Recombinant protein as well as coding gene, preparation method and application thereof in detection of horse epidemic lymphangitis

Abstract

The invention discloses a recombinant protein as well as a coding gene, a preparation method and application thereof in detection of horse epidemic lymphangitis, and belongs to the technical field of gene engineering. The recombinant protein comprises an amino acid sequence as shown in SEQ ID No. 2 and / or an amino acid sequence as shown in SEQ ID No. 6. A colloidal gold test strip prepared from the recombinant protein disclosed by the invention is used for detecting an antibody of the histoplasma septentotrichum on the basis of a double-antigen sandwich gold-labeled method, has the advantages of high sensitivity and specificity and good stability, is simple and convenient to operate, and is suitable for being used as a conventional detection means for the equine epidemic lymphangitis.

Description

Technical Field

[0001] The present application relates to the field of genetic engineering technology, and in particular to a recombinant protein and its encoding gene, a preparation method and application in detecting equine epidemic lymphangitis. Background Art

[0002] Equine epidemic lymphangitis (EL), also known as pseudo-epizodes, is caused by Histoplasma capsulatum ( Histoplasma farciminosum A disease of horses caused by HF (Hypericytinemia) is a chronic contagious disease of horses and other equines. Its primary clinical feature is suppurative, ulcerative, and multifocal inflammation of the subcutaneous tissue, subcutaneous lymphatic vessels, and lymph nodes. Affected horses develop nodules, swelling, suppuration, and subsequent ulceration in the skin and subcutaneous connective tissue of the wrist and appendage joints, as well as on the head.

[0003] Equine epidemic lymphangitis is a chronic disease characterized by purulent inflammation of local lymph nodes and subcutaneous lymphatic vessels. Mild cases can render draft animals unable to work, while severe cases can lead to death, severely impacting agricultural production. Equine epidemic lymphangitis is primarily found in pastoral and agricultural areas, rarely occurring in forested areas. Once established, it has a distinct regional distribution and is difficult to eradicate.

[0004] Immunological methods primarily include the direct agglutination test (DAT), indirect immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), and protein blotting. While the direct agglutination test is economical and easy to perform, its specificity and sensitivity are suboptimal. Furthermore, it requires incubation and the preparation of serially diluted serum, making it unsuitable for testing large quantities of samples. The indirect immunofluorescence method can be used for epidemiological surveys, clinical diagnosis, and evaluation of treatment outcomes. However, it requires skilled operators and expensive equipment, making it unsuitable for grassroots application. ELISA and protein blotting offer high sensitivity and specificity, but their demanding experimental conditions and complex operation make them suitable only for laboratory research and not for grassroots application.

[0005] Molecular biology methods include PCR and qRT-PCR, which have good specificity and sensitivity, but still have some limitations, such as the need for a thermal cycler and optimized experimental conditions. Product detection is cumbersome and requires professional personnel to operate, which is not conducive to on-site use. Summary of the Invention

[0006] The present application aims to overcome the shortcomings of existing detection methods, such as high equipment requirements, long detection time, and unsuitability for routine detection, and provides a recombinant fusion protein HM for equine epidemic lymphangitis and its encoding gene, preparation method and application. The protein can be used to prepare colloidal gold detection reagent strips. The labeling technology has high sensitivity, good specificity, strong stability, and simple operation, and is suitable as a routine detection method for equine epidemic lymphangitis.

[0007] In order to achieve the above objectives, this application adopts the following technical solutions: In a first aspect, the present application provides a recombinant protein comprising the amino acid sequence shown in SEQ ID No. 2 or the amino acid sequence shown in SEQ ID No. 6.

[0008] Among them, the amino acid sequence shown in SEQ ID No. 2 was designed based on the amino acid sequence of Histoplasma capsulatum M protein, and the amino acid sequence shown in SEQ ID No. 6 was designed based on the amino acid sequence of Histoplasma capsulatum H protein.

[0009] In some embodiments of the present application, the recombinant protein includes the amino acid sequence shown in SEQ ID No. 2 and the amino acid sequence shown in SEQ ID No. 6. In this embodiment, the two proteins are fused and expressed, and the resulting fusion protein is used for detection, which can not only improve the sensitivity of diagnosis but also reduce cross-reactions with other pathogens.

[0010] In the present application, there are two ways to fuse the two proteins according to the order, namely, M protein first and H protein second, or H protein first and M protein second.

[0011] In some preferred embodiments of the present application, a flexible linker sequence is further included between the amino acid sequence shown in SEQ ID No. 2 and the amino acid sequence shown in SEQ ID No. 6. The flexible linker includes but is not limited to GSGSG, GSGSGSG, (GGGGS)n (when n>2, the domain is separated), (G) n (Common n=6 / 8).

