CN116925198B - Recombinant protein of microsporidian polar tube protein EcPTP and preparation method and application thereof - Google Patents
Recombinant protein of microsporidian polar tube protein EcPTP and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a recombinant protein of microsporidian polar tube protein EcPTP and a preparation method and application thereof, belonging to the technical field of genetic engineering. The invention provides a recombinant protein of microsporidian polar tube protein EcPTP, and the amino acid sequence of the recombinant protein is shown as SEQ ID No. 1. The recombinant protein can be obtained by expression of an expression system. The invention utilizes a prokaryotic expression system to prepare the recombinant protein, has better antigenicity when the recombinant protein is used as a diagnostic antigen, can rapidly and efficiently capture target molecules in serum, achieves the purpose of rapidly and accurately detecting microsporidian infection, and lays a foundation for developing microsporidian diagnostic reagents and vaccines. Meanwhile, the method is also suitable for other microsporidia in the same genus, and has wide application range.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a recombinant protein of microsporidian polar tube protein EcPTP and a preparation method and application thereof.
Background
Microsporidia are a typical class of single-cell eukaryotes with obligate intracellular parasitism. Microsporidians are widely found in nature, with over 1200 species of microsporidians being classified in the phylum microsporidian. Microsporidian infects all groups of vertebrates and invertebrates and is an important animal husbandry, agriculture and human pathogen. It is currently accepted that four species of microsporidia of two genera are predominant among those capable of infecting humans. Wherein the genus encephalitis microsporidian (Encephalitozoon) is rabbit encephalitis microsporidian (E.cuniculi), sea encephalitis microsporidian (E.hellem) and enteroencephalitis microsporidian (E.intestinalis). The infection of animals with the encephalitis microsporidian is usually followed by the spread of the animal and accumulation of all organs. Animals with normal immunity are usually asymptomatic after infection with microsporidia encephalitis, but are mostly accompanied by multifocal granuloma and necrosis, and pathogens are detected in their kidneys, liver, spleen and liver. In contrast to a normal-immune host, an immune immature animal shows severe clinical symptoms after infection with encephalitis microsporidia, which is often fatal, and after infection, can exhibit meningitis, nephritis, and arteritis, and can detect pathogens in its brain, kidney, spleen, and liver. At present, no specific vaccine and medicine for treating the microsporidian disease exist, so that it is very important to diagnose the encephalitis microsporidian disease in time and control the infectious source of the encephalitis microsporidian disease.
Microsporidians have a unique infection mechanism and can transport microsporidian sporogenes into host cells through highly specialized polar tubes. The polar tube is connected with an anchor disk at the front end of the spore and spirals around the sporopouenin of microsporidian. When microsporidian is stimulated by a proper external environment, the polar tube can pop out quickly to form a hollow pipeline with the length of 50-500 mu m, and sporogenes are conveyed into host cells. The polar tube provides a bridge for the transportation of the sporogenes to the host cells, and simultaneously prevents the sporogenes from being damaged by the external extreme environment in the transportation process. It was found that polar tube proteins exhibit strong immunogenicity, both under experimental conditions and under natural infection conditions.
The detection method of microsporidian mainly comprises etiology detection, molecular biology detection and immunology detection. Immunological detection of microsporidia is a sensitive and specific detection method, and serological detection is generally an important method for diagnosing microsporidia disease in living animals or humans. At present, microsporidian collected from infected animals or cultured by tissues and cells is mainly used as an antigen for detecting serum-specific antibodies, but the antigen has low purity and complex components, is easy to cause cross reaction, and finally affects the detection effect.
Disclosure of Invention
The invention aims to provide a recombinant protein of microsporidian polar tube protein EcPTP and a preparation method and application thereof, which can accurately detect a large number of samples at the same time, adapt to the detection requirement of basic microsporidian and have good application prospect.
The invention provides a recombinant protein of microsporidian polar tube protein EcPTP, and the amino acid sequence of the recombinant protein is shown as SEQ ID No. 1.
The invention also provides a primer pair for amplifying the gene for encoding the recombinant protein, which comprises an upstream primer with a nucleotide sequence shown as SEQ ID No.2 and a downstream primer shown as SEQ ID No. 3.
