Recombinant protein of microsporidia polar tube protein EbPTP2 and its preparation method and application

Abstract

The present invention belongs to the field of genetic engineering and immunoassay technology, and specifically relates to a recombinant protein of the polar tube protein EbPTP2 of microsporidia, and a preparation method and application thereof. The present invention has found that the polar tube protein exhibits strong immunogenicity both under experimental conditions and under natural infection conditions, which provides a potential target for establishing accurate diagnosis and effective prevention and treatment of microsporidiosis. The recombinant protein of the present invention can be used as an antigen for detecting microsporidia, and has good antigenicity, accurately captures specific antibodies in serum, and achieves the purpose of accurately detecting microsporidia infection. At the same time, the recombinant protein is obtained by expressing the optimized gene sequence through an in vitro expression system. After sequence optimization, the recombinant protein in the present application has a higher protein yield, and the operation steps are simple and the cost is low, which provides technical support for the development of microsporidia diagnostic reagents and vaccines.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and immune detection, and particularly relates to a recombinant protein of microsporidia polar tube protein EbPTP2 and a preparation method and application thereof. Background Art

[0002] Microsporidia are a class of single-celled eukaryotic organisms that are widely distributed in nature and are obligate intracellular parasites. Their hosts range from protozoa to invertebrates and vertebrates, such as insects, fish, and mammals, including humans. To date, over 200 genera and 1,500 species of microsporidia have been reported, 17 of which have been reported to infect humans and cause related diseases. Among these 17 species, Enterocytozoon bieneusi is the most prevalent in humans. Numerous genotypes of Enterocytozoon bieneusi have been identified, including those that infect humans exclusively, those that can infect both humans and animals, and those that infect only animals. These findings suggest that Enterocytozoon bieneusi has multiple modes of infection and transmission, including zoonotic, interzootic, and human-to-human transmission. Typical clinical symptoms of Enterocytozoon bieneusi infection include chronic diarrhea, anorexia, weight loss, and abdominal distension. Epidemiological studies of immunocompromised individuals, such as those who have undergone kidney, lung, liver, and bone marrow transplants, have revealed a prevalence of 1%-10%, with clinical manifestations including watery, non-bloody diarrhea, nausea, diffuse diarrhea, and fever. In immunocompetent patients, infection typically manifests as a transient illness. Currently, there are no effective vaccines or medications for microsporidiosis, making timely diagnosis of Enterococcus bieneusi and control of the source of infection crucial.

[0003] The main methods for detecting microsporidia include etiological testing, molecular biology testing, and immunological testing. Immunological testing for microsporidia is sensitive and highly specific, and serological testing can effectively detect specific antibodies produced by microsporidia infection. Currently, the detection of serum-specific antibodies mainly relies on microsporidia collected from infected animals or cultured in tissues and cells as antigens. However, the resulting microsporidia are of low purity and complex composition, which can easily cause cross-reactions, affecting detection effectiveness and significantly reducing the accuracy of test results. Summary of the Invention

[0004] The present invention aims to provide a recombinant protein of the microsporidian polar tube protein EbPTP2, a preparation method thereof, and an application thereof. The recombinant protein can be used as an antigen for detecting microsporidia, has good antigenicity, accurately captures specific antibodies in serum, and achieves the purpose of accurately detecting microsporidian infection.

[0005] The present invention provides a recombinant protein of microsporidium polar tube protein EbPTP2, the amino acid sequence of the recombinant protein is shown in SEQ ID NO.1.

[0006] The present invention also provides a gene encoding the recombinant protein described in the above technical solution, and the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0007] The present invention also provides a method for preparing the recombinant protein described in the above technical solution, comprising: expressing the recombinant protein using an in vitro protein expression system, wherein the in vitro protein expression system includes a prokaryotic expression system, a eukaryotic expression system or a plant expression system.

[0008] Preferably, the step of expressing the recombinant protein using the prokaryotic expression system comprises:

[0009] connecting the gene encoding the recombinant protein to a prokaryotic expression vector to obtain a recombinant prokaryotic expression vector;

[0010] Transforming the recombinant prokaryotic expression vector into prokaryotic cells to obtain recombinant engineered bacteria;

[0011] The recombinant engineered bacteria are induced to express with IPTG, and the obtained induced bacteria contain the recombinant protein.

