Recombinant mycobacterium tuberculosis fusion protein EMC and preparation method thereof

Abstract

The invention provides a mycobacterium tuberculosis recombinant fusion protein EMC, and the connection sequence of the mycobacterium tuberculosis recombinant fusion protein EMC is as follows: EAST6-MPT64-CFP10. The fusion protein formed by the EAST6 protein, the MPT64 protein and the CFP10 protein has better antigenicity, it is found that effective series connection can more effectively promote mutual complementation of the EAST6 protein, the MPT64 protein and the CFP10 protein in the antigenicity aspect, the number of antigen epitopes is increased, and then the antigenicity of the fusion protein is remarkably improved; and the use dosage of the fusion protein as a detection antigen for immunological diagnosis and an antigen for vaccine development is effectively reduced. The invention also provides a novel tuberculosis intradermal diagnostic reagent and a preparation method thereof.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and mainly relates to recombinant Mycobacterium tuberculosis fusion protein EMC and its preparation method. Background Technology

[0002] Diagnostic methods for tuberculosis targeting pathogens include acid-fast staining microscopy, bacterial culture, and molecular biological diagnostic methods based on PCR amplification. However, these pathogen-based methods are not suitable for diagnosing smear-negative tuberculosis patients. Although the incidence of tuberculosis in my country is slowly decreasing, the number of patients with smear-negative tuberculosis is increasing (reaching 80%). These patients urgently need immunological diagnostic methods to differentiate between Mycobacterium tuberculosis infection and non-tuberculous mycobacterial infection. Management of the tuberculosis-infected population is necessary, including the development of new vaccines for prevention and treatment, and the establishment of diagnostic methods for latent tuberculosis infection. Immunological diagnostic methods are based on immunological principles to diagnose the body's immune status and are divided into immunoserological diagnosis and immunocytological diagnosis. The value of serological diagnosis for tuberculosis is questioned, and the WHO does not recommend serological diagnosis. Immunocytological diagnostic methods include the traditional tuberculin skin test (TST) and the newly established interferon release assay (IGRA). The TST involves intradermal injection of a purified protein derivative of Mycobacterium tuberculosis (Mondu method), and the transverse and longitudinal diameters of the redness or induration reaction at the injection site are measured 72 hours later. IGRA is a method for detecting IFN-γ secreted by peripheral blood or isolated mononuclear lymphocytes in vitro after stimulation by the RD1 region protein polypeptide of Mycobacterium tuberculosis. The WHO first recommended TST or IGRA as a diagnostic method for latent tuberculosis infection in its 2018 TB report (WHO, TBreport, 2018).

[0003] IGRA is divided into two types: ELISA IGRA and ELISPOT IGRA. The in vitro stimulation epitope peptides used are synthesized using instruments, making them relatively expensive. ELISA IGRA involves stimulating peripheral blood cells in vitro with RD1 region antigenic epitope peptides, and then detecting the IFN-γ content in the culture medium using ELISA. ELISPOT IGRA involves extracting peripheral blood mononuclear lymphocytes, stimulating them in vitro with RD1 region antigenic epitope peptides, and then detecting the number of cells secreting IFN-γ using an enzyme-linked dot assay. ELISPOT IGRA is more complex and expensive, requiring equipment such as a biosafety cabinet, centrifuge, cell counter, CO2 incubator, and dot assay, as well as reagents and consumables such as lymphocyte separation medium, cell culture medium, blood, and centrifuge tubes. The experiment takes about 6 hours. ELISA IGRA requires a biosafety cabinet, centrifuge, incubator, and ELISA reader, and requires the purchase of an ELISA IFN-γ kit. ELISPOTIGRA is highly sensitive but not suitable for large-scale screening. ELISA IGRA is suitable for large-scale screening, but some data are in the gray area, making it difficult to distinguish between positive and negative results. IGRA testing is expensive and requires highly skilled equipment and operators. The ELISA skin test only requires injection and measurement, and trained personnel can quickly master it, making it suitable for both on-demand and large-scale screening.