The invention also provides a method for expressing the recombinant protein, which comprises the following steps: connecting the gene for encoding the recombinant protein to an expression vector, and constructing to obtain a recombinant expression vector;
Transforming an expression host by using the recombinant expression vector, and constructing to obtain a recombinant expression host;
Inducing the recombinant expression host to express the recombinant protein.
The invention also provides a method for preparing the recombinant protein by using the prokaryotic expression vector, which comprises the following steps: amplifying the primer pair to obtain a gene for encoding the recombinant protein, and connecting the gene to a prokaryotic expression vector to construct a recombinant expression vector;
Transforming prokaryotic cells by using the recombinant expression vector to obtain recombinant engineering bacteria;
And inducing the recombinant engineering bacteria to express by using IPTG.
Preferably, the prokaryotic expression vector comprises pET-32a (+).
Preferably, the prokaryotic cells comprise e.coli Rosetta competent cells.
Preferably, after completion of the expression, purification is also included.
The invention also provides application of the recombinant protein or the recombinant protein expressed by the method or the recombinant protein prepared by the method in preparation of reagents for detecting microsporidian.
Preferably, when microsporidian is detected by immunological methods, the recombinant protein is used as an antigen.
The invention also provides a kit for detecting microsporidian, which comprises antigens and other immunological detection reagents;
The antigen is the recombinant protein or the recombinant protein expressed by the method or the recombinant protein prepared by the method.
The beneficial effects are that: the invention provides a recombinant protein of microsporidian polar tube protein EcPTP, and the amino acid sequence of the recombinant protein is shown as SEQ ID No. 1. The recombinant protein is obtained by intercepting the B cell epitope of a soluble expression part based on the prediction of the B cell epitope and expressing the B cell epitope by an expression system.
In the embodiment of the invention, the prokaryotic expression system is utilized to prepare the recombinant protein, so that the method has the advantages of high protein yield, simplicity in operation, low cost and the like. When the recombinant protein provided by the invention is used as a diagnostic antigen, the recombinant protein has better antigenicity, can rapidly and efficiently capture target molecules in serum, achieves the purpose of rapidly and accurately detecting microsporidian infection, and lays a foundation for developing microsporidian diagnostic reagents and vaccines. Meanwhile, the method is also suitable for other microsporidia in the same genus, and has wide application range.
Drawings
FIG. 1 is a diagram (A) showing the result of PCR amplification electrophoresis of EcPTP gene and a diagram (B) showing the result of PCR electrophoresis of pET-32a (+) -EcPTP1 recombinant plasmid; lane 1 in a: DL5000 DNAMARKER; lane 2: ecPTP1 gene fragment after PCR amplification; lane 1 in B is pET-32a (+) empty; lane 2 is pET-32a (+) -EcPTP1 recombinant expression plasmid;
FIG. 2 is a graph showing the results of pET-32a (+) -EcPTP1 induced expression of a recombinant protein; lane M is a protein molecular weight standard; lane 1 is pET-32a (+) -EcPTP1 non-induced total protein supernatant; lanes 2-4 are total protein supernatants of pET-32a (+) -EcPTP1 induced by different concentrations of IPTG; lanes 5-8 are the pellet from centrifugation of the supernatant corresponding to lanes 1-4;
FIG. 3 is a graph showing the results of purification of recombinant protein EcPTP 1; a: SDS-PAGE analysis of purified recombinant protein EcPTP-His, lane 1: ultrasonically crushing the supernatant; lane 2: flowing through liquid after sample addition; lanes 3-9: imidazole eluted target proteins at different concentrations; b: his antibody detection expressed recombinant protein result diagram;
FIG. 4 is a diagram of the result of Westernblot detection of E.cutuli serum infection by the recombinant protein; a: a Westernblot analysis result diagram of infection E.cuniculi serum; b: western blot analysis result graphs of injected 1 XPBS serum;
FIG. 5 is a graph showing the Western blot detection results of the recombinant proteins on E.hellem and E.intestinalis infected sera; a: western blot analysis result diagram of E.hellem serum infection; b: westernblot analysis result diagram of infection E.intestinalis serum; c: westernblot analysis results of 1 XPBS serum injection.