[0012] In some specific embodiments of the present application, the recombinant protein includes the amino acid sequence shown in SEQ ID No. 9 or SEQ ID No. 11.

[0013] In a second aspect, the present application provides a gene for any recombinant protein according to the first aspect of the present application, encoding the amino acid sequence shown in SEQ ID No. 2 including the nucleotide sequence shown in SEQ ID No. 4.

[0014] In some embodiments of the present application, the sequence encoding the amino acid sequence shown in SEQ ID No. 6 includes the nucleotide sequence shown in SEQ ID No. 8.

[0015] In some embodiments of the present application, the nucleotide sequence of the gene is shown as SEQ ID No. 10, or as SEQ ID No. 12.

[0016] In this application, in order to express the recombinant protein in E. coli, codons have been optimized according to the preference of E. coli for codons. Different species have different usage frequencies of synonymous codons, and this codon preference has an impact on the translation process. If an mRNA has a lot of rare codons in clusters, this will have a negative impact on the movement speed of the ribosome, greatly reducing the level of protein expression. Here, the gene sequence is codon optimized, which is applicable to E. coli expression and can improve protein expression efficiency.

[0017] The third aspect of the present application provides an expression vector comprising any gene described in the second aspect of the present application.

[0018] In some embodiments of the present application, the expression vector is pET32a, which has ampicillin resistance, and the expressed fusion protein has a histidine (His) tag.

[0019] The fourth aspect of the present application provides a host cell containing the expression vector described in the third aspect of the present application.

[0020] Furthermore, the host cell is a eukaryotic host cell or a prokaryotic host cell.

[0021] In some embodiments of the present application, the host cell is a prokaryotic host cell. Preferably, the host cell is Escherichia coli, and more preferably, the Escherichia coli is BL21. Using Escherichia coli for expression has the advantages of short cycle time, low cost, and high expression yield.

[0022] The fifth aspect of the present application provides a method for preparing any recombinant protein described in the first aspect of the present application, comprising the step of expressing the protein in the corresponding host cell according to the fourth aspect of the present application.

[0023] Furthermore, the host cell is a eukaryotic host cell or a prokaryotic host cell.

[0024] In some embodiments of the present application, the host cell is a prokaryotic host cell. Preferably, the host cell is Escherichia coli, and more preferably, the Escherichia coli is BL21. Using Escherichia coli for expression has the advantages of short cycle time, low cost, and high expression yield.

[0025] In some specific embodiments of the present application, the step of inducing Escherichia coli to express protein is: S1, culturing the Escherichia coli in LB medium containing 50 μg / mL ampicillin at 37°C, S2, when the OD600 of the E. coli culture reached 0.5-0.7, IPTG was used to induce expression at a final concentration of 1 mM. The induction conditions were: 28-32°C, 150-250 rpm, for 3-5 h. S3, after induction, centrifuge and disrupt the cells to obtain the supernatant and purify and renature the expressed recombinant protein.

[0026] Preferably, in step S2, induction is performed when the OD600 of the E. coli culture solution reaches 0.6.

[0027] Preferably, in order to better maintain the active sites of the recombinant protein, the present application selects relatively mild culture and induction conditions. The induction temperature during expression is 30°C, the induction speed is 200 rpm, and the induction IPTG concentration is 1 mM. This allows the recombinant protein to be expressed more slowly and has sufficient time to form a spatial conformation.

[0028] Preferably, in step S3, the disruption is performed by ultrasonication at 400W for 3 seconds with an interval of 6 seconds. The disruption method of the present application avoids excessive disruption that may cause loss of recombinant protein.

[0029] In the present application, the recombinant protein can be purified by various methods, such as ion exchange chromatography, gel filtration chromatography, affinity chromatography, etc. In some embodiments of the present application, affinity chromatography is selected because a His tag is added to the recombinant protein, and a one-step purification can achieve a higher purity.

[0030] Preferably, during purification, the loading buffer in the column buffer is 50 mM Tris, 0.15 M NaCl, pH 8.0, and the elution buffer is 50 mM Tris, 0.15 M NaCl, 0.5 M Imidazole, pH 8.0; after purification, the collected target protein is dialyzed with 10 mM PBS, pH 7.4, and the dialysate is changed every 12 hours for a total of 3 times. After dialysis, the protein solution is sterilized by filtering with a 0.22 μm filter, and the protein concentration is determined by the Bradford method and stored at -20°C for future use.