[0012] Preferably, the prokaryotic expression vector comprises a plasmid vector;

[0013] The prokaryotic cells include competent Escherichia coli cells.

[0014] Preferably, the preparation method further comprises purifying the recombinant protein in the induced bacteria to obtain the recombinant protein.

[0015] The present invention also provides a recombinant expression vector, which includes a gene encoding the recombinant protein in the above technical solution and a starting vector.

[0016] The present invention also provides an engineered bacterium, which comprises a gene encoding the recombinant protein described in the above technical solution or a recombinant expression vector described in the above technical solution.

[0017] The present invention also provides the use of the recombinant protein, the gene, the recombinant protein prepared by the preparation method, the recombinant expression vector or the engineered bacteria described in the above technical solution in the preparation of a reagent for detecting microsporidia and / or microsporidia infection.

[0018] Preferably, the recombinant protein is used as an antigen.

[0019] Beneficial effects:

[0020] The present invention provides a recombinant protein of the microsporidian polar tube protein EbPTP2, the amino acid sequence of which is shown in SEQ ID NO.1. The infection organs of microsporidia (spore wall, polar tube, sporoplasm and polar membrane) successfully transform this highly diverse species into an intracellular parasite that can infect almost all types of cells. The polar tube, as a unique infection structure of microsporidia, not only provides a bridge for the transport of sporoplasm to host cells, but also prevents the sporoplasm from being destroyed by the external extreme environment during the transport process. Therefore, the polar tube plays an important role in the microsporidian infection process. The present invention has found that the polar tube protein exhibits strong immunogenicity both under experimental conditions and under natural infection conditions, which provides a potential target for establishing accurate diagnosis and effective prevention and treatment of microsporidiosis. The recombinant protein of the present invention can be used as an antigen for detecting microsporidia, and has good antigenicity, and can accurately capture specific antibodies in serum, achieving the purpose of accurately detecting microsporidian infection.

[0021] At the same time, the recombinant protein is obtained by expressing the optimized gene sequence through a prokaryotic expression system. After sequence optimization, the recombinant protein in this application has a higher protein yield, and the operation steps are simple and the cost is low, providing technical support for the development of microsporidia diagnostic reagents and vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0023] Figure 1 This is a graph showing the results of the recombinant protein induced by pET-32a(+)-EbPTP2 in Example 1;

[0024] Figure 2 This is a diagram showing the expression and purification results of the recombinant protein EbPTP2 in Example 1;

[0025] Figure 3 This is a diagram showing the results of SDS-PAGE analysis of the recombinant protein EbPTP2-His purified before optimization in Example 1;

[0026] Figure 4 This is a diagram showing the results of Western blot analysis of the polyclonal antibody against the microsporidian polar tube protein EbPTP2 in Example 2;

[0027] Figure 5 Figure 3 shows the stool genome results of E.bieneusi-specific primers for positive samples (A) and the Western blot results of EbPTP2 recombinant protein for positive sample serum (B) in Example 4. DETAILED DESCRIPTION

[0028] The present invention provides a recombinant protein of microsporidium polar tube protein EbPTP2. The amino acid sequence of the recombinant protein is shown in SEQ ID NO.1, specifically: MQVGTAPQQQLLNTAAVDPTGLVEH LKELQREAQDAREKAAAECRAKTEQNNQTIEGRPLVTFDPVEIQKCIAEKKEKLSKIATSIKQLISKEMSKPSSSSSSSKPCVTKLDQKSKACYTKTALQQWIKDPRFSVDVHNNEAEIWEGDTLLMMIVHESPKYVIMKPPKICRTFFKKPNYKMDINRAGDMITSEGKPNQKKPQELNCKPCSDFLNTTEGKFANCDNSCSPINILTSVKDTITEENSKVIDKKLTKNVEKENKEE.

[0029] The present invention also provides a gene encoding the recombinant protein described in the above technical solution. The nucleotide sequence of the gene is shown in SEQ ID NO.2, specifically: 5'-ATGCAAGTTGGTACTGCACCACA -3'; the nucleotide sequence shown in SEQ ID NO.2 is obtained by optimizing the microsporidian polar tube protein EbPTP2 gene sequence (XM_001827820.1) in GenBank; through the optimization, the expression level of the recombinant protein can be increased.