[0004] The earliest skin test for tuberculosis used the old tuberculin (OT) skin test, which was prone to side effects and non-specific reactions. Second-generation skin test reagents are purified protein derivatives of tuberculin (TB-PPD or BCG-PPD). Compared to OT, TB-PPD or BCG-PPD skin tests have fewer side effects: TB-PPD or BCG-PPD is obtained by culturing Mycobacterium tuberculosis or BCG for 8-10 weeks, sterilizing the culture at 121°C for 30 minutes, precipitating the filtrate with trichloroacetic acid and saturated ammonium sulfate, and then dialyzing the precipitate after redissolving it. In addition to the protein derivative, it also contains metabolites of Mycobacterium tuberculosis or BCG, such as bacterial polysaccharides, nucleic acids, lipids, and some components of the culture medium. However, the PPD antigen shares common antigens with almost all mycobacteria; therefore, the PPD skin test still suffers from low specificity. The genes encoding the EAST6 and CFP1O proteins in the RD1 region and the MPT64 protein in the RD2 region of Mycobacterium tuberculosis are deleted in the genomes of BCG strains and most NTM strains. The sensitivity and specificity of tuberculosis immunodiagnosis are highly variable, especially the effect of single antigens is extremely unsatisfactory. Therefore, the combined use of RD region protein antigens can significantly improve the sensitivity and specificity of detection, and has great application value for the diagnosis and prevention of tuberculosis. Summary of the Invention

[0005] To address the technical problems existing in the prior art, the purpose of this invention is to provide a recombinant fusion protein EMC of Mycobacterium tuberculosis. This protein has high antigenic immunogenicity, and using a low dose of this fusion protein can achieve effective immunological diagnosis of tuberculosis and screening for latent tuberculosis infection (including skin test, cytokine release, and serological diagnosis).

[0006] Firstly, this invention provides a recombinant fusion protein EMC from Mycobacterium tuberculosis, linked in the following order: EAST6-MPT64-CFP10. The fusion protein formed by these three proteins exhibits better antigenicity. This invention discovers that effective tandem linkage can more effectively promote the mutual complementarity of EAST6, MPT64, and CFP10 proteins in terms of antigenicity, increasing the number of antigenic epitopes and thus significantly improving the antigenicity of the fusion protein. This effectively reduces the dosage of the fusion protein used as a detection antigen for immunological diagnosis and as an antigen in vaccine development.

[0007] Secondly, the present invention also provides a target protein expression nucleic acid sequence and a protein sequence. Those skilled in the art can design different nucleotide sequences that can encode the above-mentioned Mycobacterium tuberculosis recombinant fusion protein EMC according to the codon preference of the expression host. All nucleic acids that can encode the Mycobacterium tuberculosis recombinant fusion protein EMC provided by the present invention are within the protection scope of the present invention.