Detailed Description
The invention provides a recombinant protein of microsporidian polar tube protein EcPTP, the amino acid sequence of which is shown as SEQ ID No.1 ：ATALCSNAYGLTPGQQGMAQ QPSYVLIPSTPGTIANCASGSQDTYSPSPAAPTSPVTPGKTSENETSPSAPAEDVGTCKIAVLKHCDAPGTTSGTTPGSGPCETPEQQQPLSVISTTPAVPVTVESAQSPSVVPVVPVVAHHQAVPGYYNNGTSGIPGQQQILSGTLPPGATLCQGQAMPSTPGQQQVPSGTLPPGVTLCQGQATPSTPGQQQVLSGTLPPGVTLCQGQATPSTPGQQQVLSGTLLPGATLCQDQGMPGTSGVPGQQGQSSGQCCAPQIPNPVMPPSMNISGNGYPSSTAYSPNLGSLGSCVDIQKTGGTSCEQKPEKSATQYAMEACATPTPTVIIGNSEYLVGPGMYNAINSPCNTA.
The polar tube protein of the microsporidian shows stronger immunogenicity in vivo and in vitro, so that the B cell epitope of a soluble expression part is intercepted by carrying out B cell epitope analysis on the polar tube protein EcPTP of the microsporidian, and the recombinant protein is constructed.
The invention also provides a primer pair for amplifying the gene for encoding the recombinant protein, which comprises an upstream primer with a nucleotide sequence shown as SEQ ID No.2 and a downstream primer shown as SEQ ID No. 3.
The invention designs a primer according to a microsporidian polar tube protein EcPTP gene sequence (NM_ 001041403.1) in Genbank, which is used for amplifying a coding gene of EcPTP1, wherein the designed primer sequences are respectively as follows:
An upstream primer: 5'-ACAAGGCCATGGCTGATATCGCAACCGCACTGTGCAGCA A-3';
a downstream primer: 5'-TGGTGCTCGAGTGCGGCCGCAGCAGTGTTGCATGGAGA GT-3'.
The invention also provides a method for expressing the recombinant protein, which comprises the following steps: connecting the gene for encoding the recombinant protein to an expression vector, and constructing to obtain a recombinant expression vector;
Transforming an expression host by using the recombinant expression vector, and constructing to obtain a recombinant expression host;
Inducing the recombinant expression host to express the recombinant protein.
The specific expression system of the expression is not particularly limited, and prokaryotic expression systems, eukaryotic expression systems, even plant expression systems and other expression systems which are well known in the art can be utilized to meet the requirements, and prokaryotic expression is taken as an example in the embodiment.
The invention also provides a method for preparing the recombinant protein by using the prokaryotic expression vector, which comprises the following steps: amplifying the primer pair to obtain a gene for encoding the recombinant protein, and connecting the gene to a prokaryotic expression vector to construct a recombinant expression vector;
Transforming prokaryotic cells by using the recombinant expression vector to obtain recombinant engineering bacteria;
And inducing the recombinant engineering bacteria to express by using IPTG.
The prokaryotic expression vector preferably comprises pET-32a (+), and the target gene is inserted into BamHI and HindIII double enzyme cutting sites to complete the construction of the recombinant expression vector.
The recombinant expression vector is preferably transformed into E.coli Rosetta competent cells according to the invention, and the transformation method is carried out by using a conventional method in the art. The invention cultures the recombinant engineering bacteria which are positive in transformation and carries out IPTG induction expression, and collects thalli, wherein the thalli crushing liquid contains the recombinant protein. The invention preferably further comprises purification after obtaining the recombinant protein, and corresponding purification is carried out according to the type of the purification tag.
The invention also provides application of the recombinant protein or the recombinant protein expressed by the method or the recombinant protein prepared by the method in preparation of reagents for detecting microsporidian.
When the microsporidian is detected by an immunological method, the recombinant protein is preferably used as an antigen, and can specifically, quickly and efficiently capture target molecules in serum and accurately and quickly detect the infection of the microsporidian.
The invention also provides a kit for detecting microsporidian, which comprises antigens and other immunological detection reagents;
The antigen is the recombinant protein or the recombinant protein expressed by the method or the recombinant protein prepared by the method.
