Mycobacterium tuberculosis and mycobacterium bovis antibody detection antigen combination, antigen preparation method, detection method and reagent

Abstract

The invention relates to a fusion expression protein antigen combination containing a mycobacterium tuberculosis antigen TB16-38 and a fusion expression protein antigen combination containing a mycobacterium tuberculosis antigen and a mycobacterium bovis antigen MPB83-CFP10-ESAT-6. The method for preparing the combined antigen comprises the following steps: connecting a mycobacterium tuberculosis 38KD gene and a 16KD gene to escherichia coli by adopting a linker, expressing to obtain a TB16-38 antigen, and connecting a mycobacterium tuberculosis ESAT-6 gene, a CFP10 gene and a mycobacterium bovis MPB83 gene to escherichia coli by adopting two linkers, and expressing to obtain an MPB83-CFP10-ESAT-6 antigen; the invention relates to a mycobacterium tuberculosis and mycobacterium bovis antibody detection method, which comprises the following steps: fixing two labeled antigens on a solid phase carrier A together, contacting the carrier with a sample to be detected, moving the obtained labeled antigen-antibody compound on a solid phase carrier B, respectively contacting with two antigens (T1 and T2) independently coated on the carrier B, and fixing at the positions of T1 (6) and T2 (7), and determining mycobacterium tuberculosis and mycobacterium bovis antibodies according to display signals of T1 (6) and T2 (7).

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of preventing veterinary infectious diseases, and relates to an immunochromatographic detection technology, in particular to a Mycobacterium tuberculosis and Mycobacterium bovis antibody detection antigen, an antigen preparation method, a detection method and a reagent. BACKGROUND

[0002] Tuberculosis mycobacterium (TM) includes Mycobacterium tuberculosis, Mycobacterium bovis M. bovis , Mycobacterium africanum, also known as Mycobacterium tuberculosis complex (MTBC). Human Mycobacterium tuberculosis infection is most common in the lungs, but all parts of the human body can be infected with Mycobacterium tuberculosis and cause diseases. Pulmonary tuberculosis is still a major infectious disease that seriously endangers human health in this century, is a global public health and social problem, and is one of the main diseases to be controlled in China. So far, there is no ideal Mycobacterium tuberculosis vaccine for tuberculosis prevention, and the BCG vaccine used in clinical practice has been proved to have limited protection, therefore, early diagnosis of Mycobacterium tuberculosis infection plays a very important role in Mycobacterium tuberculosis infection. Traditional early laboratory diagnosis techniques for tuberculosis include PPD (tuberculin protein derivative) skin test, acid-fast staining and Mycobacterium tuberculosis isolation and culture. Since the antigen contained in PPD is pathogenic Mycobacterium, non-pathogenic Mycobacterium and BCG, it cannot distinguish whether the PPD skin test is positive for pathogenic Mycobacterium, non-pathogenic Mycobacterium infection or BCG vaccination. Therefore, its sensitivity and specificity are not high (Wang QZ et al., Chinese Journal of Veterinary Medicine, 2015, 56-59), and the sensitivity of acid-fast staining and Mycobacterium tuberculosis isolation and culture is low (Cambau E, et alClin Microbiol, 1999, 37: 2013-2015. Chen Xueke et al., Chinese Journal of Medical Laboratory Science, 1996, 19: 229. Liu Lihua et al., Chinese Journal of Medical Laboratory Science, 2000, 80: 506). Immunodiagnosis, represented by tuberculosis antibody detection and gamma interferon test, is a diagnostic technique for Mycobacterium tuberculosis that has received considerable attention in recent years. Because Mycobacterium tuberculosis is an intracellular parasite, its infection mechanism and the body's immune response mechanism are quite complex. Various immunodiagnostic methods for tuberculosis each have their advantages and disadvantages. Among numerous detection and diagnostic techniques, tuberculosis antibody detection technology has significant advantages. The specificity and sensitivity of the detection reagent depend on the antigen used and the design of the detection method. Currently, the antigens used in Mycobacterium tuberculosis antibody detection reagents mainly include Mycobacterium tuberculosis somatic proteins, Mycobacterium tuberculosis cell wall lipopolysaccharide (LPS), and Mycobacterium tuberculosis secreted proteins. While LPS has high detection sensitivity, its specificity is not strong. Mycobacterium tuberculosis secretory proteins are a group of antigenic proteins secreted by Mycobacterium tuberculosis during its reproduction. These proteins can stimulate the body to produce specific antibodies, and the detection of Mycobacterium tuberculosis secretory protein-specific antibodies has high diagnostic value for tuberculosis, especially active tuberculosis. Therefore, Mycobacterium tuberculosis membrane proteins and secretory proteins have become the main diagnostic antigens for Mycobacterium tuberculosis. Existing literature reports that among the many Mycobacterium tuberculosis antigens, membrane proteins 38kD and 16kD, and secretory proteins ESAT-6 and CFP10 have greater diagnostic value.