[0008] The sequence of the nucleic acid of the fusion protein EMC (SEQ ID NO.1): The amino acid sequence of the fusion protein EMC (SEQ ID NO.2): Met Thr Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser AlaIle Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly Lys Gln Ser LeuThr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser Glu Ala Tyr Gln Gly Val GlnGln Lys Trp Asp Ala Thr Ala Thr Glu Leu Asn Asn Ala Leu Gln Asn Leu Ala ArgThr Ile Ser Glu Ala Gly Gln Ala Met Ala Ser Thr Glu Gly Asn Val Thr Gly MetPhe Ala Ala Pro Lys Thr Tyr Cys Glu Glu Leu Lys Gly Thr Asp Thr Gly Gln AlaCys Gln Ile Gln Met Ser Asp Pro Ala Tyr Asn Ile Asn Ile Ser Leu Pro Ser TyrTyr Pro Asp Gln Lys Ser Leu Glu Asn Tyr Ile Ala Gln Thr Arg Asp Lys Phe LeuSer Ala Ala Thr Ser Ser Thr Pro Arg Glu Ala Pro Tyr Glu Leu Asn Ile Thr SerAla Thr Tyr Gln Ser Ala Ile Pro Pro Arg Gly Thr Gln Ala Val Val Leu Lys ValTyr Gln Asn Ala Gly Gly Thr His Pro Thr Thr Thr Tyr Lys Ala Phe Asp Trp AspGln Ala Tyr Arg Lys Pro Ile Thr Tyr Asp Thr Leu Trp Gln Ala Asp Thr Asp ProLeu Pro Val Val Phe Pro Ile Val Gln Gly Glu Leu Ser Lys Gln Thr Gly Gln GlnVal Ser Ile Ala Pro Asn Ala GlyLeu Asp Pro Val Asn Tyr Gln Asn Phe Ala ValThr Asn Asp Gly Val Ile Phe Phe Phe Asn Pro Gly Glu Leu Leu Pro Glu Ala AlaGly Pro Thr Gln Val Leu Val Pro Arg Ser Ala Ile Asp Ser Met Leu Ala Met AlaGlu Met Lys Thr Asp Ala Ala Thr Leu Ala Gln Glu Ala Gly Asn Phe Glu Arg IleSer Gly Asp Leu Lys Thr Gln Ile Asp Gln Val Glu Ser Thr Ala Gly Ser Leu GlnGly Gln Trp Arg Gly Ala Ala Gly Thr Ala Ala Gln Ala Val Val Arg Phe GlnGlu Ala Ala Asn Lys Gln Lys Gln Glu Leu Asp Glu Ile Ser Thr Asn Ile Arg GlnAla Gly Val Gln Tyr Ser Arg Ala Asp Glu Glu Gln Gln Gln Ala Leu Ser Ser GlnMet Gly Phe* Thirdly, the present invention also provides a novel intradermal diagnostic reagent for tuberculosis; EMC is an in vivo diagnostic product for tuberculosis infection screening, tuberculosis auxiliary diagnosis, and differentiation of BCG vaccination and tuberculosis infection. EMC can effectively distinguish between people who are not infected with Mycobacterium tuberculosis, people who have been vaccinated with BCG, people with latent tuberculosis infection, and patients with active tuberculosis. Fourthly, the present invention also provides a method for preparing a novel intradermal diagnostic reagent for tuberculosis.

[0009] Fermentation expression: The recombinant plasmid was transformed into BL21(DE3) competent cells to prepare engineered bacteria; the bacterial strain was inoculated at a 1:1000 ratio in vials and cultured overnight at 37°C at 220 rpm; the next day, it was transferred to a large vial (1:100 ratio) and cultured at 37°C for 6-8 hours; the temperature was lowered to 25°C, and the cells were induced overnight at 180 rpm with 0.5 mM IPTG; harvesting: the bacterial cells were harvested by centrifugation at 8000g for 30 min. Extraction and purification: Bacterial cell disruption (10:1); centrifugation at 12000 rpm, 4℃ for 1 hour, collection of clear supernatant; addition of saturated ammonium sulfate to the supernatant to 30%, salting out for 1 hour, centrifugation, loading of the supernatant onto Phenyl hydrophobic chromatography, gradient elution, followed by SuperQ chromatography with gradient elution. The eluent was ultrafiltered to obtain the stock solution; vector construction and enzyme digestion verification chromatograms, electrophoretic assay chromatograms of the purified stock solution, and HPLC assay chromatograms of the purified stock solution were obtained.

[0010] The beneficial effects of this invention are as follows: (1) Through research, this invention has found that EMC protein can detect tuberculosis and latent tuberculosis infection, and can distinguish between BCG vaccination and tuberculosis infection, which means it can be used as a new reagent for tuberculosis diagnosis; (2) Further research by the present invention has revealed that EMC protein requires a lower dosage and has better specificity than TB-PPD.

[0011] (3) The fusion proteins obtained by the three protein tandem methods have stronger immunogenicity and diagnostic effect.

[0012] (4) Innovative expression and purification methods.

[0013] (5) Iterative new products for in vivo tuberculosis diagnosis. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 To construct an enzyme digestion verification map for the vector, including: Lane from left to right.