[0030] The present invention also provides a method for preparing the recombinant protein described in the above technical solution, comprising: expressing the recombinant protein using an in vitro protein expression system, wherein the in vitro protein expression system includes a prokaryotic expression system, a eukaryotic expression system or a plant expression system, preferably a prokaryotic expression system.

[0031] The steps of expressing the recombinant protein using the prokaryotic expression system of the present invention include: connecting the gene encoding the recombinant protein to a prokaryotic expression vector to obtain a recombinant prokaryotic expression vector;

[0032] Transforming the recombinant prokaryotic expression vector into prokaryotic cells to obtain recombinant engineered bacteria;

[0033] The recombinant engineered bacteria are induced to express with IPTG, and the obtained induced bacteria contain the recombinant protein.

[0034] The present invention connects the gene encoding the recombinant protein to a prokaryotic expression vector to obtain a recombinant prokaryotic expression vector. The nucleotide sequence of the gene encoding the recombinant protein of the present invention is preferably as shown in SEQ ID NO.2; when performing the connection, the nucleotide sequence of the gene encoding the recombinant protein is preferably prepared by a gene synthesis method, and the preparation steps are not particularly limited, and the steps of conventional gene synthesis in the art can be adopted. The prokaryotic expression vector of the present invention preferably includes a plasmid vector, more preferably pET-32a(+); when the prokaryotic expression vector is pET-32a(+), the gene encoding the recombinant protein is preferably inserted between the Bam HI and Hind III double enzyme cutting sites. The present invention does not specifically limit the specific steps of constructing the recombinant prokaryotic expression vector, and the steps of constructing conventional recombinant expression vectors in the art can be adopted.

[0035] After obtaining the recombinant prokaryotic expression vector, the present invention transforms the recombinant prokaryotic expression vector into prokaryotic cells to obtain recombinant engineered bacteria. The prokaryotic cells of the present invention preferably include competent Escherichia coli cells, more preferably competent E. coli Rosetta cells. The present invention does not particularly limit the method for transforming the recombinant prokaryotic expression vector into prokaryotic cells, and conventional procedures in the art can be used.

[0036] After obtaining the recombinant engineered bacteria, the present invention induces the expression of the recombinant engineered bacteria with IPTG to obtain induced bacteria. The final concentration of IPTG in the bacterial solution of the recombinant engineered bacteria is preferably 0.1 to 0.5 mM / L, more preferably 0.1 to 0.3 mM / L. The OD of the bacterial solution of the recombinant engineered bacteria of the present invention is 600 The value is preferably 0.5 to 0.7, more preferably 0.6. The present invention has no particular limitation on the specific steps of inducing expression with IPTG, and conventional IPTG induction steps in the art can be used.

[0037] After obtaining the induced bacteria, the present invention preferably performs a first resuspending of the induced bacteria in Buffer A, and then ultrasonically disrupting and centrifuging the resuspended bacteria to obtain a precipitate; the Buffer A of the present invention is an aqueous solvent, preferably further comprising 100mM NaCl and 10mM Tris-HCl, and the pH value is preferably 8.0. The present invention does not specifically limit the ultrasonic disruption and centrifugation steps, and conventional ultrasonic disruption and centrifugation steps in the art can be used.

[0038] After obtaining the precipitate, the induced cells are preferably resuspended a second time in Buffer C, and the resuspended cells are ultrasonically disrupted and centrifuged to obtain a supernatant as a cell disruption solution, which contains the recombinant protein. Buffer C in the present invention is an aqueous solvent, preferably further comprising 100 mM NaCl, 10 mM Tris-HCl, and 8 M Urea, with a pH of preferably 8.0. The ultrasonic disruption and centrifugation steps are not particularly limited in the present invention, and conventional ultrasonic disruption and centrifugation steps in the art can be used.

[0039] After obtaining the bacterial cell disrupted liquid, the present invention preferably purifies the bacterial cell disrupted liquid. The specific type of the purification is not particularly limited, and the corresponding purification can be performed according to the type of purification tag used.