[0003] The Mycobacterium tuberculosis 38kD protein is a phosphate transporter protein, a surface membrane protein of the tuberculosis metabolite (MTB), possessing two MTB-specific B-cell epitopes. It is expressed only in the Mycobacterium tuberculosis complex, and the amount of 38kD protein synthesized by BCG is only one-tenth that of Mycobacterium tuberculosis. ELISA detection of 38kD protein-specific antibodies in serum from tuberculosis patients from different countries showed a sensitivity of 49%-89% and a specificity of 88%-98% (Nakamura RM). et al . Int J Tuberc. Luung Dis, 1998, 2(7): 541). The 38kD protein was also the earliest developed antigen for commercial diagnostic kits, and it is very effective in diagnosing active pulmonary tuberculosis, but its sensitivity varies greatly among different populations.

[0004] The 16kD structural protein of Mycobacterium tuberculosis, also known as the 14kD protein, is found only in the Mycobacterium tuberculosis complex. It is a member of a small heat shock protein family. This protein carries a MTB-specific B-cell epitope and can induce a specific humoral immune response to produce specific antibodies (Agrewala JN). et al. Clin Exp Immunol, 1998, 114(3):392-397), the primary tuberculosis patients and sputum smear-positive tuberculosis patients close contacts of the serum, anti-16kD protein antibody titers increased, suggesting that 16kD protein in the early infection has a significant immunogenicity (Bothamley GH, et al Thorax, 1992, 47(4):270). In addition, it also has chaperone activity, plays an important role in the survival and stability of MTB, with the characteristics of the dominant expression in the MTB dormant period (Yan M in Hu, et al. FEMS Microbiol Lett, 1998, 158 (1): 139). 16kD protein specific, with other mycobacteria essentially no common antigen components, is the ideal diagnostic antigen for early infection and dormant MTB carriers of tuberculosis.

[0005] ESTA-6 and CFP10 are encoded by RD1 (BCG in vitro passage period of deletion of gene fragments - difference zone) gene, RD1 only exists in Mycobacterium tuberculosis and Mycobacterium bovis, BCG deletion. Using ESTA-6 or CFP10 alone as an antigen to detect specific antibodies in the serum of tuberculosis patients, the sensitivity is low, when combined with 38kD protein, ESTA-6 or CFP10 can detect specific antibodies in the serum of tuberculosis patients that lack response to 38kD antigen, which can make up for the lack of 38kD in detecting smear-negative tuberculosis patients on the basis of maintaining high specificity (Dillon DC, et al. J Clin Microbiol, 2000, 38(9):3285).

[0006] Bovine tuberculosis is caused by Mycobacterium bovis, a zoonosis, which can be transmitted from cattle to humans by inhaling aerosols containing bacteria or consuming contaminated dairy products, seriously affecting the development of animal husbandry and human health. Some human tuberculosis is caused by Mycobacterium bovis, and there is a growing trend, which has caused serious public health problems. Therefore, the control of bovine tuberculosis is a very important link in the prevention and treatment of human tuberculosis. In addition to being able to infect cattle, Mycobacterium bovis can also spread among humans, animals and animals, posing new challenges to tuberculosis prevention and control. Literature reports that about 0.5% to 1.5% of tuberculosis patients in developed countries are caused by Mycobacterium bovis infection, and the proportion of tuberculosis patients in developing countries is 15% to 25%, of which 30% of children tuberculosis is caused by Mycobacterium bovis (Rodwell, T.C, et al. Emerging Infectious diseases,2008;14:909). In addition to M. bovis, which can infect humans, M. tuberculosis can also infect cattle, and a certain proportion of bovine tuberculosis is caused by M. tuberculosis or mixed infection of M. bovis and M. tuberculosis. This cross-infection also brings certain technical difficulties to the prevention and control of tuberculosis. At present, the bovine tuberculosis skin sensitivity test (TST) is mainly carried out by inoculating animals with M. bovis tuberculin purified protein derivative (PPD). TST test not only takes a long time (48 hours), but also has certain human error in measuring skin thickness, and PPD contains non-pathogenic environmental mycobacteria and BCG common antigens, which can easily produce false positives. Therefore, the development of specific and sensitive diagnostic reagents is the key to the prevention and control of bovine tuberculosis. M. bovis is an intracellular parasite, which first induces T cell immune response after infecting the body, and then induces B cell immune response, and there is a certain time difference between the two. The TST detection method based on cellular immune response has low sensitivity for open tuberculosis and systemic tuberculosis in the late stage of infection, and this period is the high incidence period of transmission. Serological antibody detection based on humoral immune response can make up for the deficiency of TST. After M. bovis infects the body, it secretes a variety of antigens to induce the body to produce antibodies, and the detection of antibodies to the corresponding antigen has important clinical value for the diagnosis of bovine tuberculosis. MPB83 protein is a secreted lipoprotein mainly expressed in M. bovis, and its expression in BCG is extremely low. MPB83 can stimulate cellular immunity and induce humoral immunity, and contains important B cell epitopes, so serum MPB83 antibody detection has important diagnostic value (Chambers, M. et al . Clinical infectious diseases,2000;30:S283-S287).

