Type I toxoplasma gondii claviform protein ROP16 truncation and application thereof

Abstract

The invention relates to the technical field of genetic engineering, in particular to an I-type toxoplasma gondii coryneform protein ROP16 truncation and application thereof. The truncated body of the I-type toxoplasma gondii claviform protein ROP16 is the 361st to 540th amino acids of the I-type toxoplasma gondii claviform protein ROP16. The invention illustrates the influence of the I-type toxoplasma gondii coryneform protein ROP16 truncation on the polarization, proliferation, apoptosis and cycle of mouse alveolar macrophages MH-S. According to the invention, toxoplasma gondii secretory protein is taken as a research object, the influence of the ROP16 molecular preparation on host cells and the action mechanism of the ROP16 molecular preparation are researched, the molecular mechanism of the ROP16 molecular preparation influencing the polarization and anti-inflammatory action of macrophages is discussed, and the research on the molecular mechanism of toxoplasma gondii infection is beneficial to clinical control of toxoplasma gondii infection and also beneficial to clinical application of the ROP16 molecular preparation. The potential application value of exploring polypeptide preparations causing polarization of the macrophages is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, in particular to a truncated form of type I Toxoplasma gondii rhoptry protein ROP16 and an application thereof. Background Art

[0002] Toxoplasma gondii is an obligate intracellular, opportunistic parasitic zoonosis that spreads globally and infects nearly all warm-blooded animals, including humans. It can severely affect organs throughout the body, leading to severe toxoplasmosis. Macrophages are the host's first line of defense against Toxoplasma infection, and their immune response determines the host cell's ability and characteristics to resist infection. The interaction between macrophages, Toxoplasma gondii, and its secreted proteins is of great significance for studying the relationship between host cells and Toxoplasma. ROP16 is a rod-shaped protein with serine / threonine kinase activity secreted by Toxoplasma gondii in host cells. It can be directly localized to the host cell nucleus and participate in the regulation of host cell signaling pathways by phosphorylating STAT. The research team previously constructed a ROP16 overexpression vector and transfected it into MH-S macrophages. The results showed that overexpression of type I ROP16 protein can upregulate the expression of anti-inflammatory factors by regulating the JAK-STAT3 signaling pathway, thereby causing M2 polarization. In rat alveolar macrophages NR8383, overexpression of ROP16 was found to affect the gene expression profile of macrophages. Key differentially expressed genes were involved in multiple signaling pathways, including JAK-STAT, To11-like receptors, and IL-17. Functional clustering was mainly manifested in cytokine-cytokine receptor interactions, cell proliferation regulation, immune regulation, and signal transduction. The specific amino acid sites that cause the changes in the host cell phenotype of type I ROP16 protein are still unknown. Summary of the Invention

[0003] In order to solve the above problems, the present invention provides type I Toxoplasma gondii rod-shaped protein ROP16 truncation and its application. The present invention first studies the specific protein truncation that regulates macrophage phenotypic changes. According to ROP16I bioinformatics analysis, it is divided into four protein truncation forms: △1-180, △181-360, △361-540, and △ROP16. By constructing a ROP16I overexpression MH-S macrophage model and a silent expression model under siRNA intervention, the expression of ROP16I in the macrophages was detected by qRT-PCR, Western-blot, and ELIS. A. Molecular biological techniques such as CCK8 flow cytometry were used to investigate the effects of ROP16I protein truncations on the JAK-STAT signaling pathway in MH-S macrophages, as well as their effects and molecular mechanisms on macrophage polarization, proliferation, apoptosis, and cell cycle. This provided a theoretical basis for subsequent studies on the specific single amino acids that cause changes in macrophages induced by ROP16 protein, and on this basis, attempts were made to develop new targets or explore new ideas for the treatment of pulmonary toxoplasmosis using the Toxoplasma gondii secretory protein ROP16 as a candidate protein, which has potential translational application value for the treatment of pulmonary toxoplasmosis.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] The present invention provides a type I Toxoplasma gondii rhoptry protein ROP16 truncation, and the amino acid sequence of the type I Toxoplasma gondii rhoptry protein ROP16 truncation is shown in SEQ ID No. 1.

