Method for interaction of parasitic protein and protein in host cell

Abstract

The invention discloses a method for interaction of parasitic protein and protein in host cells, and belongs to the technical field of genetic engineering. The invention provides an isospora hyocystis ENO2 protein and pig intestine epithelial cell waveform protein interaction method, interaction conditions are optimized, the influence of the dosages of the two proteins on the interaction conditions is explored, conditions suitable for interaction of parasites and host cells are found more accurately, the success rate of experiments is increased, and the experiment cost is reduced. Therefore, a theoretical basis is provided for researching isospora hyocystis ENO2 and unknown proteins of the epithelial cells of the pig intestines, and the problem that interaction conditions of parasitic proteins and proteins in host cells are difficult to succeed at present is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly to a method for interacting a parasite protein with a protein in a host cell. Background Art

[0002] Currently, the methods for detecting protein interaction mainly include immunoprecipitation, yeast two-hybrid method, GST pull-down assay and other techniques. Each of the above detection methods has its own advantages and disadvantages, which are specifically as follows:

[0003] (1) Immunoprecipitation

[0004] Immunoprecipitation is a classical technique that uses antibodies to capture target proteins, their interacting proteins and complexes from samples (cells), and can specifically enrich the target protein of interest. Since non-denaturing conditions are used in the process, the intracellular state of the interaction and the complex is retained. Compared with methods such as yeast two-hybrid and GST pull-down, one of its characteristics is that the captured protein interaction occurs in the specific tissue cells under study, preserving the "endogenous" state of the interacting proteins and complexes. Therefore, it can reflect the interacting protein relationship of the protein in the natural tissue cell state; however, there are also some false positive problems. Therefore, specificity is very important in the selection of antibodies. It is best to select monoclonal antibodies with higher specificity. Specific antibodies can exclude non-specific binding and thus exclude false positives. At the same time, Co-IP reverse verification can be combined to further exclude false positives. If the antibody concentration is too high, the antibody concentration is reduced; if the antibody specificity is not good, the antibody must be replaced. In addition, it cannot be determined whether the interaction between the bait protein and the target protein is a direct interaction or an indirect interaction mediated by other proteins. In terms of experimental difficulty, because the solid-phase complex has many components, requires a non-denaturing environment, and has high requirements for the quality of the bait protein antibody, the whole experiment is relatively difficult.

[0005] (2) Yeast two-hybrid method

[0006] The yeast two-hybrid system is a system that clones (fuses) two proteins to be studied into the DNA-binding domain (DNA-BD) and activation domain (activation domain, DNA-AD) of a transcriptional activator (such as GAL4, etc.) of a yeast expression plasmid respectively to construct a fusion expression vector and analyzes the interaction between the two proteins from the expression products. The yeast two-hybrid system can measure the binding of proteins in vivo and has high sensitivity. This is mainly due to the following reasons: ① The expression vector with high copy number and strong promoter is used to overexpress the hybrid protein; ② The signal measurement is carried out under natural equilibrium concentration conditions, and the signal intensity will not be reduced due to steps such as multiple washing required in physical methods such as immunoprecipitation; ③ The stability between hybrid proteins can be enhanced by the binding of the activation domain and the binding domain to form a transcriptional initiation complex, and the latter binds to the promoter DNA. This ternary complex makes the binding of each component tend to be stable; ④ The signal is amplified by the production of multiple stable enzymes through mRNA. However, in the yeast two-hybrid method, the protein produced by overexpression of a single gene transfection interacts with the total protein of the host cell, and the band background is high, and interference and false positives are likely to occur, that is, some protein interactions may be misreported and need further verification. In addition, the yeast two-hybrid technique is carried out in yeast cells, and it cannot fully restore the situation of protein interaction in the real cell environment. Moreover, the technical threshold of gene cloning involved is high and time-consuming, and the colony screening process is also very time-consuming and laborious.

[0007] (3) GST pull-down assay

[0008] This experiment is an effective in vitro test technique for verifying the yeast two-hybrid system. The GST pull-down assay is generally used for in vitro experiments. Since the bait protein is a recombinant protein that needs to be tagged with GST, it is a verification under non-physiological conditions, and the structure and form of the protein may be somewhat different from the natural state. It is generally used to verify the direct interaction between two known proteins in vitro; the fused GST tag has a relatively long peptide chain and may also change the original folding structure of the original target protein. In terms of experimental difficulty, the recombinant bait protein generally uses a prokaryotic expression system, which has highly efficient commercial vectors and fewer solid-phase complex components, and there are commercial GSH agarose beads, so the whole experiment is relatively less difficult. The general operation method is to affinity-solidify the target protein-GST fusion protein on glutathione affinity resin as a support for binding to the target protein, acting as a "bait protein". The target protein solution is passed through the column, and the "capture protein" (target protein) that interacts with it can be captured from it. After eluting the binding substance, SDS-PAGE electrophoresis analysis is carried out to confirm the interaction between the two proteins or screen the corresponding target protein.