[0007] Because the antigens of the Mycobacterium tuberculosis complex are numerous and complex, the antibody spectrum of tuberculosis patients and infected cattle is diverse. Studies have found that the detection of specific anti-tuberculosis antibodies in the serum of tuberculosis patients using 10 kinds of recombinant Mycobacterium tuberculosis protein antigens showed that nearly 90% of the patient's serum contained at least one anti-tuberculosis antibody, and the number, type and level of antibody expression in the body can vary with the patient's immune background, different stages of the disease and different strains of Mycobacterium tuberculosis (Lyashchenko K, et al . Infect Immun,1998,66(8):3936). Due to the non-simplicity of the pathogen of bovine tuberculosis, the single "antigen-antibody" serum antibody detection mode also has the defect of missed detection. SUMMARY

[0008] The present application aims to solve the above technical problems. To this end, the present application provides a mycobacterium tuberculosis and mycobacterium bovis antibody detection antigen combination, an antigen preparation method and a detection method.

[0009] The present application detects the TB16-38 and MPB83-CFP10-ESAT-6 fusion expression protein antibodies of the mycobacterium tuberculosis and mycobacterium bovis infection positive bovine serum by the "double antigen sandwich" immunochromatographic detection, and finds that (1) the 38KD, 16KD, ESAT-6, CFP10 and MPB83 protein antibodies of the mycobacterium tuberculosis and mycobacterium bovis infection positive bovine serum have strong complementary effects, and the detection of the 38KD, 16KD, ESAT-6, CFP10 and MPB83 protein antibodies of the mycobacterium tuberculosis infection positive bovine serum can improve the detection sensitivity, and does not increase the false positive problem; (2) the TB16-38 and MPB83-CFP10-ESAT-6 fusion expression protein forms are combined and used, the five target points are synchronously detected by the "double antigen sandwich" immunochromatographic detection, the mycobacterium tuberculosis and mycobacterium bovis antibodies can be simultaneously detected, the missed detection of the bovine infection mycobacterium tuberculosis and the bovine infection mycobacterium bovis can be effectively prevented, and the false positive problem is not increased, and the detection method can be used for the detection of the mycobacterium tuberculosis and mycobacterium bovis antibodies.

[0010] According to the embodiment of the present application, the TB16-38 is a recombinant fusion protein artificially expressed in E. coli, and the expression method is that the human mycobacterium tuberculosis 38KD gene and the 16KD gene are connected by a linker and cloned in a prokaryotic expression vector, E. coli is transfected, and the obtained renaturation and purification after induction expression. The MPB83-CFP10-ESAT-6 protein is also a recombinant fusion protein artificially expressed in E. coli, and the expression method is that the human mycobacterium tuberculosis ESAT-6 gene, the CFP10 gene and the mycobacterium bovis MPB83 gene are connected by a linker and cloned in a prokaryotic expression vector, E. coli is transfected, and the obtained renaturation and purification after induction expression.