[0006] Preferably, the nucleotide sequence corresponding to the truncation of the type I Toxoplasma gondii rhoptry protein ROP16 is shown as SEQ ID No. 2.

[0007] The present invention also provides the use of the type I Toxoplasma gondii rhoptry protein ROP16 truncation described in the above technical solution in the preparation of a drug for treating pulmonary toxoplasmosis by regulating macrophage phenotype.

[0008] Preferably, the type I Toxoplasma gondii rhoptry protein ROP16 truncation promotes macrophage polarization.

[0009] Preferably, the type I Toxoplasma gondii rhoptry protein ROP16 truncation promotes the proliferation of macrophages.

[0010] Preferably, the type I Toxoplasma gondii rhoptry protein ROP16 truncation inhibits macrophage apoptosis.

[0011] Preferably, the type I Toxoplasma gondii rhoptry protein ROP16 truncation induces macrophage S / G2 phase cell cycle arrest.

[0012] Beneficial effects of the present invention:

[0013] The present invention provides a small molecule formulation of a type I Toxoplasma gondii rhoptry protein ROP16 truncation and its application, illustrating the effects of type I Toxoplasma gondii rhoptry protein ROP16 truncation on the polarization, proliferation, apoptosis, and cycle of mouse alveolar macrophages (MH-S). This invention fills the gap in the study of the effects of ROP16 molecular formulations on host cells and their mechanisms of action using Toxoplasma secretory proteins as research objects, exploring the molecular mechanisms of their effects on macrophage polarization and anti-inflammatory effects. This study of the molecular mechanisms of Toxoplasma infection is not only beneficial for the clinical control of Toxoplasma infection, but also has the potential application value of exploring polypeptide formulations that induce macrophage polarization. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] 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.

[0015] Figure 1 The hydrophilicity, signal peptide and transmembrane structure predictions of type I ROP16 protein. Note: A: hydrophilicity prediction; B: signal peptide prediction; C: transmembrane structure prediction;

[0016] Figure 2 Prediction of phosphorylation sites for type I ROP16 protein;

[0017] Figure 3 Prediction of the secondary and tertiary structures of type I ROP16 protein. Note: A: ROP16 protein secondary structure; B: ROP16 protein tertiary structure;

[0018] Figure 4 Validation of type I ROP16 protein in MH-S macrophages. Note: 1. MH-S 2. MH-S-EP 3. 1-1804, 181-3605, 361-5406. Full-length ROP16; ns: P>0.05 compared with the MH-S group; *: P<0.05 compared with the MH-S group; **: P<0.01 compared with the MH-S group; ***: P<0.001 compared with the MH-S group; ****: P<0.0001 compared with the MH-S group; A: WB analysis of ROP16 protein expression in macrophages; B: ImageJ software analysis of protein bands in A; C: qRT-PCR analysis of ROP16 mRNA expression in macrophages;

[0019] Figure 5Effects of type I ROP16 protein truncations on the polarization of MH-S macrophages. Notes: 1. MH-S 2. MH-S-EP 3. 1-1804. 181-3605. 361-5406. Full-length ROP16; ns: P>0.05 compared with the MH-S group; *: P<0.05 compared with the MH-S group; **: P<0.01 compared with the MH-S group; ***: P<0.001 compared with the MH-S group; ****: P<0.0001 compared with the MH-S group; A: WB analysis of the expression of polarization proteins in macrophages; B: ImageJ software analysis of the protein bands in A; C: qRT-PCR analysis of the expression of polarization factor mRNA in macrophages;

[0020] Figure 6 Figure 1: Effects of type I ROP16 protein truncations on inflammatory factors in MH-S macrophages. Notes: 1. MH-S 2. MH-S-EP 3. 1-1804. 181-3605. 361-5406. Full-length ROP16; ns: P>0.05 compared with the MH-S group; *: P<0.05 compared with the MH-S group; **: P<0.01 compared with the MH-S group; ***: P<0.001 compared with the MH-S group; ****: P<0.0001 compared with the MH-S group; A: WB analysis of inflammatory factor protein expression in macrophages; B: ImageJ software analysis of protein bands in A; C: qRT-PCR analysis of inflammatory factor mRNA expression in macrophages; D: ELISA analysis of inflammatory factor content in macrophages;