[0040] The present invention also provides a recombinant expression vector comprising a gene encoding the recombinant protein described in the above technical solution and a starting vector. The starting vector of the present invention preferably comprises a prokaryotic expression vector, more preferably a plasmid vector, and more preferably pET-32a(+). When the starting vector is pET-32a(+), the gene encoding the recombinant protein is preferably inserted between the Bam HI and Hind III double restriction sites. The present invention does not particularly limit the specific steps for constructing the recombinant expression vector; conventional steps for constructing recombinant expression vectors in the art can be used.

[0041] The present invention also provides an engineered bacterium comprising a gene encoding the recombinant protein described in the above technical solution or a recombinant expression vector described in the above technical solution. The initial strain used to prepare the engineered bacterium in the present invention preferably comprises Escherichia coli, more preferably E. coli Rosetta. The present invention does not particularly limit the method for preparing the engineered bacterium; conventional methods for preparing engineered bacteria in the art may be employed.

[0042] The present invention has discovered that polar tubular proteins exhibit strong immunogenicity both under experimental and natural infection conditions, providing a potential target for the accurate diagnosis and effective prevention and treatment of microsporidiosis. The recombinant protein described herein can be used as an antigen for detecting microsporidia, exhibiting good antigenicity and accurately capturing specific antibodies in serum, thereby accurately detecting microsporidian infection. Furthermore, the recombinant protein is expressed using a prokaryotic expression system using a sequence-optimized gene sequence. This sequence optimization results in a higher protein yield, simple production steps, and low cost.

[0043] Based on the above advantages, the present invention also provides the use of the recombinant protein, gene, recombinant protein produced by the preparation method, recombinant expression vector, or engineered bacteria described in the above technical solutions in the preparation of reagents for detecting microsporidia and / or microsporidian infections, more preferably in the preparation of reagents for detecting microsporidia and microsporidian infections. The reagents of the present invention preferably include immunological reagents, and more preferably include but are not limited to reagents for Western blot detection. The recombinant protein of the present invention is preferably used as an antigen. The microsporidia of the present invention preferably include but are not limited to Enterosporida bieneusi.

[0044] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1

[0046] The preparation method of the recombinant protein of microsporidia polar tube protein EbPTP2 comprises the following steps:

[0047] 1. Preparation of pET-32a(+)-EbPTP2 recombinant plasmid

[0048] The microsporidian polar tube protein EbPTP2 gene sequence (SEQ ID NO. 2) was synthesized by gene synthesis and ligated between the BamHI and HindIII restriction sites of the pET-32a(+) prokaryotic expression vector.

[0049] 2. Inducible expression of pET-32a(+)-EbPTP2 recombinant plasmid

[0050] The monoclonal clone transformed into E. coli Rosetta competent cells was inoculated into 5 mL of LB medium containing ampicillin and cultured at 37°C with shaking. 600When the concentration of IPTG was about 0.6), IPTG with a final concentration of 0.1mM / L, 0.3mM / L, and 0.5mM / L was added for induction, and an uninduced bacterial solution was set as a control. The bacterial solution after 4 hours of induction was centrifuged at 12000rpm for 5min, and the bacterial precipitates were collected and resuspended with Buffer A (100mM NaCl, 10mM Tris-HCl, pH=8.0) solution. After ultrasonic disruption, the solution was centrifuged at 4°C and 12000rpm for 10min, and the supernatant was collected. The precipitate was resuspended with Buffer C (100mMNaCl, 10mMTris-HCl, 8M Urea, pH=8.0) solution. After ultrasonic disruption, the solution was centrifuged at 4°C and 12000rpm for 10min to obtain the total protein supernatant after induction with different concentrations of IPTG, and the precipitate after centrifugation of the bacterial solution after induction with different concentrations of IPTG. Finally, the solubility of the target protein was analyzed by SDS-PAGE electrophoresis. The results are shown in FIG. Figure 1 As shown, in Figure 1 In the figure, lane M is a protein molecular weight standard; lane 1 is the total protein supernatant of pET-32a(+)-EbPTP2 without induction; lanes 2-4 are the total protein supernatants of pET-32a(+)-EbPTP2 after induction with 0.1 mM / L, 0.3 mM / L, and 0.5 mM / L IPTG; lanes 5-8 are the precipitates after centrifugation of the supernatants corresponding to lanes 1-4.