[0009] Therefore, in view of the problems existing in the current methods for detecting protein-protein interactions, it is very necessary to provide a method for efficiently detecting protein-protein interactions. Summary of the Invention

[0010] The object of the present invention is to provide a method for the interaction between a parasite protein and a protein in a host cell, so as to solve the problems existing in the above-mentioned prior art. By optimizing the interaction conditions, the conditions suitable for the interaction between the parasite and the host cell can be accurately found, the success rate of the experiment can be improved, and thus a theoretical basis can be provided for studying the interaction between Isospora suis ENO2 and unknown proteins in porcine intestinal epithelial cells.

[0011] To achieve the above object, the present invention provides the following solutions:

[0012] The present invention provides a method for the interaction between a parasite protein and a protein in a host cell, comprising the following steps:

[0013] After connecting the parasite protein coding gene sequence with a first expression vector to construct a recombinant expression vector, it is transferred into Escherichia coli for induced expression, the cells are lysed, and then purified by a chromatography column to prepare a first fusion protein with a TrxA-His tag;

[0014] After connecting the host cell protein coding gene sequence with a second expression vector to construct a recombinant expression vector, it is transferred into Escherichia coli for induced expression, the cells are lysed, and purified by a chromatography column to prepare a second fusion protein with a GST tag;

[0015] The second fusion protein with a GST tag and the first fusion protein with a TrxA-His tag are interacted through a GST pull-down experiment, and then the interaction relationship is determined according to the binding strength of the two proteins;

[0016] Wherein, the parasite protein is a key metalloenzyme in the glycolysis process isolated from Isospora suis, and the parasite protein coding gene sequence is as shown in SEQ ID NO: 1;

[0017] The host cell protein is vimentin isolated from porcine intestinal epithelial cells, and the host cell protein coding gene sequence is as shown in SEQ ID NO: 2.

[0018] Optionally, the first expression vector includes pET32(a), and the second expression vector includes pGEX-4T-1.

[0019] Optionally, in the interaction system, as the dosage of the first fusion protein with a GST tag increases, its binding strength with the second fusion protein with a TrxA-His tag becomes stronger.

[0020] Optionally, in the interaction system, the mass ratio of the first fusion protein with a GST tag to the second fusion protein with a TrxA-His tag is (2-3):2.

[0021] Optionally, the interaction relationship includes unbinding, moderate binding, and strong binding.

[0022] The present invention also provides an isolated parasite protein, which is a key metalloenzyme in the glycolysis process isolated from Isospora suis, and the coding gene sequence of the parasite protein is shown as SEQ ID NO: 1.

[0023] The present invention also provides a recombinant vector, which contains the coding gene of the parasite protein.

[0024] The present invention also provides a recombinant bacterium, in which the genome is integrated with the expression of the recombinant vector.

[0025] The present invention also provides the application of the parasite protein, the recombinant vector, or the recombinant bacterium in the preparation of a detection reagent for the interaction mechanism between Isospora suis and porcine intestinal epithelial cells.

[0026] Optionally, the interaction mechanism between Isospora suis and porcine intestinal epithelial cells is explored by the interaction between the parasite protein and vimentin in porcine intestinal epithelial cells. The amino acid sequence of vimentin is shown as SEQ ID NO: 4, and the coding gene sequence is shown as SEQ ID NO: 2.

[0027] The present invention discloses the following technical effects:

[0028] The present invention provides a GST pull-down assay with a relatively clean background and a single protein band. The proteins are purified separately before interaction, which is different from the traditional GST pull-down assay where the GST fusion protein interacts with the total protein of the entire intracellular system, resulting in defects such as one-to-many, relatively messy bands, and easy occurrence of false positives. In addition, the present invention can reflect different interaction levels according to the protein dosage, and more accurately achieve the purpose of successful interaction.