[0021] Figure 7 CCK-8 assay was used to detect the effects of type I ROP16 protein truncations on the proliferation of MH-S macrophages. Note: 1. MH-S 2. MH-S-EP 3. 1-1804, 181-3605, 361-5406. Full-length ROP16; ns: P>0.05 compared with the MH-S group; ***: P<0.001 compared with the MH-S group; A: Expression of MH-S macrophage proliferation; B: Analysis of MH-S macrophage activity;

[0022] Figure 8 Flow cytometric analysis of apoptosis in MH-S cells using type I ROP16 protein truncations. Note: 1. MH-S 2. MH-S-EP 3. 1-1804. 181-3605. 361-5406. Full-length ROP16; ns: P>0.05 compared with the MH-S group; ***: P<0.001 compared with the MH-S group; A: Flow cytometric analysis of cell apoptosis; B: Statistical analysis of cell apoptosis;

[0023] Figure 9Figure 2: Expression of apoptotic factors by type I ROP16 protein truncations in MH-S cells. Note: 1. MH-S 2. MH-S-EP 3. 1-1804. 181-3605. 361-5406. Full-length ROP16; ns: P>0.05 compared with the MH-S group; **: P<0.01 compared with the MH-S group; ***: P<0.001 compared with the MH-S group; A: Western blot analysis of apoptotic factor protein expression in macrophages; B: ImageJ software analysis of protein bands in A; C: qRT-PCR analysis of apoptotic factor mRNA expression in macrophages;

[0024] Figure 10 Flow cytometric analysis of the cell cycle of type I ROP16 protein truncations in MH-S cells. Note: 1. MH-S 2. MH-S-EP 3. 1-1804. 181-3605. 361-5406. Full-length ROP16; ns: compared with the MH-S group, P>0.05;; *: compared with the MH-S group, P<0.05; **: compared with the MH-S group, P<0.01; ***: compared with the MH-S group, P<0.001; A: Flow cytometric analysis of cell cycle; B: Statistical analysis of cell cycle;

[0025] Figure 11 Figure 2: Expression of cycle factors in MH-S cells by type I ROP16 protein truncations. Note: 1. MH-S 2. MH-S-EP 3. 1-1804. 181-3605. 361-5406. Full-length ROP16; ns: P>0.05 compared with the MH-S group; *: P<0.05 compared with the MH-S group; **: P<0.01 compared with the MH-S group; ***: P<0.001 compared with the MH-S group; A: Western blot analysis of cycle factor protein expression in macrophages; B: ImageJ software analysis of protein bands in A; C: qRT-PCR analysis of cycle factor mRNA expression in macrophages;

[0026] Figure 12 Western blotting analysis of the mechanism by which type I ROP16 protein truncations regulate MH-S cells. Note: 1. MH-S 2. MH-S-EP 3. 1-1804, 181-3605, 361-5406. Full-length ROP16; ns: P>0.05 compared with the MH-S group; **: P<0.01 compared with the MH-S group; ***: P<0.001 compared with the MH-S group; A: Western blotting analysis of inflammatory factor protein expression in macrophages; B: ImageJ software analysis of protein bands in A; C: ImageJ software analysis of protein bands in A.

[0027] Figure 13The silencing effect of MH-S cells after siRNA-STAT3 transfection. Note: ****: P < 0.0001 compared with the MH-S group; A: WB analysis of STTA3 protein expression in macrophages; B: ImageJ software analysis of protein bands in A; C: qRT-PCR analysis of STAT3 mRNA expression in macrophages;

[0028] Figure 14 Effects of siRNA-STAT3 on the polarization of MH-S macrophages expressing type I ROP16 protein truncations. Notes: 1. MH-S 2. MH-S-EP 3. 1-1804, 181-3605, 361-5406. Full-length ROP16; ns: P>0.05 compared with the MH-S group; *: P<0.05 compared with the MH-S group; ***: P<0.001 compared with the MH-S group; A: Western blotting analysis of polarization protein expression in macrophages; B: ImageJ analysis of protein bands in A; C: qRT-PCR analysis of polarization factor mRNA expression in macrophages;