[0051] Depend on Figure 1 It can be concluded that the EbPTP2 recombinant protein is expressed in a soluble form in E. coli Rosetta, and the expression levels of the recombinant protein are basically the same under the induction of 0.1mM / L, 0.3mM / L, and 0.5mM / L IPTG. Finally, it was decided to use 0.1mM / L IPTG for subsequent studies.

[0052] 3. Inducible expression and purification of recombinant protein EbPTP2

[0053] The preserved bacterial suspension was inoculated into 300 mL of LB medium containing ampicillin and cultured in a shaking incubator at 37°C and 180 rpm. 600 When the concentration (pH 5.0) was 0.6, 300 μL of 100 mM IPTG was added and induced at 37°C for 4 h. The induced bacterial suspension was collected and the pellet was resuspended in Buffer A (100 mM NaCl, 10 mM Tris-HCl, pH 8.0). Ultrasonic disruption was performed on ice for 30 min. After disruption, the pellet was centrifuged at 12,000 rpm at 4°C for 30 min. The protein supernatant was transferred to a 50 mL centrifuge tube.

[0054] Mix 50% Ni-NTA and draw 1mL from it to load the column. Add 20mL of sterile water for rinsing, and then add 20mL of Buffer A (100mM NaCl, 10mM Tris-HCl, pH=8.0) for equilibrium. Then combine the protein supernatant with the nickel column and control the flow rate to 1 drop per 3 seconds. First use 20mM and 50mM imidazole to remove impurities, and then elute the target protein with 200mM imidazole. All the liquids added to the column need to be filtered through a 0.22μm filter membrane. The results are as follows Figure 2 The SDS-PAGE analysis results of the recombinant protein EbPTP2-His purified in Figure A are shown, wherein lane 1: supernatant after ultrasonic disruption; lane 2: flow-through after sample loading; lane 3: 20 mM imidazole eluate; lane 4: 50 mM imidazole eluate; lane 5: 100 mM imidazole eluate; lanes 6-9: 200 mM imidazole eluate.

[0055] Depend on Figure 2 From Figure A, it can be concluded that after purification, the recombinant target protein with high purity was obtained at 55 kDa.

[0056] The gene sequence of EbPTP2 was synthesized according to the sequence disclosed in XM_001827820.1, and the expression and purification of the unoptimized EbPTP2 protein were performed according to the steps 1 to 3 in Example 1. The results are as follows: Figure 3 As shown, lane 1: supernatant after ultrasonic disruption; lane 2: flow-through after sample addition; lane 3: 20 mM imidazole eluate; lane 4: 50 mM imidazole eluate; lane 5: 100 mM imidazole eluate; lanes 6-9: 200 mM imidazole eluate.

[0057] Depend on Figure 3 It can be concluded that the expression level of EbPTP2 protein is significantly increased after optimization in the present invention.

[0058] 4. Detection of EbPTP2 recombinant protein with His antibody

[0059] (1) Electrophoresis: Mix 40 μL of the purified protein from step 3 with 10 μL of 5× SDS-PAGE loading buffer, boil for 10 min, and perform SDS-PAGE on 10 μL of the mixture.

[0060] (2) Transfer: Place filter paper soaked in transfer buffer on a semi-dry transfer apparatus, followed by the PVDF membrane and PAGE gel, and finally the filter paper soaked in transfer buffer. The transfer apparatus parameters are set to 25 V voltage, 1.5 A current, and 15 min transfer time.

[0061] (3) Blocking: After electroporation, place the PVDF membrane in 20 mL of 5% skim milk powder blocking solution and block at 37°C for 2 h.

[0062] (4) Mouse anti-His antibody (His mouse monoclonal antibody, purchased from abcam) was diluted with primary antibody diluent at a working concentration of 1:1000 (v / v). The antibody was incubated with the PVDF membrane at room temperature for 2 h or at 4°C overnight, and then cleared with 1× TBST three times for 10 min each time.

