A recombinant protein and its preparation method and application in bovine tuberculosis detection

Abstract

The present invention relates to the field of biotechnology and provides a recombinant protein, a preparation method thereof, and its application in bovine tuberculosis detection. The amino acid sequence of the recombinant protein is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the recombinant protein is shown in SEQ ID NO.2. The recombinant protein is obtained by serially combining ESAT-6, CFP-10, and BFH using a linker in the manner of ESAT-6-CFP-10-BFH. Through the above technical solution, the problem of low sensitivity of the EC method for detecting bovine tuberculosis in the prior art is solved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant protein, a preparation method thereof, and application thereof in bovine tuberculosis detection. Background Art

[0002] Bovine tuberculosis (TB) is a chronic disease caused by Mycobacterium bovis, a member of the genus Mycobacterium bovis. It is characterized by tuberculoid granulomas and caseous, calcified, necrotic lesions in tissues and organs. It is highly contagious and is classified as a Category II animal disease in my country. In the late 19th century, Koch designed the famous tuberculin skin test, which is currently recognized by the World Organization for Animal Health as a means of detecting bovine TB and is widely used in my country for the diagnosis and epidemiological investigation of bovine TB. However, the classic PPD (polypeptide-deoxyribonucleic acid) test suffers from poor specificity due to its complex antigenic composition. Most antigens are shared by mycobacteria and cross-react with environmental mycobacteria, which can easily lead to false-positive test results. Furthermore, tuberculin, a secondary product of M. tuberculosis, requires a production cycle of at least six months. Cultures of M. tuberculosis require multiple in vitro passages, which can lead to bacterial inactivation, gene deletions, and unclear genetic backgrounds. This can result in variable efficacy between batches of tuberculin tests, all of which can affect the results of the tuberculin skin test.

[0003] With the progress of the times, the production process of tuberculin has been continuously optimized, which has increased the output while ensuring the stability of the product. However, the essence of tuberculin has not changed. It still contains more than 200 antigens, resulting in poor diagnostic specificity. Most antigens are shared by BCG, and it is impossible to distinguish between naturally infected cattle and BCG-vaccinated cattle, which seriously interferes with the determination of positive results of bovine tuberculosis. BCG is widely vaccinated worldwide. This measure will interfere with the detection of tuberculin skin test, resulting in the elimination of animals with false positive results, causing serious economic losses to farmers.

[0004] Tuberculin has a history of nearly a century. With the increasing understanding of tuberculosis, it has been discovered that the naturally occurring CFP10 and ESAT6 proteins possess strong cellular immune activity. Literature reports indicate that ESAT-6 and CFP-10 are highly conserved, reinforcing their application in the diagnosis of bovine tuberculosis. Furthermore, studies have shown that both proteins can induce delayed-type hypersensitivity reactions, demonstrating their potential as diagnostics. Importantly, the ESAT6 and CFP10 genes are absent from the BCG gene. Using these two proteins, they can distinguish between Mycobacterium tuberculosis infection and BCG infection, demonstrating high specificity in in vitro diagnosis, reaching 93% compared to tuberculin. However, this sensitivity is only 73%, resulting in suboptimal results for the ESAT6-CFP10 recombinant protein (EC) in detecting bovine tuberculosis.

[0005] Currently, a test product using the EC method has been discovered in China, namely Chongqing Zhifei Biotechnology's "Yika," which uses the recombinant protein ESAT6-CFP10 for detecting Mycobacterium tuberculosis infection in humans. This product fuses the early secretory low molecular weight proteins ESAT6 and CFP10 of Mycobacterium tuberculosis and expresses the recombinant protein ESAT6-CFP10 via a prokaryotic expression system. This product only expresses the recombinant protein ESAT6-CFP10, but does not address the low sensitivity issue. The effectiveness of ESAT-6 and CFP-10 for detecting bovine tuberculosis remains inferior to tuberculin in detecting bovine tuberculosis. Summary of the Invention

[0006] The present invention provides a recombinant protein, a preparation method thereof and application in bovine tuberculosis detection, which solves the problem of low sensitivity of the EC method in the prior art in detecting bovine tuberculosis.