[0029] Figure 15 Figure 3 Effects of siRNA-STAT3 on inflammatory factors in MH-S macrophages expressing type I ROP16 protein truncations. Notes: 1. MH-S 2. MH-S-EP 3. 1-1804, 181-3605, 361-5406. Full-length ROP16; ns: P>0.05 compared with the MH-S group; *: P<0.05 compared with the MH-S group; **: P<0.01 compared with the MH-S group; ***: P<0.001 compared with the MH-S group; ****: P<0.0001 compared with the MH-S group; A: WB analysis of polarization protein expression in macrophages; B: ImageJ software analysis of protein bands in A; C: qRT-PCR analysis of polarization factor mRNA expression in macrophages;

[0030] Figure 16 Effects of siRNA-STAT3 on apoptotic factors in MH-S macrophages expressing type I ROP16 protein truncations. Note: 1. MH-S 2. MH-S-EP 3. 1-1804, 181-3605, 361-5406. Full-length ROP16; ns: P>0.05 compared with the MH-S group; **: P<0.01 compared with the MH-S group; ***: P<0.001 compared with the MH-S group; A: Western blot analysis of polarization protein expression in macrophages; B: ImageJ software analysis of protein bands in A; C: qRT-PCR analysis of polarization factor mRNA expression in macrophages;

[0031] Figure 17Figure 3 Effects of siRNA-STAT3 on cell cycle factors in MH-S macrophages expressing type I ROP16 protein truncations. Notes: 1. MH-S 2. MH-S-EP 3. 1-1804, 181-3605, 361-5406. Full-length ROP16; ns: P>0.05 compared with the MH-S group; *: P<0.05 compared with the MH-S group; **: P<0.01 compared with the MH-S group; ***: P<0.001 compared with the MH-S group; A: Western blot analysis of polarization protein expression in macrophages; B: ImageJ software analysis of protein bands in A; C: qRT-PCR analysis of polarization factor mRNA expression in macrophages;

[0032] Figure 18 This is a map of the pCDNA3.1-CMV-MCS-EF1-ZsGreen-T2A-puro vector. DETAILED DESCRIPTION

[0033] The present invention provides an amino acid sequence of a truncated form of the rhoptry protein ROP16 of type I Toxoplasma gondii as shown in SEQ ID No. 1, specifically as follows:

[0034] NERVMMINANGVPIALYNRGHLGSGHFGAVIKASLDDGTLYAAKVPYSQIVPNADATSAELEAGISSSARAELVKTIRQELDVRDKLVAKGLTLTETVSQYGLPLCQMTLTLPENKATVVRRGSRLFVVSKEVMLLPLIDGSALNSLVQSQPPFLFQRAVAREAILALAKLHELGFAHGDV.

[0035] In the present invention, the nucleotide sequence corresponding to the truncated form of the type I Toxoplasma gondii rhoptry protein ROP16 is shown in SEQ ID No. 2, which is as follows:

[0036] .

[0037] The present invention also provides the use of the type I Toxoplasma gondii rhoptypic protein ROP16 truncation described in the above technical solution in the preparation of a drug for treating pulmonary toxoplasmosis by regulating the phenotype of macrophages. In the present invention, the type I Toxoplasma gondii rhoptypic protein ROP16 truncation preferably promotes the polarization of macrophages. In the present invention, the type I Toxoplasma gondii rhoptypic protein ROP16 truncation preferably promotes the proliferation of macrophages. In the present invention, the type I Toxoplasma gondii rhoptypic protein ROP16 truncation preferably inhibits the apoptosis of macrophages. In the present invention, the type I Toxoplasma gondii rhoptypic protein ROP16 truncation preferably induces macrophage S / G2 phase cell cycle arrest.

[0038] In order to further illustrate the present invention, the present invention is described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1

[0040] Part I: Construction of target plasmids (△1-180; △181-360; △361-540);

[0041] 1. Vector and target gene information

[0042] 1.1 Destination vector information

[0043] See the pCDNA3.1-CMV-MCS-EF1-ZsGreen-T2A-puro vector map for details. Figure 18 .

