7000rpm at 4℃for 10min to collect the cells.

EXAMPLE 3 purification and renaturation of the equine infectious anemia Virus p26-gp90 fusion protein

50mL of a Binding Buffer (50mM Tris,0.2M Nacl,pH8.0) was used to break the cells with 50mL of a loading Buffer; then, carrying out ultrasonic crushing under the condition of 550w for 2s at intervals of 5s for 100 times; finally, the supernatant was collected by centrifugation at 12000rpm for 30min at 4℃and the target protein was contained in the supernatant. Then purifying the mixture by a Ni column, and eluting the target protein by an Elution Buffer (50mM Tris,0.2M Nacl,0.5M Imidazole,pH8.0). The target protein was detected by PAGE gel electrophoresis, and the results are shown in FIG. 1.

As can be seen from FIG. 1, the purified fusion protein had high purity, and the purified recombinant protein was dialyzed against dialysis buffer (50mM Tris,0.2M Nacl,pH8.0) with 3 changes of dialysis solution every 12 hours. The dialyzed protein solution was taken out, filtered through a 0.22 μm filter, and the concentration was measured by BCA method, followed by storage at-20℃for further use.

Example 4 double antigen sandwich Jin Biaofa detection of equine infectious anemia Virus antibody

Preparation of 1 double antigen sandwich gold-labeled method detection strip

1.1 firing of colloidal gold

1000mL of ultrapure water is added into the triangular flask, the mixture is heated to boiling on a magnetic heating stirrer, then 4mL of 10% chloroauric acid (sigma) is added, 6mL of 10% trisodium citrate solution is added, heating and boiling are continued for 5min, then the mixture is cooled to room temperature, and after the colloidal gold is filtered by a 0.22 mu m filter, the mixture is placed at 4 ℃ for standby.

1.2 labelling of recombinant equine infectious anemia Virus p26-gp90 fusion proteins

100mL of colloidal gold solution is taken and put into a beaker, and 0.2. 0.2M K is added by stirring ₂ CO ₃ The pH of the gold water is regulated to 7.5, 1.5mg of purified recombinant equine infectious anemia virus p26-gp90 fusion protein is added after stirring, stirring is carried out for 15min at room temperature, 1mL of 10% BSA solution is added, stirring is carried out for 15min at room temperature, centrifugation is carried out at 12000rpm for 10min, the supernatant is carefully sucked out and discarded, and the precipitate is fixed to 1mL by gold standard diluent (20mM Tris,1%BSA,0.03%Proclin300,pH8.0), thus obtaining the labeled recombinant equine infectious anemia virus p26-gp90 fusion protein colloidal gold complex.

1.3 murine IgG markers

100mL of colloidal gold solution is taken and put into a beaker, and 0.2. 0.2M K is added by stirring ₂ CO ₃ The pH of the gold water was adjusted to 7.0, 1mg of mouse IgG (product number: AS00901, hangzhou Longji biotechnology Co., ltd.) was added after stirring, stirring was performed at room temperature for 15 minutes, 1mL of 10% BSA solution was added, stirring was performed at room temperature for 15 minutes, centrifugation was performed at 12000rpm for 10 minutes, the supernatant was carefully aspirated and discarded, and the precipitate was fixed to 1mL with gold-labeled diluent (20mM Tris,1%BSA,0.03%Proclin300,pH8.0), which was a labeled mouse IgG colloidal gold complex.

Diluting the gold-labeled compound by 100 times with gold-labeled diluent, mixing with the colloidal gold compound of the equine infectious anemia virus p26-gp90 fusion protein diluted in the step 1.2, soaking glass fibers, and drying at 37 ℃ for 4 hours to prepare the gold-labeled pad.