[0016] Lane 1: undigested Lane 2: digested with HpaI Lane 3: ladder.

[0017] Figure 2 The HPLC chromatogram of the purified stock solution is shown, in which: Lane 1: Protein marker Lane 2: EMC stock solution.

[0018] Figure 3 The HPLC chromatogram is for the purification of the original solution.

[0019] Figure 4 Images of guinea pigs sensitized with EMC to Mycobacterium tuberculosis during their 24-hour DTH period.

[0020] Figure 5 Animal images of guinea pigs sensitized by BCG and exposed to DTH for 24 hours.

[0021] Figure 6 and Figure 7 In a double-blind method, the longitudinal and transverse diameters (mm) of redness and / or induration at the injection site were measured at 24 and 48 hours, respectively. The average of the longitudinal and transverse diameters was used as the skin test reaction diameter of the injection sample at that point. Detailed Implementation

[0022] To make the technical problem to be solved, the technical solution, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only for explaining the content of this invention and are not intended to limit this invention.

[0023] Example 1

[0024] We provide a recombinant fusion protein EMC from Mycobacterium tuberculosis. This protein has high antigenic immunogenicity, and using low doses of this fusion protein can achieve effective immunological diagnosis of tuberculosis and screening for latent tuberculosis infection (including skin tests, cytokine release, and serological diagnosis).

[0025] This embodiment provides a recombinant fusion protein EMC from Mycobacterium tuberculosis, with the following linkage order: EAST6-MPT64-CFP10. The fusion protein formed by these three proteins exhibits better antigenicity. This invention has found that effective tandem linkage can more effectively promote the mutual complementarity of EAST6, MPT64, and CFP10 proteins in terms of antigenicity, increase the number of antigenic epitopes, and thus significantly improve the antigenicity of the fusion protein, effectively reducing the dosage of the fusion protein used as a detection antigen for immunological diagnosis and as an antigen for vaccine development.

[0026] This embodiment also provides the target protein expression nucleic acid sequence and protein sequence. Those skilled in the art can design different nucleotide sequences that can encode the above-mentioned Mycobacterium tuberculosis recombinant fusion protein EMC according to the codon preference of the expression host. All nucleic acids that can encode the Mycobacterium tuberculosis recombinant fusion protein EMC provided by the present invention are within the protection scope of the present invention.

[0027] The sequence of the nucleic acid of the fusion protein EMC (SEQ ID NO.1): The amino acid sequence of the fusion protein EMC (SEQ ID NO.2): Met Thr Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser AlaIle Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly Lys Gln Ser LeuThr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser Glu Ala Tyr Gln Gly Val GlnGln Lys Trp Asp Ala Thr Ala Thr Glu Leu Asn Asn Ala Leu Gln Asn Leu Ala ArgThr Ile Ser Glu Ala Gly Gln Ala Met Ala Ser Thr Glu Gly Asn Val Thr Gly MetPhe Ala Ala Pro Lys Thr Tyr Cys Glu Glu Leu Lys Gly Thr Asp Thr Gly Gln AlaCys Gln Ile Gln Met Ser Asp Pro Ala Tyr Asn Ile Asn Ile Ser Leu Pro Ser TyrTyr Pro Asp Gln Lys Ser Leu Glu Asn Tyr Ile Ala Gln Thr Arg Asp Lys Phe LeuSer Ala Ala Thr Ser Ser Thr Pro Arg Glu Ala Pro Tyr Glu Leu Asn Ile Thr SerAla Thr Tyr Gln Ser Ala Ile Pro Pro Arg Gly Thr Gln Ala Val Val Leu Lys ValTyr Gln Asn Ala Gly Gly Thr His Pro Thr Thr Thr Tyr Lys Ala Phe Asp Trp AspGln Ala Tyr Arg Lys Pro Ile Thr Tyr Asp Thr Leu Trp Gln Ala Asp Thr Asp ProLeu Pro Val Val Phe Pro Ile Val Gln Gly Glu Leu Ser Lys Gln Thr Gly Gln GlnVal Ser Ile Ala Pro Asn Ala GlyLeu Asp Pro Val Asn Tyr Gln Asn Phe Ala ValThr Asn Asp Gly Val Ile Phe Phe Phe Asn Pro Gly Glu Leu Leu Pro Glu Ala AlaGly Pro Thr Gln Val Leu Val Pro Arg Ser Ala Ile Asp Ser Met Leu Ala Met AlaGlu Met Lys Thr Asp Ala Ala Thr Leu Ala Gln Glu Ala Gly Asn Phe Glu Arg IleSer Gly Asp Leu Lys Thr Gln Ile Asp Gln Val Glu Ser Thr Ala Gly Ser Leu GlnGly Gln Trp Arg Gly Ala Ala Gly Thr Ala Ala Gln Ala Val Val Arg Phe GlnGlu Ala Ala Asn Lys Gln Lys Gln Glu Leu Asp Glu Ile Ser Thr Asn Ile Arg GlnAla Gly Val Gln Tyr Ser Arg Ala Asp Glu Glu Gln Gln Gln Ala Leu Ser Ser GlnMet Gly Phe* This embodiment also provides a novel intradermal diagnostic reagent for tuberculosis; EMC is an in vivo diagnostic product for tuberculosis infection screening, tuberculosis auxiliary diagnosis, and differentiation of BCG vaccination and tuberculosis infection. EMC can effectively distinguish between people who are not infected with Mycobacterium tuberculosis, people who have been vaccinated with BCG, people with latent tuberculosis infection, and patients with active tuberculosis.