[0029] More specifically, the present invention provides a method for the interaction between Isospora suis ENO2 protein and porcine intestinal epithelial cell (IPEC-J2) vimentin, optimizes the interaction conditions, explores the influence of the dosage of the two proteins on the interaction conditions, more accurately finds the conditions suitable for the interaction between the parasite and the host cell, improves the success rate of the experiment, and further provides a theoretical basis for studying the unknown proteins of Isospora suis ENO2 and porcine intestinal epithelial cells, solving the problem that it is difficult to succeed in the interaction conditions between parasite proteins and host cell proteins at present. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0031] Figure 1 It is the SDS-PAGE diagram after immunoprecipitation with the flag monoclonal antibody; M is the standard protein molecule, and 1-3 are the samples transfected with pcDNA3.1-flag into cells respectively; 4-6 are the samples transfected with pcDNA3.1-flag-eno2 into cells respectively;

[0032] Figure 2 It is the electrophoresis diagram for detecting the target gene by agarose gel electrophoresis; (A) is the PCR identification of the pET32(a)-eno2 recombinant plasmid, and (B) is the PCR identification of the pGEX-4T-1-vimentin recombinant plasmid;

[0033] Figure 3 It is the SDS-PAGE electrophoresis diagram after purification of the recombinant protein; (A) is the TrxA-His-ENO2 fusion protein, (B) is the GST protein, and (C) is the GST-Vimentin fusion protein;

[0034] Figure 4 It is the verification of the GST pull-down assay with GST or GST-Vimentin (10 μg, 20 μg, 30 μg) and TrxA-His-ENO2; (A) 10 μg, (B) 20 μg, (C) 30 μg. DETAILED DESCRIPTION OF THE INVENTION

[0035] Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0036] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0037] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to those documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0038] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the description of this invention, which will be obvious to those skilled in the art. Other embodiments obtained from the description of this invention will be obvious to those skilled in the art. The description of this invention and the examples are merely exemplary.

[0039] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0040] Example 1

[0041] 1. Cloning of pcDNA3.1-flag-eno2 gene and construction of overexpression vector

[0042] 1.1 Experimental materials

[0043] pcDNA3.1-flag was prepared or stored in the laboratory. Competent cell DH5α was purchased from Takara Biotechnology (Beijing) Co., Ltd. The transfection reagent was purchased from Thermo Fisher scientific. Nhe1 and Xba1 restriction enzymes were purchased from Shanghai Sangon Biotech Co., Ltd.

[0044] 1.2 Experimental methods

[0045] 1.2.1 Cloning of eno2 gene

[0046] (1) PCR amplification

[0047] Using the cDNA after reverse transcription extracted from Isospora suis as a template, and using the primers of eno2 gene (eno2-F: 5’-GCT AGC ATGGAAATCTTCTTCTTCTTTGC-3’ (Nhe1) (SEQ ID NO: 5); eno2-R: 5’- TCTAGAPCR amplification was performed using 5'-TTATTTCGGATTACGAAAATCCG-3’ (Xba1) (SEQ ID NO: 6) as the primer. The PCR reaction system was set with a total volume of 50 μL: 2 μL of template was added, 1 μL of each forward and reverse primer, 0.5 μL of Taq enzyme, 4 μL of dNTP, 5 μL of 10× Buffer, and ddH2O was added to make up 50 μL. The eno2 gene fragment was amplified by conventional PCR. The reaction procedure was: pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 sec, annealing at 52 °C for 30 sec, extension at 72 °C for 90 sec, for 30 cycles; finally, extension at 72 °C for 10 min.

[0048] (2) Restriction enzyme digestion, ligation and transformation

[0049] The PCR products of pcDNA3.1-flag and eno2 gene were double digested with two restriction enzymes, Nhe1 and Xba1. After gel extraction and recovery, ligation and transformation were carried out. After obtaining colonies, plasmids were extracted and verified by both PCR and sequencing.

[0050] 2. Overexpression vector large-scale expression, extraction of pcDNA3.1-flag-eno2 and transfection of IPEC-J2 cells

[0051] 2.1 Experimental materials

[0052] The QIAGEN plasmid kit was purchased from UNIV-BIO. IPEC-J2 cells were purchased from Qingqi Biotechnology Development Co., Ltd.