[0011] According to the embodiment of the present application, the multi-target "double antigen sandwich" immunochromatographic detection method is innovated on the conventional "double antigen sandwich" immunochromatographic detection method which is easily obtained by ordinary skilled technicians, and the specific innovative method is as follows: (1) antigen labeling, the recombinant Mycobacterium tuberculosis fusion expression protein antigens TB16-38 and MPB83-CFP10-ESAT-6 are labeled respectively, and the labeling material can be selected from any one of colloidal gold, colored latex microspheres and fluorescent microspheres. After labeling, the antigens are solidified on non-woven fabric or glass fiber. (2) Antigen NC membrane coating, referring to the NC membrane protein coating method which is easily obtained by ordinary skilled technicians, T1 and T2 detection lines are coated on the NC membrane detection line position at the same time, the distance between T1 and T2 lines is 0.3 cm, T1 is coated with TB16-38 fusion protein, and T2 line is coated with MPB83-CFP10-ESAT-6 fusion expression protein. (3) Detection reaction mode, when serum, plasma or whole blood sample is detected, any one of the Mycobacterium tuberculosis antigen 38kD, 16kD, ESAT-6, CFP10 and MPB83 protein antibodies existing in the sample will produce antigen-antibody reaction with the corresponding protein to form a "labeled antigen-antibody" complex, the complex climbs along the NC membrane to the detection line T1 and T2 to produce antigen-antibody reaction again with the corresponding antigens coated on the detection line to form a "labeled antigen-antibody-antigen" complex, the labeled antigen is fixed on the detection line T1 or T2, and the label will develop color or fluorescence, and the sample is determined to be Mycobacterium tuberculosis antibody positive by direct observation and qualitative judgment, or the content of the antibody is quantitatively analyzed by an instrument. If any one of the above Mycobacterium tuberculosis antigen protein antibodies does not exist in the sample, the detection line T1 and T2 do not develop color or fluorescence at the end of the detection reaction.

[0012] Figure 1 The mode of the detection method and detection reagent of the present application is shown According to the embodiment of the present application, the Mycobacterium tuberculosis antibody and bovine Mycobacterium tuberculosis detection method provided by the present application can be used for Mycobacterium tuberculosis antibody and bovine Mycobacterium tuberculosis antibody detection, can simultaneously improve the sensitivity of Mycobacterium tuberculosis antibody and bovine Mycobacterium tuberculosis antibody, and does not increase the false positive problem of detection results. The detection sample can be serum, plasma or whole blood.

[0013] The present application provides a Mycobacterium tuberculosis and bovine Mycobacterium tuberculosis antibody multi-target detection reagent in a second aspect. For the reagent of the present application, it is preferred that the labeled recombinant Mycobacterium tuberculosis fusion protein TB16-38, the labeled MPB83-CFP10-ESAT-6 and the labeled murine antibody (monoclonal antibody or polyclonal antibody) are prepared respectively.

[0014] The reagent of the present application is composed of a detection strip and an outer shell, and the detection strip and the outer shell are shown in Figure 2The reagent needs to be matched with other components. For example, sample diluent is needed in double antigen sandwich immunochromatographic detection. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is the structural diagram of the detection card of the present application

[0016] 1-detection card 2-sample hole 3-observation hole

[0017] Figure 2 is the internal structure diagram of the detection card of the present application

[0018] 4-sample end water absorption layer 5-gold mark pad 6-detection line T1 7-detection line T2

[0019] 8-control line 9-detection layer 10-water absorption end water absorption layer 11-liner

[0020] Best embodiment of the present application 1 TB16-38, MPB83-CFP10-ESAT-6 fusion protein recombinant expression of target gene amplification: using Mycobacterium tuberculosis H37RV international standard strain (inactivated) as template, PCR amplification hspX (TB16KD), pstS1 (38KD), esat-6 and cfp10 gene, overlap PCR amplification TB16-38 gene and cfp10-esat-6 gene. According to the known sequence of GenBank bovine Mycobacterium tuberculosis mpb83 gene, the gene is synthesized, and the primer is designed, and the mpb83-cfp10-esat-6 gene is amplified by overlap PCR. hspX (16kD) gene amplification primer: PF1: 5'-CCGGATCCATGGCCACCACCCTTCC-3'; PR2: 5'-GCTGCCGCCACCGCCGCTTCCGCCACCGCCGCTTCCACCGCCACCG TTGGTGGACCGGA-3'. pstS1 (38kD) gene amplification primer: PF3: 5'-GGTGGCGGTGGAAGCGGCGGTGGCGGAAGCGGCGGTGGCGGCAGCATGGGCTCGAAACCACCGAGCG-3'; PR4: 5'-GGCAAGCTTCTAGCTGGAAATCGTCGCGA-3'. Cfp10 gene amplification primer: PF5: 5'-GGAGGCGGTGGATCAGGTGGATTCGGTTCTGGTGGCGGAGGCTCTATGGCAGAGATGAAGACCGA-3'; PR6: 5'-GCTGCCGCCACCGCCGCTTCCGCCACCGCCGCTTCCACCGCCACCGAAGCCCATTTGCGAGGACAG-3'. Esat-6 gene amplification primers: PF7: 5'-GGTGGCGGTGGAAGCGGCGGTGGCGGAAGCGGCGGTGGCGGCAGCATGGCAGAGATGAAGACCGA-3'; PR8: 5'-GGCAAGTGCGAAVATCCCAGTGACGT-3'. Mpb83 gene amplification primers: PF9: 5'-CCGGATATGATCAACGTTCAGGCCAA-3'; PR10: 5'-AGAGCCTCCGCCACCAGAACCGCCTCCACCTGATCCACCGCCTCCCTGTGCCGGGGGCATCAGCA-3'.