[0007] The technical solutions of the present invention are as follows:

[0008] The present invention provides a recombinant protein, the amino acid sequence of the recombinant protein is shown in SEQ ID NO.1.

[0009] The present invention also provides a gene encoding the recombinant protein, and the nucleotide sequence of the gene is shown as SEQ ID NO.2.

[0010] As a further technical solution, the recombinant protein includes ESAT-6, CFP-10 and BFH in a series combination.

[0011] As a further technical solution, the amino acid sequence of ESAT-6 is shown as SEQ ID NO.3, the amino acid sequence of CFP-10 is shown as SEQ ID NO.4, and the amino acid sequence of BFH is shown as SEQ ID NO.5.

[0012] As a further technical solution, the ESAT-6, CFP-10 and BFH are combined in series using a linker in the manner of ESAT-6-CFP-10-BFH. The amino acid sequence of the linker is shown in SEQ ID NO.6.

[0013] The present invention also provides a method for preparing the recombinant protein, comprising the following steps:

[0014] (1) The amino acid sequences of ESAT-6, CFP-10, and BFH were codon optimized respectively;

[0015] (2) Use Linker to combine the optimized ESAT-6, CFP-10, and BFH in series in the manner of ESAT-6-CFP-10-BFH to obtain the synthetic gene EC-BFH;

[0016] (3) Inserting the synthetic gene EC-BFH into the prokaryotic expression vector pET 28a to obtain the EC-BFH expression vector;

[0017] (4) The EC-BFH expression vector was transferred into the protein expression strain, and the recombinant protein was obtained after expression and purification.

[0018] As a further technical solution, the protein expression strain is a BL21 (DE3) Escherichia coli strain.

[0019] As a further technical solution, urea denaturation and renaturation are used during the purification.

[0020] As a further technical solution, the urea deformation and renaturation comprises the following steps:

[0021] (1) Cell disruption and protein denaturation: Resuspend the cells obtained after expression in a binding buffer containing urea, disrupt the cells with ultrasonication using a cell disruptor, centrifuge, and collect the supernatant;

[0022] (2) Protein purification: Affinity chromatography is used for protein purification: the supernatant is incubated with the filler for 5-20 minutes, then washed with washing buffer to remove impurities, and the protein is eluted with elution buffer;

[0023] (3) Protein refolding: The urea in the eluate is removed by dialysis to obtain the recombinant protein.

[0024] As a further technical solution, the binding buffer has a formula of: 20 mM PB, 200 mM NaCl, 10 mM imidazole, 2-6 M urea, and a solution pH of 7.4;

[0025] The formula of the washing buffer is: 20 mM PB, 200 mM NaCl, 150 mM imidazole, 2-6 M urea, pH 7.4;

[0026] The elution buffer has a formula of 20 mM PB, 200 mM NaCl, 400 mM imidazole, 2-6 M urea, and a solution pH of 7.4.

[0027] As a further technical solution, the specific operation of removing urea from the eluate by dialysis is as follows: first, the dialysis bag is treated with treatment solution 1 and treatment solution 2 in sequence, and then the eluate is transferred to the dialysis bag and placed in the refolding solution for dialyzing.

[0028] As a further technical solution, the formula of the treatment solution 1 is: 2% sodium bicarbonate, 1 mM EDTA, solution pH 8.0;

[0029] The formula of the treatment solution 2 is: 10 mM EDTA, solution pH 8.0;

[0030] The formula of the refolding solution is: 20 mM PB, wherein the formula of 0.1M PB is: 77.4 mL 1M Na2HPO4, 22.6 mL 1M NaH2PO4, and deionized water is diluted to 1 L, and the solution pH is 7.4.

[0031] The invention also proposes the use of the recombinant protein in preparing a reagent for detecting or diagnosing bovine tuberculosis.

[0032] As a further technical solution, the reagent for detecting or diagnosing bovine tuberculosis includes the recombinant protein and low molecular weight hyaluronic acid.

[0033] The invention also proposes the use of the recombinant protein in preparing a bovine tuberculosis detection kit.