The immunological detection of the invention preferably comprises Westernblot or ELISA and the like.
In order to further illustrate the present invention, the following examples are provided to describe in detail a recombinant protein of Microsporum polar tube protein EcPTP, its preparation method and application, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Construction of pET-32a (+) -EcPTP1 prokaryotic expression vector
Primers were designed based on the gene sequence of microsporidian polar tube protein EcPTP in GenBank (NM-001041403.1). The primers used to amplify EcPTP gene sequences were: an upstream primer (SEQ ID No. 2) and a downstream primer (SEQ ID No. 3).
The extracted E.cuniculi genome is used as a template, and a EcPTP gene fragment is amplified by conventional PCR, and the result is shown as A in figure 1, and the size is 1107bp; bamHI and HindIII double enzyme digestion pET-32a (+) are unloaded, and the PCR product and double enzyme digestion product are recovered by gel and then carried on the target fragment 1:8, connecting EcPTP target fragments into a pET-32a (+) double-enzyme-digested vector to construct a pET-32a (+) -EcPTP1 recombinant expression plasmid, and sequencing and identifying the constructed recombinant expression plasmid, wherein the result is shown as B in figure 1.
PCR reaction System (50. Mu.L): 2X PRIMER STAR Max Premix 25. Mu.L, forward primer (10. Mu.M) 1. Mu.L, REVERSE PRIMER (10. Mu.M) 1. Mu.L, E.cuniculi genomic DNA 1. Mu.L and the balance ddH 2 O.
PCR reaction procedure: pre-denaturation at 98℃for 3min; denaturation at 98℃for 30s, annealing at 60℃for 15s, elongation at 72℃for 15s,35 cycles; finally, the extension is carried out for 7min at 72 ℃.
2. Construction of engineering bacteria
Gently mixing recombinant expression plasmid with correct sequence and premelted competent strain E.coli Rosetta, and incubating on ice for 30min; then heat-shocking at 42 ℃ for 90s, and then ice-bathing for 3min; then 500 mu L of LB liquid medium without ampicillin is added for resuscitating for 40min at 37 ℃; then the bacterial liquid is coated on an LB culture medium plate containing ampicillin, and is cultured for 12 hours at 37 ℃; and (3) picking up the monoclonal bacterial plaque, inoculating the monoclonal bacterial plaque into 500 mu L of liquid LB culture medium containing ampicillin, and culturing at 37 ℃ and 180rpm to obtain seed solution of the prokaryotic expression strain.
Example 2
EcPTP1 expression and purification of recombinant protein and detection of His antibody to express recombinant protein.
1. Inducible expression of pET-32a (+) -EcPTP1 recombinant plasmid
The monoclonal transformed into E.coli Rosetta competent cells was inoculated into 5mL of LB medium containing ampicillin, shake cultured at 37℃and induced by adding IPTG at final concentrations of 0.1mM, 0.3mM and 0.5mM, respectively, when grown to the logarithmic phase (OD 600 is about 0.6), and the uninduced bacterial liquid was set as a control. After 4h induction, the bacterial liquid was centrifuged at 12000rpm for 5min, bacterial pellet was collected, resuspended in BufferA (100mM NaCl,10mM Tris-HCl, ph=8.0) solution, sonicated, centrifuged at 12000rpm for 10min at 4 ℃, supernatant was collected, pellet was resuspended in Buffer C (100mM NaCl,10mM Tris-HCI,8m urea, ph=8.0) solution, sonicated, centrifuged at 12000rpm for 10min at 4 ℃, and finally analyzed for target protein solubility by SDS-PAGE electrophoresis.
As a result, as shown in FIG. 2, ecPTP1 recombinant proteins were expressed in soluble form in E.coli Rosetta, and the expression levels of the recombinant proteins were substantially uniform under the induction of 0.1mM, 0.3mM, and 0.5mM IPTG, and finally, it was confirmed that 0.1mM IPTG was used for the subsequent study.