[0021] Single gene amplification reaction system: ddH2O 33.5 µl dNTP mixture (2.5 mM) 4 µl Template 4 µl 10x PCR Buffer 5 µl Forward primer 1.5 µl Reverse primer 1.5 µl rpolymerase 0.5 µl Total 50 µl Amplification procedure: 94℃ 10 min; 94℃ 1 min, 55℃ 1 min, 72℃ 1 min, 30 cycles; 72℃ 10 min; 4℃ storage. Take 5 μl PCR product for agarose gel electrophoresis to detect the amplification results of the target gene. Use Axygen DNA gel recovery kit to recover the PCR amplification product, and refer to the kit instructions for operation steps.

[0022] hspX - pstS1, mpb83-cfp10-esat-6 PCR amplification of fusion gene: Use the single gene recovered by gel to amplify the fusion gene by Over-lap PCR. The reaction system is: ddH2O 36.5 µl dNTP mixture (2.5 mM) 3.5 µl 10x PCR Buffer 5 µl Gene 1 1.5 µl Gene 2 1.5 µl Total 47 µl 47 μl in total. Amplification was performed according to the following procedure: 94℃ 10 min; 94℃ 1 min, 55℃ 1 min, 72℃ 1 min, 5 cycles. PCR tubes were removed and rTaq polymerase, PF1 / PR4 (amplifying hspX-pstS1), PF5 / PR8 (amplifying cfp10-esat-6), PF9 / PR8 (amplifying mpb83-cfp10-esat-6) were added, and the total volume of the PCR reaction was 50 μl, and the reaction system was as follows: rpolymerase 1 µl F primer 1 µl R primer 1 µl Amplification was performed according to the following procedure: 94℃ 10 min; 94℃ 1 min, 55℃ 1 min, 72℃ 1 min, 30 cycles; 72℃ 10 min; 4℃ storage. The target fragment was recovered using an Axygen DNA gel recovery kit, and the operation was performed according to the kit instructions.

[0023] hspX - pstS1, mpb83-cfp10-esat-6 Double enzyme digestion of the fusion gene and pET-30a empty vector hspX-pstS1, mpb83-cfp10-esat-6 The fusion gene and pET-30a empty vector were respectively subjected to double enzyme digestion, and the total volume was 50 μl, and the reaction system was as follows: ① hspX-pstS1 、 mpb83-cfp10-esat-6 Fusion gene: Fusion gene 30 µl ddH2O 9 µl 10x Buffer 5 µl I 3 µl I 3 µl Total 50 µl ② Double enzyme digestion of pET-30a empty vector Vector plasmid 30 µl ddH2O 9 µl 10x Buffer 5 µl I 3 µl I 3 µl Total 50 µl 37℃ enzyme digestion overnight. The enzyme digestion product was subjected to agarose gel electrophoresis, and the enzyme digestion product was recovered using an Axygen gel recovery kit, and the operation was performed according to the kit instructions.

[0024] Ligation of the target gene and pET-30a empty vector The total volume of the ligation reaction was 25 μl, and the reaction system was as follows: pET30a vector recovered from gel 6 µl ddH2O 13.5 µl 10x T4 DNA ligase Buffer 2.5 µl Gene of interest recovered from gel 2 µl T4 DNA ligase 1 µl Total 25 µl Mixed uniformly, 16℃ ligation for 4 h or 4℃ overnight.

[0025] Transformation of the ligation product: 100 μl E. coliAdd 10 μl ligation product to BL21(DE3) competent cells, ice bath for 30 min, 42℃ heat shock for 45 s, ice bath for 4 min. Add 300 μl LB liquid medium, 37℃ incubate for 50 min. Take 200 μl transformation product and evenly spread on LB selective medium containing the corresponding antibiotic (concentration of 100 μg / ml), 37℃ inverted culture overnight. The selective medium is LB solid medium containing 100 μg / ml Kana.