[0044] 1.2 Target gene sequence information (insert the target gene sequence into the pCDNA3.1-CMV-MCS-EF1-ZsGreen-T2A-puro vector):

[0045] (1) The base sequence corresponding to amino acids 1-180 of the Δ1-180I gene is as follows (SEQ ID No. 3):

[0046] The amino acid sequence of the Δ1-180I gene at positions 1-180 is as follows (SEQ ID No. 4):

[0047] MKVTTKGLAFALALLFCTRCATARYMSFEEAQKASEAAKRQIATLPSPDSPLSNPGSRHRNRGGSPTAGQPSQSTLQPEQAAAEVGLGAGGSTQGQGRTGGSAGAREERRSPSPESAYPATSSASLRGYQTQLSPSHLPPHSSGPGGWFPTESIYTLWSSPPQRLTHRKPSLSGVVVTEF。

[0048] (2) The base sequence corresponding to amino acids at positions 181 - 360 of the △181 - 360 type I gene is as follows (SEQ ID No. 5):

[0049] ATGCAAGAGCCACAAGAACAGTATGGCGCAGCGAGCAGTCTTGCGTCCTCGCCAAAGGGATACGTCGGTGGCGCAAGCTCTAGTGCATTGTCAGGAAAGGCGGTGCCGACGCCTGCGTCGCTTGGTCAAGAAAATCCTCTTTTTCCTGGTCAGAGCGCTACATTGGATTCAGGAATACAGTCTCCGGCACAAAAGCGTCGGGGATCCCCTCAAAGACAGAGTGCGATGCCGACCGGAAATCCAGCAGATAGCGGCGCCTCGCAGCTTGCCTTCAGTCATTCTAGTTATGTATCAGTACAAGCTTCTCTTGCGAAACGTTCAGAACGCATCCGGCGCGTTCGACTTTCAGAAGAGGGTCTGGAAGAAGTTCAGCAGCTGAAAGCAGCTGCCGCACAGCTTCTCGTAGCGGTTCCGGACTATGAGGCAATGCGGGCTGTTCTGCAAGAGGCGGTCCTCTCAGAACAGAGGGTTGCTGCCCGTAAGCGGAAGAGAAAGCAACCTCCAGGAGCGGTGGAGTCAGCTGTTGACGAAGTGTTTCCTCCATAG;

[0050] Amino acids at positions 181 - 360 of the △181 - 360 type I gene (SEQ ID No. 6):

[0051] QEPQEQYGAASSLASSPKGYVGGASSSALSGKAVPTPASLGQENPLFPGQSATLDSGIQSPAQKRRGSPQRQSAMPTGNPADSGASQLAFSHSSYVSVQASLAKRSERIRRVRLSEEGLEEVQQLKAAAAQLLVAVPDYEAMRAVLQEAVLSEQRVAARKRKRKQPPGAVESAVDEVFPP。

[0052] (3) The base sequence corresponding to amino acids at positions 181 - 360 of the △361 - 540I gene is as follows (SEQ ID No.2):

[0053] ATGCATCATCACCATCACCATAATGAGCGTGTCATGATGATAAATGCCAACGGAGTGCCGATCGCTCTATACAATCGTGGGCACCTCGGCAGTGGACATTTCGGGGCTGTCATCAAGGCCAGCTTAGACGATGGGACGCTGTATGCAGCGAAGGTGCCGTACAGCCAGATCGTCCCGAATGCTGATGCCACGTCAGCAGAACTGGAGGCGGGAATTTCCTCAGCTAGGGCGGAGTTGGTAAAGACAATTCGACAGGAGTTGGATGTTCGGGATAAGCTTGTGGCTAAAGGGCTCACACTTACAGAGACTGTGAGCCAATACGGTCTGCCATTGTGCCAAATGACTTTAACGCTTCCTGAGAACAAAGCAACCGTGGTACGTCGAGGTTCTCGACTCTTTGTCGTGTCCAAAGAAGTCATGCTGCTGCCATTAATTGATGGCTCCGCATTGAACAGTCTAGTCCAGTCGCAACCACCATTTCTCTTCCAGCGAGCTGTGGCAAGGGAAGCAATTCTTGCATTGGCCAAGCTTCACGAACTTGGATTCGCGCATGGAGATGTTTAG;

[0054] △360-540I type gene 181-360 amino acids (SEQ ID No.1): NERVMMINANGVPIALYNRGHLGSGHFGAVIKASLDDGTLYAAKVPYSQIVPNADATSAELEAGISSSARAELVKTIRQELDVRDKLVAKGLTLTETVSQYGLPLCQMTLTLPENKATVVRRGSRLFVVSKEVMLLPLIDGSALNSLVQSQPPFLFQRAVAREAILALAKLHELGFAHGDV.