[0053] 2.2 Experimental methods

[0054] According to the QIAGEN plasmid kit instruction manual, pcDNA3.1-flag and pcDNA3.1-flag-eno2 plasmids were extracted in large quantities, and then cell transfection was carried out. The specific transfection steps are as follows:

[0055] (1) Inoculate 5×10 6 IPEC-J2 cells into 10 10-cm cell culture dishes containing DMEM culture medium;

[0056] (2) Add 10 μg of pcDNA3.1-flag and pcDNA3.1-flag-eno2 plasmids into 1 mL of serum-free DMEM respectively, and vortex gently several times;

[0057] (3) Add 30 μL of Turbofect Transfection Reagent into the serum-free DMEM containing plasmid DNA in step (2), and vortex again;

[0058] (4) Let it stand at room temperature for 20 minutes;

[0059] (5) Drop the above-mentioned liquid clockwise into the cell culture dish;

[0060] (6) After 24 hours, collect the cells and perform cell lysis.

[0061] 3. Immunoprecipitation with cell lysate

[0062] 3.1 Experimental materials

[0063] Immunoprecipitation kit (Catchand v2.0 Reversible Immunoprecipitation System) was purchased from Merck. Flag monoclonal antibody was purchased from Cell Signaling Technology.

[0064] 3.2 Experimental methods

[0065] (1) Add 1 mL of cell lysate to every 100 mg of cell precipitate, vortex, and then break the cells with an ultrasonic crusher;

[0066] (2) Centrifuge at 12,000 rpm for 1 min and collect the supernatant;

[0067] (3) Add 0.7 mL of cell lysate, 7 μL of PMSF, and 1 μg of Flag antibody to a new 1.5 mL microcentrifuge tube;

[0068] (4) Incubate overnight on a horizontal shaker at 4°C;

[0069] (5) Add 30 μL of protein A / G beads to the filter column, wash with 700 μL of 1×PBS, centrifuge at 4,000 rpm, and wash 4 times.

[0070] (6) Add 700 μL of 1×IP buffer to the filter column and centrifuge at 12,000 rpm to wash the beads 6 times; wash the beads 6 times with 700 μL of 0.1×IP buffer, and centrifuge the filter column at 12,000 rpm for 30 s;

[0071] (7) Add 50 μL of 1×loading buffer to the protein A beads and stir gently (without vortexing);

[0072] (8) Insert each filter column into a 2 mL microcentrifuge tube;

[0073] (9) Centrifuge at 12,000 rpm for 30 s;

[0074] (10) Collect the samples and heat the samples at 95 °C for 5 minutes for protein denaturation.

[0075] 4. SDS-PAGE Analysis and Identification by Liquid Chromatography-Mass Spectrometry

[0076] (1) Perform 12% SDS-PAGE analysis on 15 μL of the cell lysate supernatant after transfection with pcDNA3.1-flag and pcDNA3.1-flag-eno2;

[0077] (2) Cut the differential protein bands, wash them three times with 50% acetone, and then wash them three times with deionized water for liquid chromatography-mass spectrometry analysis.

[0078] The results of SDS-PAGE analysis are as Figure 1 shown. The results show that transfection with pcDNA3.1-flag-eno2 resulted in one more protein band (at the position of 55 - 70 kDa) than pcDNA3.1-flag. This indicates that the pcDNA3.1-flag-eno2 overexpression vector was successfully constructed and expressed successfully.

[0079] 5. Cloning, Expression and Purification of the Differential Protein Vimentin Coding Gene

[0080] 5.1 Experimental Materials

[0081] Trizol reagent was purchased from Invitrogen; HiScript III 1st Strand cDNA Synthesis Kit was purchased from Nanjing Vazyme Biotech Co., Ltd. (Vazyme); Restriction enzymes were purchased from Shanghai Sangon Biotech Co., Ltd.

[0082] 5.2 Experimental Methods

[0083] 5.2.1 Cloning of the vimentin Gene

[0084] 5.2.1.1 Total RNA Extraction

[0085] (1) Take 1 petri dish of IPEC-J2 cell samples cultured to 70 - 90% confluence, digest them with trypsin, wash them twice with 1×PBS, and place them in a sterile 1.5 mL microcentrifuge tube without RNase;

[0086] (2) Add 1000 μL of Trizol, mix well, and let stand at room temperature for 5 min;

[0087] (3) Add 200 μL of chloroform, mix well, let stand at room temperature for 5 min, and centrifuge at 12000 rpm at 4 °C for 15 min;

[0088] (4) After centrifugation, transfer the supernatant to a new 1.5 mL microcentrifuge tube, add an equal volume of isopropanol, mix well, and precipitate overnight at -80 °C;

[0089] (5) Centrifuge at 4 °C and 12,000 rpm for 30 min;

[0090] (6) Discard the supernatant, add 600 μL of 70% ethanol (prepared with DEPC water), centrifuge at 4 °C and 12,000 rpm for 15 min, and discard the supernatant;

[0091] (7) Repeat step (6);

[0092] (8) Aspirate the residual liquid with a 20 μL pipette tip;

[0093] (9) Air dry in a laminar flow hood for 10 minutes;

[0094] (10) Add 50 μL of RNase-free ddH2O to dissolve the precipitate;

[0095] (11) Run a gel to identify the quality of the RNA;

[0096] (12) Store the samples in a -80 °C refrigerator for later use.