[0026] Induced expression of recombinant bacteria: Take the preserved strain from the -20℃ refrigerator, ice bath for 30 min, take 50 μl bacterial solution and add to 15 ml LB liquid medium containing 100 μg / ml Kana, 37℃ 200 rpm culture overnight. According to the ratio of 1:100, inoculate in 200 ml LB liquid medium containing 100 μg / ml Kana, 37℃ 200 rpm culture until the OD value is 0.5, take 1 ml uninduced bacterial solution from each bottle as a blank control, adjust the induction temperature to 27℃, 33℃, 42℃, add IPTG with a final concentration of 1 mmol, 37℃ induce expression for 5 h, and store at 4℃ for standby.

[0027] Purification of recombinant protein Purification of soluble protein: Equilibrate the nickel column with 10 ml pH 7.4 PBS buffer (containing 0.5 M NaCl, 20 mM imidazole). Take the supernatant collected after ultrasonic bacterial disruption, and load at a speed of 0.5 ml / min, control the flow rate at 1 ml / min. Add 15 ml PBS equilibration buffer to wash away the unbound protein, control the flow rate at 1 ml / min. Add 5 ml pH 7.4 PBS elution buffer containing 10 mM imidazole, 20 mM imidazole, 40 mM imidazole, 80 mM imidazole, 100 mM imidazole, 200 mM imidazole in sequence, wash away the bound protein on the nickel column, control the flow rate at 1 ml / min, and collect the flow sample in 2 ml / tube. Collect the 100 mM imidazole eluate, detect the protein concentration by ultraviolet spectrophotometer, and store at -20℃ for standby.

[0028] Purification of inclusion body protein: Equilibrate the nickel column with 10 ml PBS equilibration buffer (0.5 M NaCl, 8 M urea, 20 mM imidazole, pH 7.4). Load the 8 M urea eluate collected after ultrasonic disruption of the bacteria at a flow rate of 0.5 ml / min and control the flow rate at 1 ml / min. Wash the unbound proteins with 15 ml PBS equilibration buffer at a flow rate of 1 ml / min. Elute the bound proteins with 5 ml PBS elution buffer (0.5 M NaCl, 8 M urea, pH 7.4) containing 10 mM imidazole, 20 mM imidazole, 40 mM imidazole, 80 mM imidazole, 100 mM imidazole, 200 mM imidazole, respectively, at a flow rate of 1 ml / min and collect the flow-through in 2 ml tubes. Collect the 100 mM imidazole eluate and determine the protein concentration by UV spectrophotometry.

[0029] Renaturation of recombinant protein: Cut the dialysis bag into 10 cm long pieces, boil in EDTA solution (pH 8.0) for 15 min, remove and wash in ddH2O. Prepare a dialysis solution containing 100 mM Tis-HCl, 1 mM EDTA, 0.1 mM oxidized glutathione, 0.2 mM reduced glutathione, pH 8.0. Add 500 ml of the dialysis solution to a beaker. Add the purified inclusion body recombinant protein to the dialysis bag at a ratio of 10 mg recombinant protein per liter of dialysis solution. Place the dialysis bag in the beaker and dialyze overnight at 4°C to renature the recombinant protein. Add 500 ml of 4 M urea, 2 M urea and PBS solution to the beaker, respectively, and dialyze overnight at 4°C. Concentrate the recombinant protein by centrifugation at 5000 rpm and determine the protein concentration in each collection tube by UV spectrophotometry.

[0030] 2. Recombinant expression of fusion protein antigen tag Preferably, the colloidal gold label is prepared as follows: i. Preparation of colloidal gold: 1.5% of citric acid trisodium salt with a concentration of 1% is added to 0.01% chloroauric acid, and boiled for 10-20 minutes to obtain a colloidal gold solution with a particle size of 25-50 nm; ii. Labeling of the recombinant expression fusion protein antigen TB16-38 with colloidal gold: the colloidal gold solution obtained in (i) is adjusted to pH 8.0-8.6 with 0.2M potassium carbonate solution, and 10-12 mg / 100 ml of the protein antigen TB16-38 is added. After stirring, 0.5 g / 100 ml of animal serum protein is added to the solution, which is then allowed to stand at 4°C for 2-4 hours; iii. The colloidal gold solution obtained in (ii) is centrifuged at 2000 rpm for 10-15 minutes, and the precipitate is removed; iv. The supernatant obtained in (iii) is centrifuged at 10000 rpm for 60 minutes, and the precipitate is removed; v. The precipitate obtained in (iv) is dissolved in 8-10 ml / 100 ml of 0.02M Tris-Hcl buffer solution (pH 7.4) containing 0.5% animal serum protein and 0.05% sodium azide, to obtain a colloidal gold solution; vi. Glass fibers or non-woven fabric are immersed in the colloidal gold solution obtained in (v), until liquid seeps out of the glass fibers or non-woven fabric, which is then dried at 37°C to obtain a colloidal gold pad.