[0031] The sixth aspect of the present application provides the use of any recombinant protein described in the first aspect of the present application in preparing a kit for detecting equine epidemic lymphangitis.

[0032] In a seventh aspect, the present application provides a kit for detecting equine epidemic lymphangitis, comprising any recombinant protein described in the first aspect of the present application.

[0033] Furthermore, the kit also includes chicken IgY and goat anti-chicken IgY.

[0034] In some embodiments of the present application, the double antigen sandwich gold label method is used to detect antibodies to Histoplasma capsulatum to further diagnose equine epidemic lymphangitis.

[0035] Colloidal gold-labeled immunoassay is a new analytical technique that has emerged and rapidly developed in recent years. It is characterized by its speed, simplicity, low cost, pollution-free nature, and the lack of training required. Compared with traditional methods, it is more suitable for on-site testing. Compared with traditional methods, it has advantages such as faster color development time and the absence of expensive instrumentation, and has broad market prospects and application value.

[0036] In some specific embodiments of the present application, the kit includes a double antigen sandwich gold label test strip, and the reagent method of the test strip is as follows: (1) Add 1000 mL of ultrapure water to a conical flask and heat to boiling on a magnetic heating stirrer. Then add 1 mL of 10% chloroauric acid (sigma) and 1 mL of 10% trisodium citrate solution. Continue heating and boiling for 15 minutes, and then cool to room temperature.

[0037] (2) Add 0.2M K2CO3 to the cooled gold water to adjust the pH to 8.0, add the recombinant antigen, stir for 15 minutes, then add the BSA solution, stir again for 30 minutes, centrifuge at 10,000 rpm for 20 minutes, and resuspend the precipitate in gold label diluent.

[0038] (3) Add 0.2 M K2CO3 to the gold water to adjust the pH to 7.0, add chicken IgY, stir for 15 minutes, then add BSA solution, stir again for 30 minutes, centrifuge at 10,000 rpm for 20 minutes, and resuspend the precipitate in gold label diluent; (4) Mixing the gold solutions labeled in steps (2) and (3) and soaking the glass fiber, and freeze-drying to obtain a gold label pad; (5) Dilute each to 0.5 mg / mL with membrane diluent, and dilute goat anti-chicken IgY polyclonal antibody to 1.0 mg / mL with the same diluent. Streak the two diluted solutions onto nitrocellulose membrane and dry at 37°C overnight. The membrane diluent is 10 mM PBS, 1% sucrose, pH 7.4; (6) Assemble the above gold label pad, coated nitrocellulose membrane and filter paper, sample pad, card shell, etc. into a colloidal gold detection reagent strip.

[0039] The gold label diluent was 20 mM Tris, 0.5% BSA, 0.1% TritonX-100, 0.01% Proclin300, pH 8.0 solution.

[0040] When using, add the horse biological sample to the sample pad and let it stand at room temperature for 10 minutes before judging the test result. The judgment criteria are as follows: ① Only one band appears on the quality control line, and no band appears in the test area, which is negative; ② Two bands appear, one in the quality control area and the other in the test area, indicating a positive result; ③ If no strip appears on the quality control line, it indicates that the test strip is damaged and a new test strip should be replaced and retested.

[0041] In some embodiments of the present application, the biological sample is serum or plasma, or any other body fluid that may contain antibodies to Histoplasma carcinomatosis.

[0042] Compared with the prior art, this application has the following beneficial effects: (1) The recombinant protein of the present application analyzed the antigenic epitopes of the full-length M protein and / or H protein, selected the most advantageous antigenic epitopes and verified them, and unexpectedly found that the M1 protein and the H1 protein had high sensitivity in detecting antibodies to Histoplasma carcinoma. The M1 protein and the H1 protein were further fused to further improve the accuracy of the detection.

[0043] The recombinant protein of the present application is obtained in an Escherichia coli expression system, which has the advantages of short production cycle, high yield, low cost and good specificity. In addition, the His tag on the recombinant protein facilitates one-step purification to achieve higher purity, and can exhibit good activity without enzyme cleavage.

[0044] The recombinant fusion protein of the present application can be used to detect antibodies to Histoplasma capsulatum using a double antigen sandwich gold label method, thereby further diagnosing equine epidemic lymphangitis.

[0045] The kit of the present application includes a colloidal gold test strip prepared using the recombinant protein, which is used to detect antibodies to Histoplasma capsulatum. It has the advantages of high sensitivity, high specificity, and good stability, and is easy to operate, and is suitable as a routine detection method for equine epidemic lymphangitis.