[0055] 2. Vector digestion

[0056] Add each reagent in the order shown in the table below, gently pipette to mix, and place in a 37°C water bath for 1-2 hours. After the enzyme digestion is complete, perform agarose gel electrophoresis to recover the target fragment.

[0057] The vector enzyme digestion system is as follows:

[0058] Table 1 Enzyme digestion system

[0059] Reagents Volume (μL) Vector DNA (1ug / uL) 1 10*buffer 4 <![CDATA[DdH2O]]> 32 EcoRI 1.5 BamHI 1.5 total 40

[0060] Note: The amount of restriction enzyme used and the digestion time can be adjusted according to the enzyme activity; if a single enzyme digestion is used, the corresponding system should be adjusted.

[0061] 3. Acquisition of target fragments

[0062] 3.1 Primer design:

[0063] Table 2 Primer sequences

[0064]

[0065]

[0066] 3.2 PCR amplification of target fragments

[0067] Prepare the following system, mix gently, and place in a PCR instrument for reaction; Fragment PCR amplification system:

[0068] Table 3 Amplification system

[0069]

[0070] Table 4 PCR program

[0071]

[0072]

[0073] Note: 1) Annealing temperature is the primer Tm value, which directly determines the amplification specificity; 2) If the amplification specificity is poor, the annealing temperature can be appropriately increased by +2°C each time; 2) Prolonging the extension time appropriately can help improve the amplification yield.

[0074] 4. Connect the target fragment to the vector

[0075] HB infusion TM One-step cloning and ligation system:

[0076] Prepare the following reaction mixture in an ice-water bath. If the liquid accidentally sticks to the tube wall, briefly centrifuge it to allow it to settle to the bottom. Incubate the ligation reaction at 50°C for 30 minutes, then place on ice for 5 minutes and immediately transform.

[0077] Table 5 Connection system

[0078] Component name Volume (μL) Target gene fragment X(≥100ng) Linearized vector Y(≥50ng) 2×HBinfusionTMMastermix 10 <![CDATA[ddH2O]]> Z(=10-XY) Total volume 20

[0079] Note: 1) When ligating 2-3 fragments, it is recommended to use a total of 0.02-0.5 pmols of DNA fragments (generally, the amount of added fragments is 100-150 ng and the amount of vector is 50-100 ng). When ligating 4-6 fragments, the total amount of DNA added is 0.2-1.0 pmols. DNA splicing efficiency gradually decreases with increasing number of spliced fragments or increasing length of spliced fragments.

[0080] 5. Conversion

[0081] 1) After taking the DH5α competent cells out of the -80℃ freezer, they should be immediately placed on ice to thaw. The competent cells should be packaged gently to minimize mechanical damage.

[0082] 2) After the competent medium has thawed, aliquot the aliquots into 50 μL aliquots per tube (20 μL is sufficient for plasmid transformation). Add the ligation product to the aliquots at a volume no greater than 1 / 10 of the competent medium volume (currently add 5 μL of ligation product) and place on ice for 20-30 minutes.

[0083] 3) Heat shock at 42°C for 90 seconds (this time must be very strict), and immediately incubate on ice for 2-3 minutes.