[0031] The labeling method of the recombinant expression fusion protein antigen MPB83-CFP10-ESAT-6 is similar to that of the recombinant expression fusion protein antigen TB16-38.

[0032] The mouse IgG antibody label, preferably a mouse-derived anti-bovine IgG monoclonal antibody, is labeled by a method similar to that of the recombinant expression fusion protein antigen TB16-38.

[0033] The recombinant expression fusion protein antigens TB16-38 and MPB83-CFP10-ESAT-6 are coated on the T1 detection line and the T2 detection line of the NC membrane, and the coating method of the quality control line and the detection line is as follows: TB16-38 and MPB83-CFP10-ESAT-6 protein a are taken, adjusted to a concentration of 1 mg / ml, and sprayed on the T1 and T2 detection lines of the NC membrane at the same time using a special membrane spraying machine, with a distance of 0.3 cm between the T1 and T2 detection lines; goat anti-mouse IgG or rabbit anti-mouse IgG antibody is taken, adjusted to a concentration of 1 mg / ml, and sprayed on the quality control line of the NC membrane at a distance of 0.5 cm from the T2 detection line using a special membrane spraying machine, with a spraying amount of 10-15 μl / ml for both the detection line and the quality control line. After spraying, the membrane is dried at 37°C for 2 hours; and then, 10% calf serum in 0.01M PBS (phosphate buffered saline) with a pH of 7.0 is used to block the membrane at 37°C for 30 minutes, the membrane is rinsed with 0.01M PBS with a pH of 7.0, and the membrane is dried at 37°C.

[0034] The reagent of the present application comprises a sample diluent, which is a 0.01M phosphate buffer solution with pH 7.2.

[0035] The reagent of the present application has sensitivity and specificity to bovine serum 15 samples of positive bovine serum infected with tuberculosis and 5 samples of healthy bovine serum are detected by using the reagent of the present application, and the corresponding sample source bovine anticoagulant blood is detected by using a Prionic tuberculosis detection kit (gamma-interferon release experiment), and the corresponding bovine nasal swab is detected by using a bovine tuberculosis mycobacterium real-time fluorescence quantitative PCR detection kit.

[0036] Results: 13 of the 15 samples of positive bovine serum infected with tuberculosis are detected to have positive antibodies by using the reagent of the present application, the positive rate is 86.66%, 5 samples of healthy bovine serum are detected to have negative antibodies, the negative rate is 100%; 13 of the 15 samples of anticoagulant blood are detected to have positive results by using the Prionic tuberculosis detection kit, the positive rate is 86.66%; 4 of the 5 samples of healthy bovine anticoagulant blood are detected to have negative results by using the Prionic tuberculosis detection kit, the negative rate is 80%; 11 of the 15 samples of positive bovine nasal swab are detected to have positive results by using the fluorescence quantitative PCR detection kit, the positive rate is 73.33%, 1 of the 5 samples of healthy bovine nasal swab is detected to have a positive result, and 4 samples are detected to have negative results, the negative rate is 80%.

[0037] The above results prove that the sensitivity of the reagent of the present application reaches 86.66%, reaches the sensitivity (86.66%) of the gamma-interferon release experiment, is higher than the sensitivity (73.33%) of the fluorescence quantitative PCR, the specificity of the present application reaches 100%, is higher than the specificity (80%) of the gamma-interferon release experiment and the specificity (80%) of the fluorescence quantitative PCR.

[0038] The antigen combination for detecting the anti-tuberculosis mycobacterium antibody and the bovine tuberculosis mycobacterium antibody according to the present application has high sensitivity and high specificity, rarely has the problem of false negative which troubles the clinic, and rarely has false positive, and thus is beneficial to the detection of the anti-tuberculosis mycobacterium antibody and the bovine tuberculosis mycobacterium antibody.

[0039] The preparation method of the tuberculosis mycobacterium antibody and the bovine tuberculosis mycobacterium of the present application can provide a multi-antigen fusion expression and structure remodeling method, the antigen has high sensitivity and high specificity, rarely has the problem of false negative which troubles the clinic, and rarely has false positive.