1.4 Point membrane of recombinant equine infectious anemia Virus p26-gp90 fusion protein

The purified p26-gp90 fusion protein was diluted to 0.9mg/mL with a spot film diluent (50 mM Tris,2% sucrose, pH 8.5) as a detection Line (Test-Line, T Line) for colloidal gold Test strips, goat anti-mouse IgG (Hangzhou Longji Biotechnology Co., ltd., product number: PS 00901) was diluted to 0.3mg/mL with the same diluent as a Control-Line (C Line) for colloidal gold Test strips, and the two diluted solutions were streaked onto nitrocellulose membranes and dried at 37℃overnight.

1.5 double antigen sandwich Jin Biaofa Assembly of test strip for detecting equine infectious anemia virus antibody

And (3) assembling the gold-labeled pad, the coated raw materials on a polyester plate of a nitrocellulose membrane (NC membrane), a filter paper, a sample pad and other mounting bottom plates to form the equine infectious anemia virus antibody double-antigen sandwich method detection test strip. The specific installation mode is as shown in fig. 2: sample pad 1, gold-labeled pad 2, NC membrane 3, and filter paper 4 are mounted on bottom plate 5, respectively. Wherein the sample pad 1 is partially superimposed on the gold-labeled pad 2, the gold-labeled pad 2 is partially superimposed on the NC membrane 3, and the filter paper 4 is partially superimposed on the NC membrane 3. The NC film 3 is divided into a test area and a quality control area, the test area is provided with a detection line 31 (T line), the quality control area is provided with a quality control line 32 (C line), the detection line 31 is close to the gold mark pad 2, and the quality control line 32 is close to the filter paper 4.

Further, the assembled test strip is cut into 3mm strips by a strip cutter, and then is put into a specially formulated plastic card, thus obtaining the mature detection reagent card.

Detection of 2 double antigen sandwich Jin Biaofa detection equine infectious anemia virus antibody test strip/card

Adding 90 mu L of sample to be detected (horse serum, plasma) to a sample adding place (S), standing at room temperature for 10min, and judging the result, wherein the judgment standard of the result is as follows (shown in figure 3):

(4) two bands appear, one of which is positioned in the quality control area and the other one of which is positioned in the test area, and is positive;

(5) only one strip appears on the quality control line, no strip appears in the test area, and the test area is negative;

(6) the quality control line has no strip, which indicates that the test strip is damaged, and whether the strip appears on the detection line or not, a new test strip should be replaced for retesting.

Detection result of 3 double antigen sandwich Jin Biaofa detection equine infectious anemia virus antibody test strip/card

A total of 20 horse infectious anemia virus infection positive horse serum (sample numbers 1-20), and 50 normal non-diseased and non-immunized horse serum (sample numbers 21-70), wherein the two lines of T line and C line represent positive detection results, and only one line of C line represents negative detection results.

The test results are shown in Table 1: positive 19 out of 20 positive sera were detected, 1 (sample No. 16) was missed, and 3 (sample No. 26, sample No. 34, and sample No. 41) false positive occurred in 50 negative sera.

TABLE 1 detection results of equine infectious anemia Virus antibody

As a result, the sensitivity and specificity of the sample detection were 99.5% and 94%, respectively, and the overall compliance was 94.3%, as shown in FIG. 4.

The result shows that the recombinant equine infectious anemia virus p26-gp90 fusion protein of the present application is used for detecting equine infectious anemia virus, has very high sensitivity and specificity, can be used as raw materials for preparing equine infectious anemia virus antibody detection test strips, and can be widely applied in clinical detection.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Sequence listing

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acctgggtga acaccattca gcagcataac ctgctgaacg aagcgagcgt gaacctgttt 120