[0034] The working principle and beneficial effects of the present invention are:

[0035] The tuberculin test is a skin test based on the principle of type IV hypersensitivity. Dendritic cells (DCs) are the most powerful professional antigen-presenting cells, possessing the unique ability to stimulate T cell proliferation and initiating the body's immune response. The intensity of this response depends on the efficiency with which subcutaneous DCs phagocytose, digest, and present antigens.

[0036] 1. In this invention, ESAT-6 and CFP-10 proteins are fused to bovine ferritin heavy chain (BFH) to form the recombinant protein EC-BFH. A 24-mer antibody formed from ferritin serves as the antigen backbone, increasing the molecular weight of the antigen, enhancing immunogenicity, and promoting DC phagocytosis, thereby improving the sensitivity of the EC assay for bovine tuberculosis detection. The use of bovine ferritin heavy chain also avoids immune reactions caused by heterologous antigens and false positives, thereby improving detection accuracy.

[0037] 2. In the present invention, urea denaturation and renaturation are used to purify the recombinant protein EC-BFH during preparation, which effectively avoids the formation of inclusion bodies during prokaryotic expression of EC-BFH. The purification method is simple to operate, which improves the purification efficiency and ensures the protein purity, so that the purity of the prepared recombinant protein reaches more than 90%.

[0038] 3. In the present invention, low molecular weight hyaluronic acid is added as an immunopotentiator. Low molecular weight hyaluronic acid can promote the maturation of DC cells and improve the ability of DC cells to present antigens, thereby enhancing the sensitivity of the EC method for detecting bovine tuberculosis infection and the reaction effect of the skin test.

[0039] 4. In the present invention, the reagent for detecting or diagnosing bovine tuberculosis includes the recombinant protein EC-BFH and low molecular weight hyaluronic acid. The recombinant protein EC-BFH has a large molecular weight, thereby improving the efficiency of promoting DC cells to phagocytize antigens. The low molecular weight hyaluronic acid promotes DC cell maturation, thereby improving the DC cell's ability to present antigens. Therefore, the combination of the two solves the problem of low sensitivity of the EC method for detecting bovine tuberculosis in the prior art from the two aspects of promoting DC cell phagocytosis and promoting DC cells to present antigens. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0041] Figure 1 Schematic diagram of the construction of the EC-BFH expression vector of the present invention;

[0042] Figure 2 This is an SDS-PAGE electrophoresis purity analysis diagram of the recombinant protein EC-BFH of the present invention;

[0043] Figure 3 This is a Western blot purity analysis diagram of the recombinant protein EC-BFH of the present invention;

[0044] Figure 4 This is a comparison chart of the skin test effects of the two recombinant proteins EC and EC-BFH in the present invention;

[0045] Figure 5 This is a graph showing the skin test results of low molecular weight sodium hyaluronate of the present invention;

[0046] Figure 6 This is a graph showing the experimental results of the enhancement of the EC-BFH skin test reaction by the low molecular weight sodium hyaluronate of the present invention. DETAILED DESCRIPTION

[0047] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0048] Example 1

[0049] Recombinant protein and encoding gene:

[0050] The amino acid sequence of the recombinant protein EC-BFH is shown in SEQ ID NO.1.

[0051] The nucleotide sequence of the gene encoding the recombinant protein EC-BFH is shown in SEQ ID NO.2.

[0052] Example 2

[0053] The method for preparing a recombinant protein comprises the following steps:

[0054] (1) The Mycobacterium bovis genome data (NCBI accession number: CP015773.2) were queried from the NCBI database, and the amino acid sequences of CFP-10 and ESAT-6 were selected. The amino acid sequence of bovine ferritin heavy chain (BFH) from Bos bortus was queried from the NCBI database (ID: NP_776487.1). The codons of the selected amino acid sequences of ESAT-6, CFP-10, and BFH were optimized to make them suitable for efficient expression in E. coli. Among them, the amino acid sequence of ESAT-6 is shown in SEQ ID NO.3, the amino acid sequence of CFP-10 is shown in SEQ ID NO.4, and the amino acid sequence of BFH is shown in SEQ ID NO.5.