[0046] It should be understood that the content described in this section is not intended to identify the key or important features of the embodiments of the present application, nor is it intended to limit the scope of the present application. Other features of the present application will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The above and other objects, features and advantages of the exemplary embodiments of the present application will become readily understood by reading the detailed description below with reference to the accompanying drawings. In the accompanying drawings, several embodiments of the present application are shown in an illustrative and non-limiting manner, in which: Figure 1 The results of SDS-PAGE gel electrophoresis of each protein in Example 2 of the present application are shown; Figure 2 The results of detecting antibodies to Histoplasma capsulatum using each single protein in Example 4 of the present application are shown; Figure 3 The results of SDS-PAGE gel electrophoresis of each fusion protein in Example 5 of the present application are shown; Figure 4 The results of detecting antibodies to Histoplasma capsulatum using single proteins M2 and H2 and fusion proteins M2-H2 and H2-M2 in Example 5 of the present application are shown. DETAILED DESCRIPTION

[0048] The terms "comprising", "including", "having" and their derivatives do not exclude the presence of any other components, steps or processes and are irrelevant to whether these other components, steps or processes are disclosed in this application. To eliminate any doubt, all compositions using the terms "comprising", "including", or "having" in this application may include any additional additives, excipients or compounds unless expressly stated otherwise. In contrast, the term "essentially consisting of" excludes any other components, steps or processes from the scope of any subsequent description of the term, except those necessary for operational performance. The term "consisting of" does not include any components, steps or processes that are not specifically described or listed. Unless expressly stated otherwise, the term "or" refers to the listed members alone or in any combination thereof.

[0049] In order to make the technical problems, technical solutions and beneficial effects solved by the present application clearer and more understandable, the present application is further described in detail below in conjunction with the embodiments.

[0050] The following examples are provided herein to illustrate preferred embodiments of the present application. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present application and, therefore, can be considered preferred embodiments of the present application. However, those skilled in the art will appreciate, based on this specification, that many modifications may be made to the specific embodiments disclosed herein while still achieving the same or similar results without departing from the spirit or scope of the present application.

[0051] 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, and the disclosures and materials cited therein are hereby incorporated by reference.

[0052] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0053] The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The instruments and equipment used in the following examples, unless otherwise specified, are all conventional laboratory instruments and equipment; the experimental materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.

[0054] Example 1 Construction of a gene expression vector for the recombinant protein of Histoplasma capsulatum In order to prepare a kit for detecting equine epidemic lymphangitis with excellent sensitivity, specificity and stability, the inventors selected parts of the M protein and the H protein to construct a fusion protein.

[0055] 1. Selection of M protein Since the full length of the M protein is relatively long, it is not suitable for direct use as an antigen in the preparation of colloidal gold test strips. In this example, a partial sequence of the M protein was selected to prepare a polypeptide as an antigen for the preparation of colloidal gold test strips. Using the full length sequence of the M protein with accession number AAB84182.2 in GeneBank as a benchmark, there are two ways to select.

[0056] The first selection method is to select the first 280aa to prepare the M1 polypeptide, whose amino acid sequence is as follows: MPSGQKGPLDRRHDTLSDPTDQFLSKFYIDDEQSVLTTDVGGPIEDQHSLKAGNRGPTLLEDFIFRQKIQHFDHERVPERAVHARGAGAHGVFTSYNNWSNITAASFLNAAGKQTPVFVRFSTVAGSRGSVDSARDIHGFAT RLYTDEGNFDIVGNNVPVFFIQDAIQFPDLIHAVKPQPDSEIPQAATAHDTAWDFLSQQPSSLHALFWAMSGHGIPRSMRHVDGWGVHTFRLVTDEGNSTLVKFRWKTLQGRAGLVWEEAQALGGKNPDFHRQDLWDA (SEQ ID No. 1) The second selection method is to select the most dominant antigenic epitopes after sequence analysis and combine them in series to prepare the M2 polypeptide, the amino acid sequence of which is as follows: RRHDTLSDPTDQFLSKFYIDDEQSVLTTDVGAVKPQPDSEIPQAATAHDTAWDFLSQQQLNRHGGPNFEQLPINRPRIPFHNNNRDGAGQMYSGADGSIFDAVIVVGGLLTSASTQYPRGR (SEQ ID No. 2) In order to express the above-mentioned M1 polypeptide and M2 polypeptide in Escherichia coli, the inventors performed codon optimization on their encoding nucleotide sequences.