ggcattctga gcgtggattg caccagcgaa gaaatgaacg cgtttctgga tgtggtgccg 180

ggccaggcgg gccagaaaca ggtgctgctg gatgcgctgg ataaaattgc ggaagattgg 240

gataaccgcc atccgctgcc gaacgcgccg ctggtggcgc cgccgcaggg cccgattccg 300

atgaccgcgc gctttattcg cggcctgggc gtgccgcgcg aacgccagat ggaaccggcg 360

tttgatcagt ttcgccagac ctatcgccag tggattattg aagcgatgac cgaaggcatt 420

aaagtgatga ccggcaaacc gaaagcgcag aacattcgcc agggcccgaa agaaccgtat 480

ccggaatttg tggatcgcct gctgagccag attgaaagcg aaggccatag caccgaaatt 540

acccgctttc tgaccgatac cctgaccatt cagaacgcga acgaagaatg ccgcaacgcg 600

atgcgccatc tgcgcccgga agatagcctg gaagaaaaaa tgtatgcgtg ccgcgatttt 660

ggcagcacca aactgagcaa aaacagcatg gcggaaagca aagaagcgcg cgatcaggaa 720

atgaacctga aagaagaaag caaagaagaa aaacgccgca acgattggtg gaaaattggc 780

atgtttctgc tgtgcctggc gggcaccacc ggcggcattc tgtggtggta tgaaggcctg 840

ccgcagcagc attatattgg cctggtggcg attggcggcc gcctgaacgg cagcggccag 900

agcaacgcga ttgaatgctg gggcagcttt ccgggctgcc gcccgtttca gaactatttt 960

agctatgaaa ccaaccgcag catgcatatg aacaacaaca ccgcgaccct gctggaagcg 1020

tatcatcgcg aaattacctt tatttataaa agcagctgca ccgatagcga tcattgccag 1080

gaatatcagt gcaaaaaagt ggatctgatt aacagcagca gcaacagcgt gcgcgtggtg 1140

gaaaacgaaa ccaccaccga atattggggc tttaaatggc tggaatgcaa ccagaccgaa 1200

aacctgaaaa ccattctggt gccggaaaac gaaatggtga acattaacga tagcgatacc 1260

tggattccga aaggctgcaa cgaaacctgg gcgcgcgtga aacgctgccc gattgatatt 1320

ctgtatggca ttcatccgat tcgcctgtgc gtgcagccgc cgttttttct ggtgcaggaa 1380

aaaggcattg cgaacaacag ccgcattagc aactgcggcc cgaccatttt tctgggcgtg 1440

ctggaagata acaaaggcgt gattcgcggc aacagcacca tttgcaaagt gaacattacc 1500

gaaattaaac gcaaagatta taccggcatt tatcaggtgc cgatttttta tacctgcaac 1560

tttaccaaca ttaccagctg caacaacgaa agcattatta gcgtgattat gtatgatacc 1620

aaccaggtgc agtatctgct gtgcaacaac aacaacagca acaactataa ctgcgtggtg 1680

cagagctttg gcgtgattgg ccaggcgcat ctggaactgc cgcgcctgaa caaacgcatt 1740

cgcaaccaga gctttaacca gtataactgc agcattaaca acaaaaccga actggaaacc 1800

tggaaactgg tgaaaaccag cggcattacc ccgctgccga ttagc 1845

Claims

1. The equine infectious anemia virus p26-gp90 recombinant protein is characterized by consisting of an amino acid sequence shown in SEQ ID NO. 1.

2. A gene encoding the recombinant protein of claim 1, comprising the nucleotide sequence set forth in SEQ ID No. 2.

3. An expression vector comprising the gene of claim 2.

4. A host cell comprising the expression vector of claim 3.

5. A method of producing the recombinant protein of claim 1, comprising the step of inducing protein expression in the host cell of claim 4.

6. The method of claim 5, wherein the expression vector is pET30a and the host cell is e.

7. The method of claim 6, wherein the step of inducing the host cell to express the protein comprises:

s2, when the OD600 of the escherichia coli culture solution is 0.5-0.7, the induction expression is carried out by using IPTG with the final concentration of 1mM, wherein the induction conditions are as follows: 25 ℃, and rotating at 200rpm for 4 hours;

s4, crushing thalli by using a Buffer solution Binding Buffer;

8. Use of the recombinant protein of claim 1 in the preparation of a kit for detecting equine infectious anemia virus antibodies.

9. A kit for detecting equine infectious anemia virus antibodies, comprising the recombinant protein of claim 1.