[0028] This embodiment also provides a method for preparing a novel intradermal diagnostic reagent for tuberculosis.

[0029] Fermentation expression: The recombinant plasmid was transformed into BL21(DE3) competent cells to prepare engineered bacteria; the bacterial strain was inoculated at a 1:1000 ratio in vials and cultured overnight at 37°C at 220 rpm; the next day, it was transferred to a large vial (1:100 ratio) and cultured at 37°C for 6-8 hours; the temperature was lowered to 25°C, and the cells were induced overnight at 180 rpm with 0.5 mM IPTG; harvesting: the bacterial cells were harvested by centrifugation at 8000g for 30 min. Extraction and purification: Cell disruption (10:1); centrifugation at 12000 rpm, 4℃ for 1 hour, collection of the clear supernatant; addition of saturated ammonium sulfate to the supernatant to 30%, salting out for 1 hour, centrifugation, loading the supernatant onto Phenyl hydrophobic chromatography, gradient elution, followed by SuperQ chromatography with gradient elution. The eluent was then subjected to ultrafiltration to obtain the stock solution; the vector construction and enzyme digestion verification chromatogram, the electrophoresis assay chromatogram of the purified stock solution, and the HPLC assay chromatogram of the purified stock solution are shown in the attached figures. Figure 1-3 As shown.

[0030] Example 2

[0031] Animal experiments verify accuracy

[0032] Healthy, untested SPF-grade white guinea pigs, weighing 300g-500g, were used. One vial of frozen Mycobacterium tuberculosis was taken out, allowed to thaw naturally at room temperature, and diluted 10-fold with physiological saline. 0.5ml of the diluted bacterial solution was subcutaneously injected into the groin area of ​​each guinea pig's hind leg. Skin tests were performed 5-6 weeks after sensitization. Hair was removed from both sides of the guinea pig's spine, and 0.1ml of various diluted EMC samples, as well as TB-PPD and EC standards, was injected intradermally. Using a double-blind method, the longitudinal and transverse diameters (mm) of redness and / or induration at the injection site were measured at 24 and 48 hours. The average of the longitudinal and transverse diameters was used as the diameter of the skin test reaction at that injection point.

[0033] The positive rate of DTH skin test induced by EMC stock solution at 5 μg / mL and the positive control (EC) at 10 μg / mL was the same, both at 4 / 4.