[0055] (2) ESAT-6, CFP-10, and BFH were designed into gene sequences using a linker according to the tandem combination scheme of ESAT-6-CFP-10-BFH. The sequences were then submitted to a third-party gene synthesis company, Shanghai Langjing Company, for gene synthesis to obtain the synthetic gene EC-BFH. The amino acid sequence of the linker is shown in SEQ ID NO.6.

[0056] (3) Insert the synthetic gene EC-BFH into the prokaryotic expression vector pET 28a to construct the EC-BFH expression vector, such as Figure 1 shown.

[0057] (4) The EC-BFH expression vector was transformed into the BL21 (DE3) Escherichia coli strain. The bacterial liquid after successful transformation was reserved as seed liquid, frozen and stored, and streaked for culture when needed for the expression and preparation of recombinant protein.

[0058] (5) Expression and purification of the recombinant protein EC-BFH, specifically including the following steps:

[0059] ① Expression of recombinant EC-BFH protein: On day 1, thaw a frozen bacterial strain at room temperature, then streak onto a solid LB plate containing Kan antibiotic and incubate at 37°C for 12 hours. On day 2, pick a single colony and transfer it to 5 mL of LB liquid medium. Add Kan antibiotic to a final concentration of 50 μg / mL. Incubate overnight at 37°C, 180 rpm, for 12 hours to allow the strain to recover. On day 3, inoculate the recovered bacterial suspension at a ratio of 1:100 into 100 mL of autoinduction medium. Add Kan antibiotic to a final concentration of 50 μg / mL. Induce overnight at 37°C, 180 rpm, for 14 hours. Centrifuge at 8000 rpm for 10 minutes to harvest the cells, weigh, label, and store in a -20°C freezer until needed.

[0060] ② Bacterial disruption and protein denaturation: Resuspend the cells in binding buffer. Add urea to a final concentration of 2 M. Disrupt the cells using a cell disruptor using sonication at 20% power for 3 seconds, followed by an 8-second pause, for 10 minutes. After thorough disruption, centrifuge the cells at 8000 rpm at 4°C for 20 minutes in a low-temperature ultracentrifuge. Collect the supernatant for later use.

[0061] ③ Protein purification: Protein purification was performed using affinity chromatography. The filler, sepharose-CL-6B-Ni, was purchased from Beijing Golden Helix Biotechnology Center. After washing with pure water, the filler was equilibrated with binding buffer to enhance its affinity for the target protein. The supernatant was incubated with the filler for 5 minutes to allow the filler to bind firmly to the target protein in the supernatant. The filler was then washed with wash buffer to remove contaminants, and the protein was eluted with elution buffer.

[0062] ④ Protein refolding: Use dialysis to remove urea from the eluate, achieving protein refolding. First, treat the dialysis bag and cut it to the appropriate size based on the dialysis volume. Boil it in Treatment Solution 1 for 10 minutes and rinse thoroughly with pure water. Then, boil it in Treatment Solution 2 for 10 minutes and rinse thoroughly with pure water to remove any harmful substances in the bag. After treatment, clamp one end of the dialysis bag with a dialysis clamp and transfer the denatured protein eluate into the dialysis bag, ensuring that the volume does not exceed 2 / 3 of the bag's volume. Then, clamp the other end of the dialysis bag with a dialysis clamp and place it in a 1 L beaker with refolding solution. Place the beaker on a magnetic stirrer and stir at low speed. Change the refolding solution every two hours, and after three changes, remove the dialysis bag and collect the refolded recombinant protein.

[0063] In steps ①-④, the formula of the binding buffer is: 20 mM PB, 200 mM NaCl, 10 mM imidazole, 2 M urea, pH 7.4; the formula of the washing buffer is: 20 mM PB, 200 mM NaCl, 150 mM imidazole, 2 M urea, pH 7.4; the formula of the elution buffer is 20 mM PB, 200 mM NaCl, 400 mM imidazole, 2 M urea, pH 7.4; the formula of the treatment solution 1 is: 2% sodium bicarbonate, 1 mM EDTA, pH 8.0; the formula of the treatment solution 2 is: 10 mM EDTA, pH 8.0; the formula of the refolding solution is: 20 mM PB; the formula of 0.1M PB is: 77.4 mL 1M Na2HPO4, 22.6 mL 1MNaH2PO4, dilute to 1 L with deionized water, pH 7.4.