[0063] (5) Goat anti-mouse IgG-HRP (goat anti-mouse IgG HRP, purchased from BioSharp) was diluted with PBS at 1:8000 (v / v), and then the secondary antibody was incubated with the PVDF membrane at room temperature for 40 min, and then washed three times with 1× TBST, each time for 10 min;

[0064] (6) After the PVDF membrane reacts with the ECL chemiluminescent substrate in the dark, the color development result of the PVDF membrane is observed using an imager.

[0065] The results are as follows Figure 2 As shown in the figure B, the His antibody detected the expressed recombinant protein. The His antibody reacted with the recombinant target protein at 55 kDa, which was consistent with the expected result.

[0066] Example 2

[0067] Western blot analysis of polyclonal antibodies against the polar tube protein EbPTP2 of microsporidia

[0068] (1) Electrophoresis: The EbPTP2 recombinant protein was subjected to SDS-PAGE;

[0069] (2) Transfer: Place filter paper soaked in transfer buffer on a semi-dry transfer apparatus, followed by the PVDF membrane and PAGE gel, and finally the filter paper soaked in transfer buffer. The transfer apparatus parameters are set to 25 V voltage, 1.5 A current, and 15 min transfer time.

[0070] (3) Blocking: After electroporation, place the PVDF membrane in 20 mL of 5% skim milk powder blocking solution and block at 37°C for 2 h.

[0071] (4) EbPTP2 mouse polyclonal antibody prepared by diluting primary antibody diluent at a working concentration of 1:1000 (v / v) was incubated with the PVDF membrane at room temperature for 2 h or at 4°C overnight, and then cleared with 1× TBST three times for 10 min each time;

[0072] The preparation process of EbPTP2 polyclonal antibodies involves immunizing KM mice with EbPTP2 recombinant protein (the recombinant protein shown in SEQ ID NO.1) as an antigen. For the first immunization, the EbPTP2 recombinant protein is mixed with a complete adjuvant in a 1:1 ratio and injected subcutaneously at multiple sites, with each mouse receiving 100 μg of protein. After the first immunization, three more immunizations are required. For the final three immunizations, the EbPTP2 recombinant protein is mixed with an incomplete adjuvant in a 1:1 ratio and injected subcutaneously at multiple sites, with each mouse receiving 100 μg of protein. Each immunization is repeated every seven days. Seven days after the fourth immunization, blood is collected from the eyeballs. After the blood is drawn, it is first placed at 37°C for one hour and then at 4°C overnight to allow the serum to precipitate naturally. The precipitated serum is the EbPTP2 polyclonal antibody and is stored in a -20°C refrigerator.

[0073] (5) Dilute goat anti-mouse IgG-HRP with PBS at 1:8000 (v / v), then incubate the secondary antibody with the PVDF membrane at room temperature for 40 min, and then wash three times with 1× TBST for 10 min each time;

[0074] (6) After the PVDF membrane reacts with the ECL chemiluminescent substrate in the dark, the color development of the PVDF membrane is observed using an imager. The results are as follows: Figure 4 As shown, A is the Western blot analysis result of EbPTP2 polyclonal antibody and EbPTP2 recombinant protein; B is the Western blot analysis result of negative serum and EbPTP2 recombinant protein.

[0075] Depend on Figure 4 It can be concluded that the EbPTP2 recombinant protein can react with the EbPTP2 polyclonal antibody but not with negative serum (serum from non-immunized mice). A: Western blot analysis results of the EbPTP2 polyclonal antibody; B: Western blot analysis results of the negative serum.

[0076] Example 3

[0077] PCR amplification analysis was performed on genomic DNA from stool samples of E. bieneusi-infected patients (from patients infected with Enterobacteria bieneusi at Chongqing Fourth People's Hospital) using E. bieneusi-specific primers. The reaction system for PCR amplification analysis is shown in Table 1. The PCR reaction program was as follows: initial denaturation at 95°C for 3 min; 35 cycles of denaturation at 95°C for 15 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s; and final extension at 72°C for 7 min.

[0078] Specific primers: upstream primer: 5'-GAAACTTGTCCACTCCTTACG-3' (SEQ ID NO. 3), downstream primer: 5'-CCATGCACCACTCCTGCCATT-3' (SEQ ID NO. 4).