[0034] Skin test in BCG-sensitized guinea pigs

[0035] Healthy, untested white SPF-grade guinea pigs, weighing 300g-500g, were selected. BCG cultured bacteria were washed off the culture medium with sterile physiological saline, centrifuged at 6000 rpm for 30 min, weighed, and diluted with physiological saline to a concentration of 50 mg / mL. Guinea pigs with negative skin tests for TB-PPD standard (10 IU / 0.2 ml per guinea pig) were injected subcutaneously with 0.2 ml of 50 mg / ml BCG bacterial solution in the groin area. Skin tests were performed 5-6 weeks after sensitization. Hair was removed from both sides of the spine, and 0.1 ml of EMC samples at various dilutions, as well as TB-PPD and EC standards, were injected intradermally. A double-blind method was used, measuring the longitudinal and transverse diameters (mm) of redness and / or induration at the injection site at 24 and 48 hours after the skin test. The average longitudinal and transverse diameters were used as the diameter of the skin test reaction at that injection point.

[0036] EMC induces DTH in guinea pigs for 24 hours, as shown in animal images. Figure 4 As shown.

[0037] EMC-induced BCG-sensitized guinea pig DTH 24-hour animal images, such as... Figure 5 As shown.

[0038] Example 3

[0039] Animal experiments to verify sensitivity Skin test of guinea pigs sensitized with live tuberculosis bacteria

[0040] Healthy, untested SPF-grade white guinea pigs, weighing 300g-500g, were used. One vial of frozen Mycobacterium tuberculosis was taken out, allowed to thaw naturally at room temperature, and diluted 10-fold with physiological saline. 0.5ml of the diluted bacterial solution was subcutaneously injected into the groin area of ​​each guinea pig's hind leg. Skin tests were performed 5-6 weeks after sensitization. Hair was removed from both sides of the guinea pig's spine, and 0.1ml of various diluted EMC samples, as well as TB-PPD and EC standards, was injected intradermally. Using a double-blind method, the longitudinal and transverse diameters (mm) of redness and / or induration at the injection site were measured at 24 and 48 hours. The average of the longitudinal and transverse diameters was used as the diameter of the skin test reaction at that injection point. Figure 6 , Figure 7 As shown.

[0041]

[0042]

[0043] It is evident that EMC protein requires a lower dosage and exhibits better specificity compared to TB-PPD. The fusion proteins obtained through the three protein tandem methods demonstrate stronger immunogenicity and diagnostic efficacy.

[0044] Example 4: To verify the effect of different protein tandem sequences on the intensity of skin test reactions: tuberculin skin test (DTH test).

[0045] Grouping: The baseline group was EMC (ESAT6-MPT64-CFP10), and three control groups were set up with different tandem sequences: ECM (ESAT6-CFP10-MPT64), CEM (CFP10-ESAT6-MPT64), and MEC (MPT64-ESAT6-CFP10). The purification process was consistent for all groups, and the detection principle, animal model construction, and operation procedures all followed the mature skin test method framework in Example 2, with only the variable changed from "EMC concentration / control reagent" to "fusion protein with different tandem sequences". The intradermal injection dose was 0.1 ml and 10 μg / mL. The average diameter of DTH and its relative activity to the baseline group were measured after 24 hours, and the results are shown in Table 5.

[0046] Relative activity (%) = (Mean diameter of DTH in control group at 24h ÷ Mean diameter of DTH in baseline group at 24h) × 100%

[0047] The test showed that the baseline group was significantly different from any control group, with p < 0.01.

[0048] As shown in Table 5, the tandem sequence of "ESAT6-MPT64-CFP10 (i.e. EMC)" is the optimal scheme among the recombinant fusion proteins of Mycobacterium tuberculosis. The intensity of the delayed-type hypersensitivity reaction (DTH) induced by it is significantly higher than that of other tandem sequences of fusion proteins, and the immunogenicity advantage is statistically significant.

[0049] Example 5

[0050] The experiment compared three fusion methods: EMC, CME, and MEC. Recombinant plasmids were constructed by inserting the pET-28a vector. Solubility, expression, and stability were compared under the same induction conditions (0.5 mM IPTG). The method is as follows: 1. Construction of CE protein: The target gene was digested with EcoRI / BamHI and then inserted into pET-28a. The protein was ligated overnight at 16°C using T4 ligase. The resulting protein was transformed into E. coli (DH5α) with 50 μg / mL Kan. -1 Plate screening. Selection of single-clone plasmids for extraction.