[0057] The optimized nucleotide sequence encoding the M1 polypeptide is shown below: ATGCCGAGCGGCCAGAAAGGCCCGCTGGATCGCCGCCATGATACCCTGAGCGATCCGACCGATCAGTTTCTGAGCAAATTTTATATTGATGATGAACAGAGCGTGCTGACCACCGATGTGGGCGGCCCGATTGAAGATCAGCATAGCCTGAAAGCGGGCAACCGCGGCCCGACCCTGCTGGAAGATTTTATTTTTCGCCAGAAAATTCAGCATTTTGATCATGAACGCGTGCCGGAACGCGCGGTGCATGCGCGCGGCGCGGGCGCGCATGGCGTGTTTACCAGCTATAACAACTGGAGCAACATTACCGCGGCGAGCTTTCTGAACGCGGCGGGCAAACAGACCCCGGTGTTTGTGCGCTTTAGCACCGTGGCGGGCAGCCGCGGCAGCGTGGATAGCGCGCGCGATATTCATGGCTTTGCGACCCGCCTGTATACCGATGAAGGCAACTTTGATATTGTGGGCAACAACGTGCCGGTGTTTTTTATTCAGGATGCGATTCAGTTTCCGGATCTGATTCATGCGGTGAAACCGCAGCCGGATAGCGAAATTCCGCAGGCGGCGACCGCGCATGATACCGCGTGGGATTTTCTGAGCCAGCAGCCGAGCAGCCTGCATGCGCTGTTTTGGGCGATGAGCGGCCATGGCATTCCGCGCAGCATGCGCCATGTGGATGGCTGGGGCGTGCATACCTTTCGCCTGGTGACCGATGAAGGCAACAGCACCCTGGTGAAATTTCGCTGGAAAACCCTGCAGGGCCGCGCGGGCCTGGTGTGGGAAGAAGCGCAGGCGCTGGGCGGCAAAAACCCGGATTTTCATCGCCAGGATCTGTGGGATGCG (SEQ ID No. 3) The optimized nucleotide sequence encoding the M2 polypeptide is shown below: CGCCGCCATGATACCCTGAGCGATCCGACCGATCAGTTTCTGAGCAAATTTTATATTGATGATGAACAGAGCGTGCTGACCACCGATGTGGGCGCGGTGAAACCGCAGCCGGATAGCGAAATTCCGCAGGCGGCGACCGCGCATGATACCGCGTGGGATTTTCTGAGCCAGCAGCAGCTGAAC CGCCATGGCGGCCCGAACTTTGAACAGCTGCCGATTAACCGCCCGCGCATTCCGTTTCATAACAACAACCGCGATGGCGCGGGCCAGATGTATAGCGGCGCGGATGGCAGCATTTTTGATGCGTGATTGTGGTGGGCGGCCTGCTGACCAGCGCGAGCACCCAGTATCCGCGCGGCCGC (SEQ IDNo. ​​4) 2. Selection of H protein For H protein, similarly, a partial sequence of H protein was selected to prepare polypeptides as antigens for preparing colloidal gold test strips. This example uses the full-length sequence of H protein with accession number AAA86880.1 in GeneBank as a benchmark, and also uses two selection methods.

[0058] First, the first 280aa were selected to prepare the H1 polypeptide, whose amino acid sequence is as follows: MRGGVLDLLPIAFASLAVATEHLTQSPPYYPSPWASGQGGWEDAVERARDFVSQLTLVEKVNLTTGVGWMQENCVGQVGSIPRMGLHSLCMQDGPLGFDLPDYVSAFPAGVNVGATFSKELAYLRGKAMGEEHRDKGVDVVL GPVVGPLGRSPDGGRNWEGFSPDPVNSGLLVAETIKGIQSAGVIACVKHFIGNEQERFRQGPEAQGYGFDISESSSSNIDDVTMHELYLWPFADAVRAGVGSVMCSYNQINNSYGCGNSYTQNKLLKAELGFQGFIMS (SEQ ID No. 5) Next, after analyzing the sequences, the most dominant epitopes were selected and combined in series to prepare the H2 polypeptide, the amino acid sequence of which is as follows: SLAVATEHLTQSPPYYPSPWASGQGGWEDAVERARDFVSQLTLIDDVTMHELYLWPFADAVRAGVGSVMCSYNQINNSYGCGNSYTQNATVSLVFVNADSGEGFISVDGNEGDRKNLTLWLSYTSFSYSDLNVEVVSSEPYMP TRGKTEPAPILGESDRNLSSYLFPEGTDRVTYYIYPWLNVTDPAKASMDSHYGLQKEDYIPPGATDGSPQELLPASGGPGGNPGLYEVLYRVTATITNTGSISGDEVPQLYVSLGGPNDAKVVLRNFDRFTLAAGEAKIW (SEQ ID No. 6) In order to express the above H1 polypeptide and H2 polypeptide in Escherichia coli, the inventors performed codon optimization on their encoding nucleotide sequences.