[0097] 5.2.1.2 cDNA Synthesis

[0098] Use the HiScript III 1st Strand cDNA Synthesis Kit to reverse transcribe the extracted cellular RNA, with a total RNA of 5 μg, to obtain cDNA by reverse transcription.

[0099] 5.2.1.3 PCR Amplification

[0100] Using the synthesized cDNA as a template and vimentin-F / R as primers:

[0101] vimentin-F: 5’-AAG GAATTC ATGTCCACCAGGACCGTGTCC-3’ (EcoR1) (SEQ ID NO: 7);

[0102] vimentin-R: 5’-T GCGGCCGC TCATTGATAACCCCTCAGGTTC-3’ (Not 1) (SEQ ID NO: 8);

[0103] The target fragment expected to be amplified is 1284 bp. The total volume of the amplification reaction is 50 μL (the same reaction system as the eno2 amplification above). The PCR reaction conditions are as follows: pre-denaturation at 95 °C for 5 min in the first round; denaturation at 95 °C for 1 min, annealing at 52 °C for 1 min, extension at 72 °C for 2 min, for 30 cycles; finally, extension at 72 °C for 10 min. The obtained product was subjected to electrophoresis on 1% agarose gel, and the experimental results were observed and recorded on an ultraviolet transilluminator.

[0104] 5.2.1.4 Gene cloning

[0105] The PCR products of pGEX-4T-1 and vimentin gene were digested with EcoR1 and Xho1 simultaneously. After gel cutting and recovery, ligation and transformation were carried out. After obtaining colonies, plasmids were extracted and verified by both PCR and sequencing.

[0106] The detection result of agar gel electrophoresis is as Figure 2 shown in (B) below. The pGEX-4T-1-vimentin recombinant plasmid can successfully amplify the target gene band.

[0107] 5.2.2 High-level expression and purification of GST-Vimentin fusion protein

[0108] Ampicillin (50 μg / mL) was added to LB liquid medium at a ratio of 1:1000, and the activated pGEX-4T-1-vimentin / BL21 bacterial solution was added at a ratio of 1:50. The culture was carried out on a shaker at 37 °C and 200 rpm. When the absorbance value (OD 600nm ) reached between 0.6 - 0.8, IPTG with a working concentration of 0.1 mmol / L was added, and the induction was carried out on a shaker at 37 °C and 200 rpm for 3 h. The bacterial solution was aliquoted into 50 mL centrifuge tubes and centrifuged at 4000 rpm for 30 min in a high-speed centrifuge at 4 °C. After removing the supernatant, the precipitated bacteria were placed in a -80 °C refrigerator for later use.

[0109] Take the bacterial cells out of the -80 °C refrigerator. Resuspend every 500 mL of bacterial cells by adding 30 mL of GST lysis buffer (50 mM Tris-HCl pH 8.0, 1 mM PMSF, 10 mM DTT, 150 mM NaCl, 0.1% sodium lauroyl-sarcosine, 1% Triton X-100). Lyse the bacteria by sonication under ice bath conditions (200 W, sonication for 10 s, interval for 10 s, working for 20 min). After disruption, aliquot the bacterial lysate into 1.5 mL microcentrifuge tubes and centrifuge at 4 °C, 12,000 rpm for 30 min to collect the protein supernatant. Filter the protein solution through a 0.45 μm filter. Take 3 mL of Beyogold GST-tag purification resin into the chromatography column and wash it twice with 10 mL of ddH2O. Add 10 mL of GST lysis buffer to balance the chromatography column twice.