2. Purification of recombinant protein EcPTP1
The preserved bacterial liquid was inoculated into 300mL of ampicillin-containing LB medium, cultured at 37℃in a shaking table at 180rpm, and when grown to a logarithmic phase (OD 600 was 0.6), 300. Mu.L of 100mM IPTG was added and induced at 37℃for 4 hours. The induced bacterial liquid was collected, bacterial pellet was resuspended in BufferA (100mM NaCl,10mM Tris-HCl, ph=8.0) solution and sonicated on ice for 30min. After disruption, the protein supernatant was transferred to a 50mL centrifuge tube by centrifugation at 12000rpm for 30min at 4 ℃. Mix well 50% Ni-NTA, draw 1mL from it and column. Rinse with 20mL of sterile water and equilibrate with 20mL BufferA (100mM NaCl,10mM Tris-HCl, ph=8.0). The protein supernatant was then combined with a nickel column and the flow rate was controlled to 1 drop for 3 seconds. The protein of interest was eluted with 20mM imidazole followed by 50mM imidazole. All the liquids added to the column described above need to be filtered through a 0.22 μm filter.
The result is shown as A in FIG. 3, and the recombinant target protein with higher purity is obtained at 70kDa after the expression product is purified.
3. His antibody detection EcPTP recombinant protein
(1) Electrophoresis: 40. Mu.L of purified protein was mixed with 10. Mu.L of 5 XSDS-PAGE loading buffer, and boiled for 10min, 10. Mu.L of which was subjected to SDS-PAGE.
(2) Transferring: and placing filter paper soaked by a transfer buffer solution on a semi-dry transfer membrane instrument, then placing PVDF membrane and PAGE protein glue, and finally placing filter paper soaked by the transfer buffer solution. The parameters of the film transfer instrument are set to be 25V, 1.5A current and 15min film transfer time;
(3) Closing: after the electric conversion is completed, placing the PVDF film into 20mL of 5% skimmed milk powder sealing liquid, and sealing for 2 hours at 37 ℃;
(4) Diluting the murine anti-His antibody with an anti-dilution at a working concentration of 1:1000 (v/v), incubating the antibody with PVDF membrane for 2h at room temperature or overnight at 4℃3 times with 1 XTBST for 10min each;
(5) Sheep anti-mouse IgG-HRP was diluted 1:8000 (v/v) with PBS, then secondary antibody was incubated with PVDF membrane for 40min at room temperature, and washed 3 times with 1 XTBST for 10min each;
(6) After the PVDF film reacts with the ECL chemiluminescent substrate in a dark place, the color development result of the PVDF film is observed by using an imager.
The results are shown in FIG. 3B, where the His antibody binds to the recombinant protein of interest at 70kDa, consistent with the expected results.
Example 3
An application of recombinant protein EcPTP1 in detecting e.cuniculi infection in serum, comprising the steps of:
1. Culture of cuniculi: e.cuniculi was cultured with rabbit kidney cell line (RK 13). RK13 is cultured in a T25 cell bottle, after the RK13 grows to be full of the bottle wall, the RK13 is infected according to the ratio of cells to spores of 1:20, the RK13 is cultured until spores are mature and released outside the cells, and culture supernatants are collected for spore purification.
2. Preparation of serum to be tested: each C57BL/6 mouse was intraperitoneally infected with E.cuniculi at a dose of 1X 10 7, respectively, and infected for two consecutive days, and the control mice were injected with 1 XPBS solution. On day 14 of infection, antisera were obtained and stored at-20 ℃.
3. Immunoblot (Westernblot) detection
(1) Placing a PVDF membrane with proper size in methanol for activation for 15s, then washing for 20s by ddH 2 O, and soaking in a membrane transfer buffer solution for later use;
(2) Firstly, putting filter paper soaked by a transfer buffer solution on a semi-dry transfer membrane instrument, then putting PVDF membrane and PAGE protein glue, and finally putting filter paper soaked by the transfer buffer solution. The parameters of the film transfer instrument are set to be 25V, 1.5A current and 15min film transfer time;
(3) After the electric conversion is completed, placing the PVDF film into 20mL of 5% skimmed milk powder sealing liquid, and sealing for 2 hours at 37 ℃;
(4) Diluting antiserum with primary anti-dilution according to the working concentration of antibody of 1:200 (v/v), incubating the antibody with PVDF membrane at room temperature for 2h or overnight at 4 ℃, and cleaning with 1 XTBST for 3 times each for 10min;
(5) Sheep anti-mouse IgG-HRP was diluted 1:8000 (v/v) with PBS, then secondary antibody was incubated with PVDF membrane for 40min at room temperature, and washed 3 times with 1 XTBST for 10min each;
(6) After the PVDF film reacts with the ECL chemiluminescent substrate in a dark place, the color development result of the PVDF film is observed by using an imager.