[0059] The optimized nucleotide sequence encoding the H1 polypeptide is shown below: ATGCGCGGCGGCGTGCTGGATCTGCTGCCGATTGCGTTTGCGAGCCTGGCGGTGGCGACCGAACATCTGACCCAGAGCCCGCCGTATTATCCGAGCCCGTGGGCGAGCGGCCAGGGCGGCTGGGAAGATGCGGTGGAACGCGCGCGCGATTTTGTGAGCCAGCTGACCCTGGTGGAAAAAGTGAACCTGACCACCGGCGTGGGCTGGATGCAGGAAAACTGCGTGGGCCAGGTGGGCAGCATTCCGCGCATGGGCCTGCATAGCCTGTGCATGCAGGATGGCCCGCTGGGCTTTGATCTGCCGGATTATGTGAGCGCGTTTCCGGCGGGCGTGAACGTGGGCGCGACCTTTAGCAAAGAACTGGCGTATCTGCGCGGCAAAGCGATGGGCGAAGAACATCGCGATAAAGGCGTGGATGTGGTGCTGGGCCCGGTGGTGGGCCCGCTGGGCCGCAGCCCGGATGGCGGCCGCAACTGGGAAGGCTTTAGCCCGGATCCGGTGAACAGCGGCCTGCTGGTGGCGGAAACCATTAAAGGCATTCAGAGCGCGGGCGTGATTGCGTGCGTGAAACATTTTATTGGCAACGAACAGGAACGCTTTCGCCAGGGCCCGGAAGCGCAGGGCTATGGCTTTGATATTAGCGAAAGCAGCAGCAGCAACATTGATGATGTGACCATGCATGAACTGTATCTGTGGCCGTTTGCGGATGCGGTGCGCGCGGGCGTGGGCAGCGTGATGTGCAGCTATAACCAGATTAACAACAGCTATGGCTGCGGCAACAGCTATACCCAGAACAAACTGCTGAAAGCGGAACTGGGCTTTCAGGGCTTTATTATGAGC (SEQ ID No. 7) The optimized nucleotide sequence encoding the H2 protein is as follows: (SEQ ID No. 8) The above nucleotide sequences were synthesized by General Biosystems (Anhui) Co., Ltd. The synthesized genes were inserted into the Escherichia coli expression vector pET32a(+) through genetic engineering methods to form a recombinant expression vector that confers ampicillin resistance.

[0060] Example 2 Protein Expression (1) The four recombinant expression vectors in Example 1 were transformed into Escherichia coli BL21 cells to obtain recombinant strains; (2) The recombinant strain was cultured in LB medium at 37°C in a shake flask until the bacterial concentration reached OD600 = 0.5-0.7. IPTG was added to a final concentration of 1 mM and induced for expression at 30°C and 200 rpm for 3-5 hours. (3) After induction, centrifuge and disrupt the cells to obtain the supernatant and purify the expressed polypeptide protein.

[0061] The E. coli BL21 expression system has the characteristics of short cycle, low cost, and high expression yield. In order to better maintain the active site of the recombinant protein, the inventors selected relatively mild culture and induction conditions. The induction temperature during expression was 30°C, the induction speed was 200 rpm, and the induction IPTG concentration was 1 mM. This allowed the recombinant protein to be expressed more slowly, allowing sufficient time for the formation of spatial conformation.

[0062] Figure 1 The SDS-PAGE gel electrophoresis results of the expressed and purified proteins showed that the sizes of the proteins were consistent with the expected sizes.

[0063] Example 3 Protein purification and refolding The bacteria were broken by ultrasonic disruption under the conditions of 400w, 3s ultrasonication, 6s interval, 50mM Tris, 0.15M NaCl, pH 8.0 loading buffer in the column buffer, and 50mM Tris, 0.15M NaCl, 0.5MImidazole, pH 8.0 elution buffer; the target protein collected after purification was dialyzed with 10mM PBS, pH 7.4, and the dialysate was changed every 12h for a total of 3 dialysates. After dialysis, the protein solution was sterilized by filtration with a 0.22μm filter, and the protein concentration was determined by the Bradford method and stored at -20℃ for future use.