[0079] Table 1 PCR reaction system

[0080]

[0081]

[0082] Using the fecal genomic DNA extracted from the positive sample as a template, the fecal genome was amplified by PCR using E. bieneusi specific primers using the above PCR reaction system and reaction procedure. The results are as follows: Figure 5 The results of PCR test positive samples are shown in Figure A.

[0083] pass Figure 5 The target band size in A was 609 bp, which was consistent with the expected result, confirming that the patient was a positive case of microsporidia infection.

[0084] Example 4

[0085] The recombinant protein EbPTP2 is used to detect E. bieneusi infection in serum. The steps are as follows:

[0086] 1. Collection of serum to be tested: Collect serum from the patient who tested positive for microsporidia infection in Example 3.

[0087] 2. Western blot detection:

[0088] (1) Cut a PVDF membrane of appropriate size and activate it in methanol for 15 seconds, then wash it with ddH2O for 20 seconds and soak it in transfer buffer for later use;

[0089] (2) First, place the filter paper soaked in transfer buffer on the semi-dry transfer apparatus, then place the PVDF membrane and PAGE gel, and finally place the filter paper soaked in transfer buffer. The transfer apparatus parameters are set to voltage 25V, current 1.5A, and transfer time 15min.

[0090] (3) After electroporation, place the PVDF membrane in 20 mL of 5% skim milk powder blocking solution and block at 37°C for 2 h.

[0091] (4) Dilute the serum to be tested with primary antibody diluent to a working concentration of 1:200 (v / v). Incubate the antibody with the PVDF membrane at room temperature for 2 h or at 4°C overnight. Clear the membrane with 1×TBST three times for 10 min each time.

[0092] (5) Goat anti-human IgG-HRP was diluted with PBS at 1:1000 (v / v), and then the secondary antibody was incubated with the PVDF membrane at room temperature for 40 min, and then washed three times with 1× TBST, each time for 10 min;

[0093] (6) After the PVDF membrane reacts with the ECL chemiluminescent substrate in the dark, the color development result of the PVDF membrane is observed using an imager.

[0094] The results are as follows Figure 5 As shown in Figure B, the Western blot analysis results of the EbPTP2 recombinant protein on the positive sample serum show that the EbPTP2 recombinant protein can react with the positive sample serum, which further indicates that the recombinant protein described in this application has high antigenicity and high accuracy.

[0095] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A recombinant protein of microsporidia polar tube protein EbPTP2, characterized in that: The amino acid sequence of the recombinant protein is shown in SEQ ID NO.

1.

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

2.

3. The method for preparing the recombinant protein according to claim 1, characterized in that: include: The recombinant protein is expressed by using an in vitro protein expression system, and the in vitro protein expression system is a prokaryotic expression system.

4. The preparation method according to claim 3, characterized in that: The steps of expressing the recombinant protein using the prokaryotic expression system include: Connecting the gene encoding the recombinant protein to a prokaryotic expression vector to obtain a recombinant prokaryotic expression vector; Transforming the recombinant prokaryotic expression vector into prokaryotic cells to obtain recombinant engineered bacteria; The recombinant engineered bacteria are induced to express with IPTG, and the obtained induced bacteria contain the recombinant protein.

5. The preparation method according to claim 4, characterized in that: The prokaryotic expression vector includes a plasmid vector; the prokaryotic cell includes an Escherichia coli competent cell.

6. The preparation method according to claim 4, characterized in that: The preparation method further comprises purifying the recombinant protein in the induced bacteria to obtain the recombinant protein.

7. A recombinant expression vector, characterized in that: The recombinant expression vector comprises a gene encoding the recombinant protein in claim 1 and a starting vector.

8. An engineered bacterium, characterized in that: The engineered bacteria comprises a gene encoding the recombinant protein described in claim 1 or a recombinant expression vector described in claim 7; the engineered bacteria is Escherichia coli.

9. Use of the recombinant protein according to claim 1, the gene according to claim 2, the recombinant protein prepared by the preparation method according to any one of claims 3 to 6, the recombinant expression vector according to claim 7 or the engineered bacteria according to claim 8 in preparing a reagent for detecting microsporidia infection; the microsporidia is Enterosporida bieneusi.

10. The use according to claim 9, characterized in that: The recombinant protein is used as an antigen.

Images