[0110] Pass the protein solution through the chromatography column and collect the eluted protein solution, and store it temporarily at -20 °C. Wash the chromatography column 10 times with 1×PBS, 10 mL each time. Collect 1 mL of the first three washing solutions into microcentrifuge tubes and store them temporarily at 4 °C. Elute the GST fusion protein with 10 mL of elution buffer (50 mM Tris-HCl pH 8.0, 10 mM GSH) and let the elution buffer act at 4 °C for 10 min. Collect the eluted protein solution into a 1.5 mL microcentrifuge tube and store it temporarily at -20 °C. Take 20 μL of each protein sample and add 5 μL of 5× protein loading buffer, and denature it at 99 °C for 10 min in a metal bath. Detect the purification of the GST fusion protein by SDS-PAGE method.

[0111] The results of SDS-PAGE detection of GST fusion protein and GST-Vimentin fusion protein are as Figure 3 shown in (B) and (C) below. The homozygous fusion protein has a single band at the expected position.

[0112] 5.2.3 Cloning of eno2 gene

[0113] Clone the eno2 gene and the pET32(a) empty vector according to the experimental procedure in 5.2.1.

[0114] The results of agarose gel electrophoresis detection are as Figure 2 shown in (A) below. The pET32(a)-eno2 recombinant plasmid can successfully amplify the target band.

[0115] 5.2.3.1 High-level expression and purification of TrxA-His-ENO2 fusion protein

[0116] The prepared bacteria pET32(a)-eno2 / BL21 were added to LB medium at a ratio of 1:50 and cultured at 37 °C and 200 rpm until the OD of the bacterial solution 600nm reached between 0.6 and 0.8. Then, induction expression was carried out at 25 °C, 200 rpm, and 0.1 mM IPTG for 8 h. The bacterial solution was centrifuged at 12000 rpm for 30 min. 2 L of the induced bacteria were collected and temporarily stored in a -80 °C refrigerator.

[0117] The 2 L of the induced bacteria temporarily stored in a -80 °C refrigerator were taken out. For every 500 mL of the bacteria, 30 mL of lysis buffer (20 mM Tris-HCl pH = 8.0, 1 mM PMSF (phenylmethylsulfonyl fluoride), 300 mM NaCl, 10% glycerol, 5 mM imidazole, 1% Triton-X-100) was added to resuspend, break, centrifuge, and filter the bacteria. The steps were the same as the above purification steps. 2 mL of His-Tag resin was taken into the chromatography column. 10 mL of ddH2O was added to wash the column twice, and 10 mL of lysis buffer was added to equilibrate the column twice. The protein solution was slowly passed through the column, and the flow-through solution was collected. The protein solution after binding was passed through the column again and collected, named "protein solution after binding", and temporarily stored at -20 °C. 12 mL of washing buffer (20 mM Tris-HCl pH = 8.0, 1 mM PMSF, 300 mM NaCl, 20 mM imidazole) was added to wash ten times, and 10 mL of elution buffer (20 mM Tris-HCl pH = 8.0, 1 mM PMSF, 300 mM NaCl, 10% glycerol, 300 mM imidazole) was added for elution. 20 μL of the protein solution before binding, the protein solution after binding, and the eluate were aliquoted respectively, and the purification situation was detected by SDS-PAGE method.

[0118] The results of SDS-PAGE detection of TrxA-His-ENO2 fusion protein are as Figure 3 shown in (A) below. The homozygous TrxA-His-ENO2 fusion protein has a single band at the expected position.

[0119] 6. Determination of the protein concentrations of differential protein GST-Vimentin and TrxA-His-ENO2

[0120] According to the instruction manual process of the BCA protein concentration assay kit (Vazyme), the protein standard solution was taken to make a standard curve. Then, the concentrations of GST-Vimentin and TrxA-His-ENO2 were calculated by the interpolation method.