[0064] Example 4 Detection of Equine Epizootic Lymphangitis Antibodies by Indirect Gold Labeling 1. Preparation of Colloidal Gold Detection Strips for Equine Epizootic Lymphangitis (1) Add 1000 mL of ultrapure water to a conical flask and heat to boiling on a magnetic heating stirrer. Then add 1 mL of 10% chloroauric acid (sigma) and 1 mL of 10% trisodium citrate solution. Continue heating and boiling for 15 minutes, and then cool to room temperature.

[0065] (2) Add 0.2M K2CO3 to the cooled gold water to adjust the pH to 8.0, add the recombinant antigen, stir for 15 minutes, then add the BSA solution, stir again for 30 minutes, centrifuge at 10,000 rpm for 20 minutes, and resuspend the precipitate in gold label diluent.

[0066] (3) Add 0.2 M K2CO3 to the gold water to adjust the pH to 7.0, add chicken IgY, stir for 15 minutes, then add BSA solution, stir again for 30 minutes, centrifuge at 10,000 rpm for 20 minutes, and resuspend the precipitate in gold label diluent; (4) Mixing the gold solutions labeled in steps (2) and (3) and soaking the glass fiber, and freeze-drying to obtain a gold label pad; (5) Dilute each to 0.5 mg / mL with membrane diluent, and dilute goat anti-chicken IgY polyclonal antibody to 1.0 mg / mL with the same diluent. Streak the two diluted solutions onto nitrocellulose membrane and dry at 37°C overnight. The membrane diluent is 10 mM PBS, 1% sucrose, pH 7.4; (6) Assemble the above gold label pad, coated nitrocellulose membrane and filter paper, sample pad, card shell, etc. into a colloidal gold detection reagent strip.

[0067] The gold label diluent was 20 mM Tris, 0.5% BSA, 0.1% TritonX-100, 0.01% Proclin300, pH 8.0 solution.

[0068] 2. Testing of equine epidemic lymphangitis with colloidal gold test strips Add 10 μL of the sample to be tested (serum, plasma) to the sample pad, then add the sample diluent. Leave it at room temperature for 10 minutes and then determine the result. The result determination criteria are as follows: ① Only one band appears on the quality control line, and no band appears in the test area, which is negative; ② Two bands appear, one in the quality control area and the other in the test area, indicating a positive result; ③ If no strip appears on the quality control line, it indicates that the test strip is damaged and a new test strip should be replaced and retested.

[0069] The positive and negative sera with consistent test results using the Equine Epizootic Lymphangitis (EL) Nucleic Acid Detection Kit (PCR electrophoresis method) GENENODE, 1431P) and the Equine Epizootic Lymphangitis ELISA Kit (Shanghai Qincheng Biotechnology Co., Ltd., QC8943) were used as validation samples.

[0070] The four proteins were spotted on the membrane and assembled into colloidal gold test strips to test positive and negative serum. The test results were as follows: Figure 2 As shown in the figure, all four test strips for positive and negative specimens met expectations, but the positive results showed four different strengths. Among them, the results obtained by the M2 and H2 dot films were the most positive, while the results obtained by the M1 and H1 dot films were weaker.

[0071] Example 5 Expression of recombinant protein and preparation of reagent strips To further improve detection sensitivity and specificity, the inventors fused the M2 and H2 proteins. During fusion, the two fragments were linked using the flexible peptide GSGSG, which allows for better folding of the expressed proteins. There are two possible fusion methods: one is to place M2 at the N-terminus and H2 at the C-terminus, resulting in an M2-H2 fusion protein; the other is to place H2 at the N-terminus and M2 at the C-terminus, resulting in an H2-M2 fusion protein.