[0064] The method for preparing the recombinant protein of Example 3 comprises the following steps:

[0065] The only difference from Example 2 is the expression and purification of the recombinant protein EC-BFH in step (5), which specifically includes the following steps:

[0066] ① Expression of recombinant EC-BFH protein: On day 1, thaw a frozen bacterial strain at room temperature. Streak the plate onto a solid LB plate containing Kan antibiotic and incubate at 37°C for 16 hours. On day 2, pick a single colony and transfer it to 5 mL of LB liquid medium. Add Kan antibiotic to a final concentration of 50 μg / mL. Incubate the plate in a constant temperature shaking incubator at 37°C and 180 rpm overnight for 16 hours to allow the strain to recover. On day 3, inoculate the recovered bacterial suspension at a ratio of 1:100 into 100 mL of autoinduction medium. Add Kan antibiotic to a final concentration of 50 μg / mL. Induce the plate overnight at 37°C and 180 rpm for 14-18 hours. Centrifuge the plate at 8000 rpm for 10 minutes, harvest the cells, weigh, label, and store in a -20°C freezer until needed.

[0067] ② Bacterial disruption and protein denaturation: Resuspend the cells in binding buffer. Add urea to a final concentration of 6 M. Disrupt the cells using a cell disruptor using sonication at 20% power for 3 seconds, followed by an 8-second pause, for 10 minutes. After thorough disruption, centrifuge the cells at 8000 rpm at 4°C for 20 minutes in a low-temperature ultracentrifuge. Collect the supernatant for later use.

[0068] ③ Protein purification: Protein purification was performed using affinity chromatography. The filler material, sepharose-CL-6B-Ni, was purchased from Beijing Golden Helix Biotechnology Center. After washing with pure water, the filler was equilibrated with binding buffer to enhance its affinity for the target protein. The supernatant was incubated with the filler for 20 minutes to allow the filler to bind firmly to the target protein in the supernatant. The filler was then washed with wash buffer to remove contaminants, and the protein was eluted with elution buffer.

[0069] ④ Protein refolding: Use dialysis to remove urea from the eluate, achieving protein refolding. First, treat the dialysis bag and cut it to the appropriate size based on the dialysis volume. Boil it in treatment solution 1 for 10 minutes and rinse thoroughly with pure water. Then, boil it in treatment solution 2 for 10 minutes and rinse thoroughly with pure water to remove any harmful substances in the bag. After treatment, clamp one end of the dialysis bag with a dialysis clamp and transfer the denatured protein eluate into the dialysis bag, ensuring that the volume does not exceed 2 / 3 of the bag's volume. Then, clamp the other end of the dialysis bag with a dialysis clamp and place it in a 1 L beaker with refolding solution. Place the beaker on a magnetic stirrer and stir at low speed. Change the refolding solution every two hours. After five changes, remove the dialysis bag and collect the refolded recombinant protein.

[0070] In steps ①-④, the formula of the binding buffer is: 20 mM PB, 200 mM NaCl, 10 mM imidazole, 6 M urea, pH 7.4; the formula of the washing buffer is: 20 mM PB, 200 mM NaCl, 150 mM imidazole, 6 M urea, pH 7.4; the formula of the elution buffer is 20 mM PB, 200 mM NaCl, 400 mM imidazole, 6 M urea, pH 7.4; the formula of the treatment solution 1 is: 2% sodium bicarbonate, 1 mM EDTA, pH 8.0; the formula of the treatment solution 2 is: 10 mM EDTA, pH 8.0; the formula of the refolding solution is: 20 mM PB; the formula of 0.1M PB is: 77.4 mL 1M Na2HPO4, 22.6 mL 1MNaH2PO4, dilute to 1 L with deionized water, pH 7.4.