As a result, as shown in FIG. 4, the recombinant target protein reacted with serum of E.cuniculi-infected mice, but not with serum of 1 XPBS-injected mice. A: a Westernblot analysis result diagram of infection E.cuniculi serum; b: westernblot analysis results of 1 XPBS serum injection.
Example 4
Application of recombinant protein EcPTP1 in indirect ELISA detection method
Purified recombinant protein EcPTP (0.5. Mu.g/well) obtained in example 1 was thoroughly mixed with ELISA coating solution and then added to an ELISA plate at 100. Mu.L per well overnight at 4 ℃.
ELISA coating liquid in the plate is dried, PBST is shaken for 3 times and 5 min/time, then 200 mu LELISA of blocking buffer is added into each hole, the mixture is placed for 2 hours at 37 ℃, and PBST is used for shaking for 3 times after the blocking is completed.
Positive and negative sera were diluted gradient 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800 with ELISA blocking buffer or PBS, then 100. Mu.L per well, 3 replicates per well, placed at 37℃for 2h, and PBST shaken 3 times after incubation.
Sheep anti-mouse lgG-HRP was diluted with ELISA blocking buffer or PBS at a ratio of 1:5000, 100. Mu.L per well was added, left at 37℃for 1-2h, and after incubation, PBST was shaken for 3 times.
TMB color development was added at 200. Mu.L per well, left at 37℃for 30min in the dark, and then quenched by the addition of H2SO4 at a concentration of 2M at 50. Mu.L per well.
And (3) placing the ELISA plate in an ELISA instrument, setting 450nm absorption wavelength for detection, and determining that the absorbance value of the hole to be detected is 2.1 times greater than that of the negative hole under the same dilution multiple to be positive.
As shown in Table 1, even though the serum was diluted to 1:3200, the purified recombinant protein EcPTP was still able to react with positive serum but not negative serum, indicating that the purified recombinant protein EcPTP1 had good sensitivity.
TABLE 1 results of indirect ELISA detection of recombinant protein EcPTP1
Example 5
The application of recombinant protein EcPTP1 in detecting E.hellem, E.intestinalis infection in serum comprises the following steps:
1. Culture of hellem, e.intelstinalis: culturing E.hellem with a rabbit kidney cell line (RK 13); e.intestinalis was cultured with human foreskin fibroblast cell line (HFF). RK13 and HFF were cultured in T25 cell flasks and after they had grown to the flask wall, they were infected at a cell to spore ratio of 1:20, cultured until the spores were matured and released outside the cells, and the culture supernatants were collected for spore purification.
2. Preparation of serum to be tested: each C57BL/6 mouse was infected with E.hellem, E.intelestinalis by intraperitoneal injection at 1X 10 7 doses per mouse, and two days of continuous infection, and control mice were injected with 1X PBS solution. On day 14 of infection, antisera were obtained and stored at-20 ℃.
3. Immunoblot (Westernblot) detection
(1) Placing a PVDF membrane with proper size in methanol for activation for 15s, then washing for 20s by ddH 2 O, and soaking in a membrane transfer buffer solution for later use;
(2) Firstly, putting filter paper soaked by a transfer buffer solution on a semi-dry transfer membrane instrument, then putting PVDF membrane and PAGE protein glue, and finally putting filter paper soaked by the transfer buffer solution. The parameters of the film transfer instrument are set to be 25V, 1.5A current and 15min film transfer time;
(3) After the electric conversion is completed, placing the PVDF film into 20mL of 5% skimmed milk powder sealing liquid, and sealing for 2 hours at 37 ℃;
(4) Diluting antiserum with primary anti-dilution according to the working concentration of antibody of 1:200 (v/v), incubating the antibody with PVDF membrane at room temperature for 2h or overnight at 4 ℃, and washing with 1 XTBE 3 times for 10min each time;
(5) Sheep anti-mouse IgG-HRP was diluted 1:8000 (v/v) with PBS, then secondary antibody was incubated with PVDF membrane for 40min at room temperature, and washed 3 times with 1 XTBST for 10min each;
(6) After the PVDF film reacts with the ECL chemiluminescent substrate in a dark place, the color development result of the PVDF film is observed by using an imager.