[0072] The amino acid sequence of the M2-H2 fusion protein is as follows: RRHDTLSDPTDQFLSKFYIDDEQSVLTTDVGAVKPQPDSEIPQAATAHDTAWDFLSQQQLNRHGGPNFEQLPINRPRIPFHNNNRDGAGQMYSGADGSIFDAV IVVGGLLTSASTQYPRGRGSGSGSLAVATEHLTQSPPYYPSPWASGQGGWEDAVERARDFVSQLTLIDDVTMHELYLWPFADAVRAGVGSVMCSYNQINNSYG CGNSYTQNATVSLVFVNADSGEGFISVDGNEGDRKNLTLWLSYTSFSYSDLNVEVVSSEPYMPTRGKTEPAPILGESDRNLSSYLFPEGTDRVTYYIYPWLNV TDPAKASMDSHYGLQKEDYIPPGATDGSPQELLPASGGPGGNPGLYEVLYRVTATITNTGSISGDEVPQLYVSLGGPNDAKVVLRNFDRFTLAAGEAKIW (SEQ ID No. 9) The nucleotide sequence of the gene encoding the M2-H2 fusion protein is as follows: The amino acid sequence of the H2-M2 fusion protein is as follows: SLAVATEHLTQSPPYYPSPWASGQGGWEDAVERARDFVSQLTLIDDVTMHELYLWPFADAVRAGVGSVMCSYNQINNSYGCGNSYTQNATVSLVFVNADSGEG FISVDGNEGDRKNLTLWLSYTSFSYSDLNVEVVSSEPYMPTRGKTEPAPILGESDRNLSSYLFPEGTDRVTYYIYPWLNVTDPAKASMDSHYGLQKEDYIPPG ATDGSPQELLPASGGPGGNPGLYEVLYRVTATITNTGSISGDEVPQLYVSLGGPNDAKVVLRNFDRFTLAAGEAKIWGSGSGRRHDTLSDPTDQFLSKFYIDD EQSVLTTDVGAVKPQPDSEIPQAATAHDTAWDFLSQQQLNRHGGPNFEQLPINRPRIPFHNNNRDGAGQMYSGADGSIFDAVIVVGGLLTSASTQYPRGR (SEQ ID No. 11) The nucleotide sequence of the gene encoding the H2-M2 fusion protein is as follows: The construction of the recombinant expression vector is described in Example 1, the expression of the fusion protein is described in Example 2, the purification and renaturation of the fusion protein is described in Example 3, and the preparation and detection of the reagent strip is described in Example 4. Figure 3 shown.

[0073] Similarly, the M2 protein and H2 protein were used as controls to judge the detection results of the two fusion proteins, such as Figure 4 As shown, the four test strips of positive and negative specimens are all in line with expectations, but the positive results show four different strengths. Among them, the result obtained by H2 dot membrane is the weakest, and the result obtained by H2-M2 fusion protein dot membrane is the strongest, which is significantly stronger than the result obtained by M2-H2 fusion protein dot membrane.

[0074] Example 6 Validation of clinical specimens of the test strip To further validate the sensitivity, specificity, and accuracy of the six test strips (M1, M2, H1, H2, M2-2H, and H2-M2), the inventors conducted a large-scale validation study (using the same two test kits). A total of 40 sera positive for equine epidemic lymphangitis and 80 sera from unaffected horses were tested. The results of the four test strips are shown in Table 1. Sensitivity is calculated as: true positives / (true positives + false negatives) × 100%, specificity is calculated as: true negatives / (true negatives + false positives) × 100%, and accuracy is calculated as: (positives + negatives) / total number of specimens × 100%.

[0075] Table 1 Validation of four colloidal gold test strips on clinical specimens

[0076] The data in Table 1 show that all four test strips of the present application have good sensitivity and specificity. Among them, the colloidal gold test strip made from the recombinant protein H2-M2 dot film has the highest accuracy, indicating that the H2-M2 recombinant fusion protein is very suitable as a raw material for preparing equine epidemic lymphangitis antibody detection test strips; and the colloidal gold test strips produced have the advantages of high sensitivity and good specificity, as well as short detection time, good stability, and simple operation. The result can be determined after only 10 minutes of standing at room temperature, making it very suitable for use as a routine detection method.

[0077] In addition, it should be understood that after reading the above teachings of this application, those skilled in the art may make various changes or modifications to this application, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A recombinant protein, characterized in that It includes the amino acid sequence shown in SEQ ID No.

2.

2. The recombinant protein according to claim 1, characterized in that It also includes the amino acid sequence shown in SEQ ID No.

6.

3. The recombinant protein according to claim 2, characterized in that A flexible linker sequence is further included between the amino acid sequence shown in SEQ ID No. 2 and the amino acid sequence shown in SEQ ID No.

6.

4. The recombinant protein according to claim 3, characterized in that It includes the amino acid sequence shown in SEQ ID No. 9 or SEQ ID No.

11.

5. A gene encoding the recombinant protein according to any one of claims 1 to 4, characterized in that: The amino acid sequence encoded by SEQ ID No. 2 includes the nucleotide sequence encoded by SEQ ID No.

4.

6. An expression vector, characterized in that Comprising the gene according to claim 5.

7. A host cell, characterized in that Contains the expression vector according to claim 6.

8. A method for preparing a recombinant protein, characterized in that: The method comprises the step of inducing the host cell according to claim 7 to express the protein.

9. Use of the recombinant protein according to any one of claims 1 to 4 in the preparation of a kit for detecting equine epidemic lymphangitis.

10. A kit for detecting equine epidemic lymphangitis, characterized in that: The invention comprises the recombinant protein according to any one of claims 1 to 4.

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