[0071] The recombinant protein EC-BFH obtained in Example 2 was subjected to the following tests:

[0072] 1. Purity test: The recombinant protein was subjected to SDS-PAGE electrophoresis and Western blot experiments respectively. The results of SDS-PAGE electrophoresis purity analysis were as follows: Figure 2 The results of Western blot purity analysis are shown in Figure 3 As shown, the purity of the recombinant protein reached more than 90%.

[0073] 2. Skin test

[0074] Preparation of experimental materials: First, prepare the allergen according to the following steps:

[0075] (1) Strain recovery: Inoculate bovine tuberculosis Mycobacterium tuberculosis C68001 and C68002 strains into P's solid culture medium and culture at 37°C for more than 20 days.

[0076] (2) Inactivation of bacteria: Take a centrifuge tube and add physiological saline, place the bacteria in the centrifuge tube, and inactivate at 121℃ for 30 minutes.

[0077] (3) Collecting bacteria: Centrifuge at 8000 rpm for 10 min to collect bacteria, dry in an oven at 100°C for 30 min, and weigh the dried bacteria.

[0078] (4) Bacterial disruption: Add an appropriate amount of physiological saline to the bacteria and freeze-thaw repeatedly at room temperature and -20°C for 3-5 times. Remove the bacteria and grind them with a ceramic grinder. Add Freund's incomplete adjuvant to make an emulsion and store at 2-8°C until use. The bacteria in the allergen are calculated by dry weight to a final concentration of 10 mg / mL.

[0079] (5) Guinea pig sensitization: The allergen obtained by the above method was injected into the inner hind leg muscle of the guinea pig at a rate of 0.5 mL / pig. After four weeks of normal feeding, the guinea pigs were plucked from their buttocks and the national standard tuberculin (PPD) was used to test whether the sensitization was successful. Guinea pigs that were successfully sensitized were used in the following experiments.

[0080] 2.1 Skin test of EC and EC-BFH recombinant proteins

[0081] 2.1.1 Experimental methods

[0082] Twelve sensitized guinea pigs were subjected to bilateral hair removal from the dorsal spine. The following day, injection solutions were prepared using 0.1 M PBS buffer at concentrations of 1 μg / mL, 10 μg / mL, 100 μg / mL, and 200 μg / mL of the recombinant proteins (EC and EC-BFH). 0.1 M PBS served as a negative control, and PPD at a concentration of 1000 UI / mL served as a positive control. The hair removal site was disinfected by wiping with an alcohol cotton ball. 0.1 mL each of the recombinant protein (EC and EC-BFH) solution and PPD were injected intradermally using the wheel-point injection method. The diameter of the swelling was measured 24 hours later (the sum of the vertical and horizontal diameters divided by 2). The recombinant protein EC was ESAT-6-CFP-10, and the recombinant protein EC-BFH was prepared in Example 2 of the present invention.

[0083] 2.1.2 Experimental Results

[0084] The experimental results are shown in Table 1 and Figure 4As shown:

[0085] Table 1 Comparison of the skin test results of EC and EC-BFH recombinant proteins (mean, n=6, unit: mm)

[0086]

[0087] As can be seen from the table, the diameter of the swelling produced by the recombinant protein EC-BFH at 24 hours was larger than that of the EC recombinant protein, and the diameter of the swelling produced at 24 hours when the recombinant protein injection dose was μg was close to that of PPD, indicating that the sensitivity of EC-BFH was significantly better than that of EC protein.

[0088] 2.2 Skin test in the presence of low molecular weight sodium hyaluronate

[0089] 2.2.1 Interference experiment of low molecular weight sodium hyaluronate on skin test reaction

[0090] (1) Experimental methods

[0091] Six sensitized guinea pigs were plucked from both sides of their abdomens. The next day, injection solutions were prepared by adding different concentrations of low-molecular-weight sodium hyaluronate to 0.1 M PBS buffer, resulting in adjuvant concentrations of 5 mg / mL, 10 mg / mL, 50 mg / mL, and 100 mg / mL, respectively. 0.1 M PBS served as a negative control, and PPD served as a positive control at a concentration of 1000 UI / mL. 0.1 mL of the solution was injected intradermally at targeted sites using a circular injection pattern. The skin was disinfected by wiping with 75% ethanol before injection. The diameter of the swelling (the sum of the vertical and horizontal diameters divided by 2) was measured 24 hours later.