As a result, as shown in FIG. 5, the recombinant target protein reacted with serum of E.hellem and E.intestinalis infected mice, but not with serum of control (1 XPBS injected) mice. A: westernblot analysis results of E.hellem mouse serum infection; b: western blot analysis results of serum of infected E.intestinalis mice; c: western blot analysis results of serum from mice injected with 1 XPBS.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. The recombinant protein of the microsporidian polar tube protein EcPTP is characterized in that the amino acid sequence of the recombinant protein is shown as SEQ ID No. 1.
2. A primer pair for amplifying a gene encoding the recombinant protein of claim 1, comprising an upstream primer having a nucleotide sequence shown in SEQ ID No.2 and a downstream primer having a nucleotide sequence shown in SEQ ID No. 3.
3. A method of expressing the recombinant protein of claim 1, comprising the steps of: connecting a gene for encoding the recombinant protein of claim 1 to an expression vector, and constructing a recombinant expression vector;
Transforming an expression host by using the recombinant expression vector, and constructing to obtain a recombinant expression host;
inducing the recombinant expression host to express the recombinant protein of claim 1.
4. A method for preparing the recombinant protein of claim 1 using a prokaryotic expression vector, comprising the steps of: amplifying the primer pair of claim 2 to obtain a gene encoding the recombinant protein of claim 1, connecting the gene to a prokaryotic expression vector, and constructing a recombinant expression vector;
Transforming prokaryotic cells by using the recombinant expression vector to obtain recombinant engineering bacteria;
And inducing the recombinant engineering bacteria to express by using IPTG.
5. The method of claim 4, wherein the prokaryotic expression vector comprises pET-32a (+).
6. The method of claim 4, wherein the prokaryotic cells comprise e.coli Rosetta competent cells.
7. The method of claim 4, further comprising purifying after said expressing is complete.
8. Use of the recombinant protein of claim 1 or expressed by the method of claim 3 or prepared by the method of any one of claims 4 to 7 in the preparation of a reagent for detecting microsporidian, wherein the microsporidian is a encephalitis microsporidian.
9. The use according to claim 8, wherein the recombinant protein is used as an antigen when detecting microsporidia by immunological methods.
10. A kit for detecting microsporidian comprising an antigen and other immunological detection reagents;
The antigen is the recombinant protein of claim 1 or the recombinant protein expressed by the method of claim 3 or the recombinant protein prepared by the method of any one of claims 4 to 7.
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| US6890536B2 (en) * | 1998-07-07 | 2005-05-10 | Centre National de la Recherche Scientifique—CNRS | Microsporidian polar tube proteins, nucleic acids coding for these proteins and their applications |
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| CN103740733A (en) * | 2014-01-12 | 2014-04-23 | 吉林大学 | Cryptosporidium parvum recombinant antigen for diagnosis |
| CN115109131A (en) * | 2022-06-28 | 2022-09-27 | 西南大学 | Application of polyclonal antibody in preparation of microsporidian identification reagent |
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| US6890536B2 (en) * | 1998-07-07 | 2005-05-10 | Centre National de la Recherche Scientifique—CNRS | Microsporidian polar tube proteins, nucleic acids coding for these proteins and their applications |
| CN101139607A (en) * | 2007-08-07 | 2008-03-12 | 西南大学 | Cultivated silkworm microsporidian polar tube protein PTP1 gene |
| CN103728458A (en) * | 2013-12-31 | 2014-04-16 | 中国农业科学院哈尔滨兽医研究所 | Applications of rabbit encephalitozoon cuniculi spore wall protein SWP1 to preparation of reagent for diagnosing or detecting rabbit encephalitozoon cuniculi infection |
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