[0121] 7. GST pull-down assay

[0122] Take a column and add GST-tag purification resin: Take two collection tubes with upper and lower layers and a stopper on the upper layer, and label them as "control group" and "experimental group". Remove the stopper, add 125 μL of GST-tag purification resin, centrifuge at 4 °C and 3000 rpm for 5 s, and then discard the waste liquid. Wash the column: Add 300 μL of ddH2O, centrifuge at 4 °C and 3000 rpm for 5 s, discard the waste liquid, and repeat twice. Equilibrate the column: Add 300 μL of binding buffer (20 mM Tris-HCl pH = 8.0, 25 mM NaCl, 1 mM PMSF, 10% glycerol), centrifuge at 4 °C and 3000 rpm for 5 s, and then discard the waste liquid. Repeat this process twice. Plug the stopper, and add three sets of GST or GST fusion proteins (GST or GST-Vimentin system: 10 μg; 20 μg; 30 μg) to the experimental group and the control group respectively. Add binding buffer to make up the volume to 500 μL, and bind at 4 °C on a shaker for 16 h. Remove the stopper, centrifuge at 4 °C and 3000 rpm for 5 s, and discard the waste liquid. Add 300 μL of binding buffer, centrifuge at 4 °C and 3000 rpm for 5 s, and repeat twice. Plug the stopper, add TrxA-His-ENO2 (20 μg) to both the experimental group and the control group, add binding buffer to make up the volume to 500 μL, and bind at 4 °C and 100 rpm on a shaker for 16 h. Then remove the stopper, centrifuge at 4 °C and 3000 rpm for 5 s, discard the waste liquid, and centrifuge twice with 350 μL of binding buffer. The centrifugation conditions are the same as above. Add 150 μL of elution buffer (100 mM GSH, 50 mM NaCl), and elute at 4 °C and 100 rpm on a shaker for 10 min. Finally, centrifuge at 4 °C and 12000 rpm for 2 min, collect in a 1.5 mL centrifuge tube, label it, and store it in a -20 °C refrigerator.

[0123] 8. Verify the results of protein interaction by Western Blot

[0124] Run the recycled interaction products on SDS-PAGE, cut the protein gel to an appropriate size, place it on a PVDF membrane, and transfer at 200 mA for 2 h. Put 5 mL each of TBST (20 mM Tris Base, 15 mM NaCl, 0.1% Tween-20) containing 5% skim milk powder in a five-compartment box, and block at 37 °C on a shaker at 100 rpm for 1 h. For the GST fusion protein interaction system, add GST or His monoclonal antibody diluted 1:3000 with TBST containing 5% skim milk powder. Place it on a shaker at 4 °C at 100 rpm for 16 h. Wash the membrane three times with TBST, 20 min each time. Dilute goat anti-mouse IgG H&L / HRP (secondary antibody) 1:3000 with TBST containing 5% skim milk powder and add it to the corresponding system, incubate at 37 °C on a shaker at 100 rpm for 1 h. Then wash with TBST three more times, 20 min each. Use an ECL chemiluminescence reagent detection kit, image in a chemiluminescence imager, and take pictures for recording.

[0125] The results of Western blot are as Figure 4 shown. As the GST / GST-Vimentin fusion protein increases, its binding ability to the TrxA-His-ENO2 protein becomes stronger (the binding ability data was analyzed using the IMAGE J analysis software after transfer; the binding ability range is: 0 < binding ability value < 1; the value 0 represents no binding, 0.5 represents moderate binding, and 1.0 represents strong binding). Thus, the optimized interaction conditions were confirmed.

[0126] As can be seen from the above examples, in the present invention, first, cells transfected with pcDNA3.1-flag (control group) and pcDNA3.1-flag-eno2 (experimental group) were used. After cell lysis, Flag monoclonal antibody was used to perform immunoprecipitation with the cell supernatant, and then SDS-PAGE was carried out for primary protein screening. This method has removed some non-specifically bound proteins. Then, the differentially banded regions on the SDS-PAGE gel were cut and collected for liquid chromatography-mass spectrometry (LC-MS) protein sequencing. These differentially banded regions are proteins that may interact with ENO2. Subsequently, intracellular RNA extraction, RT-PCR, and other steps were carried out to synthesize cDNA. Primers were designed to clone the gene of this possible interacting protein, Vimentin, into the pGEX-4T-1 expression vector. After the GST fusion protein was highly expressed, protein interaction experiments were carried out with different amounts of the GST fusion protein and TrxA-His-ENO2, which can be used to explore the interaction between unknown pathogen antigens and intracellular proteins of host cells.

[0127] The eno2 gene sequence (1482 bp, SEQ ID NO: 1):

[0128]

[0129] Vimentin gene sequence (1284 bp, SEQ ID NO: 2):

[0130]

[0131] ENO2 protein sequence (493 AA, SEQ ID NO: 3):