[0051] 2. Expression: The recombinant plasmid was transformed into BL21(DE3) and cultured in 5 mL of LB medium at 37°C and 200 rpm until OD600 ≈ 0.6. The culture was then diluted 1:100 with 400 mL of antibiotic-free LB medium and cultured at 37°C and 200 rpm until OD600 ≈ 0.8. 0.5 mM IPTG was added for induction at 30°C for 6 h. The cells were collected by centrifugation at 6000 g for 20 min and stored at -80°C.

[0052] 3. Lysis: 1g of bacterial cells were resuspended in 20mL of 50mM Tris-HCl pH8.0 and 50mM NaCl, homogenized by high-pressure homogenization (800 bar, 3 times, 4℃), and the supernatant was collected at 12000g for 30min.

[0053] 4. Purification: The supernatant from bacterial lysis was linearly eluted using anion exchange QHP (A: 50 mM Tris-HCl pH 8.0, 50 mM NaCl; B: 50 mM Tris-HCl pH 8.0, 500 mM NaCl). The elution sites were determined by electrophoresis and the elution was prepared again. The QHP-eluted protein solution was purified a second time using a Superdex 75 molecular sieve to obtain the target protein with a purity ≥90% as confirmed by SDS-PAGE.

[0054] The experimental results are shown in Table 6. It was found that EMC had a soluble expression rate of up to 35% compared to CME and MEC, and its stability was significantly better than the latter two.

[0055]

[0056] The embodiments described above are merely illustrative of specific implementations of the present invention, and while the descriptions are detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A recombinant fusion protein EMC from Mycobacterium tuberculosis, characterized in that: The connection order is as follows: EAST6-MPT64-CFP10.

2. The Mycobacterium tuberculosis recombinant fusion protein EMC according to claim 1, characterized in that: The nucleotide sequence of the Mycobacterium tuberculosis recombinant fusion protein EMC is shown in SEQ ID NO 1.

3. The Mycobacterium tuberculosis recombinant fusion protein EMC according to claim 1, characterized in that: The amino acid sequence of the recombinant fusion protein EMC from Mycobacterium tuberculosis is shown in SEQ ID NO 2.

4. A novel intradermal diagnostic reagent for tuberculosis, characterized in that, It contains the Mycobacterium tuberculosis recombinant fusion protein EMC as described in claims 1-3.

5. The intradermal diagnostic reagent according to claim 4, wherein, The EMC is an in vivo diagnostic product used for tuberculosis infection screening, tuberculosis auxiliary diagnosis, BCG vaccination and tuberculosis infection differentiation.

6. A method for preparing a novel intradermal diagnostic reagent for tuberculosis, characterized in that, The steps are as follows: Fermentation expression: The recombinant plasmid was transformed into BL21(DE3) competent cells to prepare engineered bacteria; the bacterial strain was inoculated at a 1:1000 ratio in vials and cultured overnight at 37°C at 220 rpm; the next day, it was transferred to a large vial (1:100 ratio) and cultured at 37°C for 6-8 hours; the temperature was lowered to 25°C, and the cells were induced overnight at 180 rpm with 0.5 mM IPTG; harvesting: the bacterial cells were harvested by centrifugation at 8000g for 30 min. Extraction and purification: Cell disruption (10:1); Centrifuge at 12000 rpm, 4℃ for 1 hour, collect the clear supernatant; Add saturated ammonium sulfate to the supernatant to 30%, salt out for 1 hour, centrifuge, and then load the supernatant onto Phenyl hydrophobic chromatography. After gradient elution, load the eluent onto SuperQ chromatography. After gradient elution, the eluent is subjected to ultrafiltration to obtain the original solution.

7. The tuberculosis intradermal diagnostic reagent prepared by the preparation method according to claim 6.

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