[0040] The tuberculosis antibody and the bovine tuberculosis bacillus antibody detection method has higher sensitivity and high specificity, rarely has false negative problem, and rarely has false positive. The sample collection and detection process is simple, the reaction sample is clinically practical, has high sensitivity, and therefore it is very beneficial to the diagnosis of tuberculosis antibody and bovine tuberculosis bacillus infection.

Claims

1. A combination of Mycobacterium tuberculosis and Mycobacterium bovis antigens comprising a fusion expression protein of Mycobacterium tuberculosis antigen TB16-38 and a fusion expression protein of Mycobacterium tuberculosis and Mycobacterium bovis antigen MPB83-CFP10-ESAT-6.

2. The combination of Mycobacterium tuberculosis and Mycobacterium bovis antigens of claim 1, wherein TB16-38 is a fusion expression protein with a molecular weight of 60KD and MPB83-CFP10-ESAT-6 is a fusion expression protein with a molecular weight of 50KD.

3. The combination of Mycobacterium tuberculosis and Mycobacterium bovis antigens of claims 1-2, wherein TB16-38 is obtained by expression in E. coli using a linker to connect the gene of 38KD protein of Mycobacterium tuberculosis and the gene of 16KD protein, and MPB83-CFP10-ESAT-6 is obtained by expression in E. coli using two linkers to connect the gene of ESAT-6 protein of Mycobacterium tuberculosis, the gene of CFP10 protein and the gene of MPB83 protein of Mycobacterium bovis.

4. A method for preparing the combination of Mycobacterium tuberculosis and Mycobacterium bovis antigens, comprising primer design and synthesis, vector construction, sequencing verification, E. coli transfection, induction of expression, protein structure renaturation and purification.

5. A method for detecting antibodies of Mycobacterium tuberculosis and Mycobacterium bovis, comprising the use of two fusion expression antigens of claims 1-3.

6. The method for detecting antibodies of Mycobacterium tuberculosis and Mycobacterium bovis of claim 5, which is to fix the two labeled antigens of claims 1-3 on a solid phase carrier A, to make the carrier contact with the sample to be tested, to make the labeled antigen-antibody complex move on a solid phase carrier B, and to make the complex contact with two antigens (T1 and T2) separately coated on carrier B and fixed on T1 and T2 positions, and to determine the antibodies of Mycobacterium tuberculosis and Mycobacterium bovis according to the signals displayed by T1 and T2.

7. The method for detecting antibodies of Mycobacterium tuberculosis and Mycobacterium bovis of claim 6, wherein the sample to be tested is bovine serum or plasma.

8. The method for detecting antibodies of Mycobacterium tuberculosis and Mycobacterium bovis of claim 5 or 6, wherein carrier A is glass fiber or non-woven fabric, and carrier B is nitrocellulose membrane.

9. The method for detecting antibodies of Mycobacterium tuberculosis and Mycobacterium bovis of claim 5 or 6, wherein the labeled antigens are labeled TB16-38 and labeled MPB83-CFP10-ESAT-6.

10. The method for detecting antibodies of Mycobacterium tuberculosis and Mycobacterium bovis of claim 5 or 6, wherein T1 is antigen TB16-38 coated on nitrocellulose membrane, and T2 is antigen MPB83-CFP10-ESAT-6 coated on nitrocellulose membrane, or T1 is antigen MPB83-CFP10-ESAT-6 coated on nitrocellulose membrane, and T2 is antigen TB16-38 coated on nitrocellulose membrane, and any of the coating methods of T1 and T2 is included in the present claim.

11. The method for detecting antibodies against M. tuberculosis and M. bovis according to claims 6 and 9, wherein the labeled antigen is a colloidal gold labeled antigen, a color microsphere labeled antigen or a fluorescent microsphere labeled antigen.

12. A test reagent for detecting antibodies against M. tuberculosis and M. bovis, comprising a combination of one M. tuberculosis antigen and one M. bovis antigen according to claims 1-5.

13. The reagent for detecting antibodies against M. tuberculosis and M. bovis according to claim 12, comprising an immobilized antigen coated on a solid support B and a labeled antigen which reacts with the antibody to be detected and is immobilized on a support A.

14. The reagent for detecting antibodies against M. tuberculosis and M. bovis according to claim 13, wherein the solid support A is a glass fiber or a non-woven fabric and the support B is a nitrocellulose membrane.

15. The reagent for detecting antibodies against M. tuberculosis and M. bovis according to claim 13, wherein the labeled antigen is a colloidal gold labeled antigen, a color microsphere labeled antigen or a fluorescent microsphere labeled antigen.

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