[0132] MEIFFFFASSCEQLLCVSSRCQLHPCLPQSSRLLLLVVCYPACYQHFNLQRIFLPCGVLSCADSRGNPTVEVDLLTDGGCFRAAVPSGASTGIYEALELRDKDPSKFLGKGVMKAVENIQKVIKPALIGKDPCDQQAIDKLMVEELDGTKNEWGWCKSKLGANAILAVSMACCRAGAAAKGMPLYKYIATLAGKPTDNMVMPVPFFNVINGGSHAGNKVAMQEFMLAPVGAKTIHEAIQIGAEVYHNLKSVIKKKYGLDATNVGDEGGFAPNISSATEALDLLVEAIKVSGHEGKVKIAADVAASEFWLGSDKVYDLDFKTPNNDKSHRKTGEQLRALYKEWSEKYPFVSIEDPFDQDDFSSYASLTSEIGAKVQIVGDDLLVTNPTRIEKALKEKACNGLLLKVNQIGTVTESIQACQLAQKNGWGVMVSHRSGETEDSFIADLVVGLRTGQIKTGAPCRSERLCKYNQLMRIEESLGSACHYAGADFRNPK。

[0133] Vimentin protein sequence (427 AA, SEQ ID NO: 4):

[0134] MSTRTVSSSSYRRMFGGPGTASRPSSSRSYVTTSTRTYSLGSALRPSTSRSLYTSSPGGVYATRSSAVRLRSSVPGVRLLQDAVDFSLADAINTEFKNTRTNEKVELQELNDRFANYIDKVRFLEQQNKILLAELEQLKGQGKSRLGDLYEEEMRELRRQVDQLTNDKARVEVERDNLAEDIMRLREKLQEETLQREEAESTLQSFRQDVDNASLARLDLERKVESLQEEIAFLKKLHDEEIQELQAQIQEQHVQIDMDVSKPDLTAALRDVRQQYESVAAKNLQEAEEWYKSKFADLSEAANRNNDALRQAKQESNEYRRQVQSLTCEVDALKGTNESLERQMREMEENFAVEAANYQDTIGRLQDEIQNMKEEMARHLREYQDLLNVKMALDIEIATYRKLLEGEESRISLPLPNFSSLNLRGYQ。

[0135] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for the interaction between a parasite protein and a protein in a host cell, characterized in that, It includes the following steps: After connecting the parasite protein coding gene sequence with the first expression vector to construct a recombinant expression vector, it is transferred into Escherichia coli for induced expression, the cells are lysed, and then purified with a chromatography column to prepare the first fusion protein with a TrxA-His tag; After connecting the host cell inner protein coding gene sequence with the second expression vector to construct a recombinant expression vector, it is transferred into Escherichia coli for induced expression, the cells are lysed, and purified with a chromatography column to prepare the second fusion protein with a GST tag; The second fusion protein with a GST tag and the first fusion protein with a TrxA-His tag are interacted through a GST pull-down experiment, and then the interaction relationship is determined according to the binding strength of the two proteins; Among them, the parasite protein is a key metalloenzyme in the glycolysis process isolated from Isospora suis, and the parasite protein coding gene sequence is as shown in SEQ ID NO: 1; The host cell inner protein is vimentin isolated from porcine intestinal epithelial cells, and the host cell inner protein coding gene sequence is as shown in SEQ ID NO:

2.

2. The method according to claim 1, wherein The first expression vector includes pET32(a), and the second expression vector includes pGEX-4T-1.

3. The method according to claim 1, wherein In the interaction system, as the dosage of the first fusion protein with a GST tag increases, its binding strength with the second fusion protein with a TrxA-His tag becomes stronger.

4. The method according to claim 1, wherein In the interaction system, the mass ratio of the first fusion protein with a GST tag to the second fusion protein with a TrxA-His tag is (2-3):

2.

5. The method according to claim 1, characterized in that, The interaction relationship includes unbound, moderately bound, and strongly bound.

6. An isolated parasite protein, characterized in that, The parasite protein is a key metalloenzyme in the glycolysis process isolated from Isospora suis, and the parasite protein coding gene sequence is as shown in SEQ ID NO:

1.

7. A recombinant vector, characterized in that, The recombinant vector contains the coding gene of the parasite protein described in claim 6.

8. A recombinant bacterium, characterized in that, The genome of the recombinant bacterium integrates and expresses the recombinant vector described in claim 7.

9. Use of the parasite protein described in claim 6, or the recombinant vector described in claim 7, or the recombinant bacterium described in claim 8 in the preparation of a detection reagent for the interaction mechanism between Isospora suis and porcine intestinal epithelial cells.

10. The application according to claim 9, characterized in that, To explore the interaction mechanism between Isospora suis and porcine intestinal epithelial cells through the interaction between the parasite protein and vimentin in porcine intestinal epithelial cells. The amino acid sequence of the vimentin is as shown in SEQ ID NO: 4, and the coding gene sequence is as shown in SEQ ID NO: 2.

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