[0092] (2) Experimental results

[0093] The experimental results are shown in Table 2 and Figure 5 As shown:

[0094] Table 2 Skin test results of low molecular weight sodium hyaluronate (unit: mm)

[0095]

[0096] As can be seen from Table 2, low molecular weight sodium hyaluronate solution has no effect on the skin test results.

[0097] 2.2.2 Experimental study on the enhancement of EC-BFH skin test reaction by low molecular weight sodium hyaluronate

[0098] (1) Experimental methods

[0099] Six guinea pigs were plucked from both sides of their abdomens. The next day, an injection solution was prepared with an EC-BFH protein concentration of 10 μg / mL and low-molecular-weight sodium hyaluronate adjuvants at concentrations of 5, 10, 50, and 100 mg / mL, respectively. PPD served as a positive control at a concentration of 1000 UI / mL, and 0.1 M PBS served as a negative control. The skin at the plucked area was disinfected with an alcohol cotton ball. 0.1 mL was injected in a circular pattern. The diameter of the swelling was measured 24 hours later (the sum of the vertical and horizontal diameters divided by 2).

[0100] (2) Experimental results

[0101] The experimental results are shown in Table 3 and Figure 6 As shown:

[0102] Table 3 Results of the enhancement experiment of low molecular weight sodium hyaluronate on EC-BFH skin test reaction (unit: mm)

[0103]

[0104] As can be seen from Table 3, when the concentration of the recombinant protein EC-BFH is fixed, as the injection amount of low molecular weight hyaluronic acid increases, the diameter of the skin redness and swelling increases. When the injection amount of low molecular weight hyaluronic acid reaches 5 mg or more, the diameter of the redness and swelling does not change much, indicating that the addition of low molecular weight hyaluronic acid enhances the skin test reaction of the recombinant protein EC-BFH, and when the injection amount of low molecular weight hyaluronic acid is 5 mg, the enhancement effect approaches saturation.

[0105] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A recombinant protein, characterized in that The amino acid sequence of the recombinant protein is shown in SEQ ID NO.

1.

2. A recombinant protein according to claim 1, characterized in that The recombinant protein includes ESAT-6, CFP-10 and BFH in a tandem combination.

3. A recombinant protein according to claim 2, characterized in that The amino acid sequence of ESAT-6 is shown in SEQ ID NO.3, the amino acid sequence of CFP-10 is shown in SEQ ID NO.4, and the amino acid sequence of BFH is shown in SEQ ID NO.

5.

4. A recombinant protein according to claim 2, characterized in that The ESAT-6, CFP-10 and BFH are combined in series using a linker in the manner of ESAT-6-CFP-10-BFH. The amino acid sequence of the linker is shown in SEQ ID NO.

6.

5. A gene encoding the recombinant protein according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

6. The method for preparing the recombinant protein according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) The amino acid sequences of ESAT-6, CFP-10, and BFH were codon optimized respectively; (2) Use Linker to combine the optimized ESAT-6, CFP-10, and BFH in series in the manner of ESAT-6-CFP-10-BFH to obtain the synthetic gene EC-BFH; (3) Inserting the synthetic gene EC-BFH into the prokaryotic expression vector pET 28a to obtain the EC-BFH expression vector; (4) The EC-BFH expression vector was transferred into the protein expression strain, and the recombinant protein was obtained after expression and purification.

7. The preparation method according to claim 6, characterized in that Urea denaturation and renaturation were used during the purification.

8. Use of the recombinant protein according to claim 1 in preparing a reagent for detecting or diagnosing bovine tuberculosis.

9. The use according to claim 8, characterized in that The reagent for detecting or diagnosing bovine tuberculosis comprises the recombinant protein and low molecular weight hyaluronic acid.

10. Use of the recombinant protein according to claim 1 in preparing a bovine tuberculosis detection kit.

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