[0084] In a clean bench, add 500 μL LB medium (note that it must be antibiotic-free LB medium) and gently invert it 3-5 times;

[0085] 5) Incubate at 37°C, 230 rpm, shaking for 45-60 min;

[0086] 6) Apply the bacterial solution evenly to a solid plate of the corresponding resistance, then incubate the plate upside down at 37°C for 12-16 hours;

[0087] 6. PCR Identification of Bacterial Liquid

[0088] 6.1 Bacterial liquid PCR identification system:

[0089] Table 6 System

[0090] Component name Volume (μL) 2xHieffPCRMasterMIX(Dye) 5 Primer 1 (upstream primer) 0.5 Primer 2 (downstream primer) 0.5 bacterial liquid 2 <![CDATA[ddH2O]]> 2 Total volume 10

[0091] Note: When configuring the mix, the proportions in the mix should be amplified in equal proportions. Number of clones to be verified; Select the verification primers according to the vector used

[0092] 6.2 Bacterial liquid PCR identification procedure

[0093] Table 7 Procedure

[0094]

[0095] 7. Sequencing

[0096] Two clones were selected from the positive clones screened out for sequencing. If the sequencing results showed that the sequencing results were consistent with the target sequence, the target plasmid was successfully constructed and stored in a -80°C refrigerator for use.

[0097] Part II: Transfection of target plasmid (MH-S mouse alveolar macrophages);

[0098] 1. Cell culture: about 0.4×10 cells per day before transfection (20-24 hours) 5 The cells were plated into six-well plates so that the cell confluence could reach about 70% on the next day.

[0099] 2. Before proceeding with the following transfection steps, replace each well of the six-well plate with fresh cell culture medium. The volume of the culture medium is approximately 2 mL.

[0100] 3. Gently mix the LipoFiter3.0 liposomal transfection reagent.

[0101] 4. For the cells in one well of the six-well plate to be transfected, take a clean sterile centrifuge tube, add 4 μg of plasmid DNA to 250 μL of DMEM solution, and gently pipette to mix.

[0102] 5. Take another clean sterile centrifuge tube, add 250μL DMEM solution, then add 10μL LipoFiter3.0, gently blow with a gun to mix, and let it stand at room temperature for 5 minutes.

[0103] 6. Combine the DNA solution from steps 4 and 5 with the LipoFiter 3.0 solution and gently pipette to mix. Note: Do not vortex or centrifuge.

[0104] 7. Incubate at room temperature for 20 minutes.

[0105] 8. Add 500 μl of the LipoFiter 3.0-DNA mixture to one well of a six-well plate. Make sure to add it evenly throughout the well. Then gently rock the plate in a figure-8 pattern to mix thoroughly. Place the plate in an incubator and incubate for 6 hours before changing the medium.

[0106] 9. After 6-12 hours of incubation in the cell culture incubator, remove the serum-free culture medium containing LipoFiter3.0-DNA and add 2 ml of fresh serum-containing cell culture medium to each well to continue incubation.

[0107] 10. After another 24-40 hours of culture, the transfection effect can be tested for subsequent experiments.

[0108] Part III: Validation of the target plasmid and stably transfected ROP16 (Li Jiaming, Wang Yixuan, Yang Ningai, et al. Effects of Toxoplasma gondii ROP16 protein on MH-S cell polarization and apoptosis and its related mechanisms [J]. Chinese Journal of Parasitology and Parasitic Diseases, 2022, 40(05): 579-586.) (MH-S mouse alveolar macrophages):

[0109] MH-S cell lines stably transfected with ROP16 were maintained in the laboratory. Total RNA and total protein were extracted from six groups of cells: normal group, negative control group, Δ1-180, Δ181-360, Δ361-540, and ROP16 full-length group. ROP16 expression in each group of cells was detected by qPCR-PCR and Western blot.

[0110] The △1-180 truncation is obtained by truncating amino acids 1-180 of the ROP16 protein to obtain its gene sequence, which is then combined with a vector to construct the corresponding plasmid;

[0111] The △181-360 truncation is obtained by truncating the amino acids 181-360 of the ROP16 protein to obtain its gene sequence, which is then combined with a vector to construct the corresponding plasmid;

[0112] The △361-540 truncation is obtained by truncating the amino acids 361-540 of the ROP16 protein, obtaining its gene sequence, combining it with a vector, and constructing the corresponding plasmid.

[0113] ROP16 full-length amino acid sequence (SEQ ID No. 13):

[0114] .

[0115] (1) Extraction of total cellular RNA:

[0116] 1. Preparation: Prepare several 15 mL centrifuge tubes and 1.5 mL EP tubes. Sterilize the pipette and pipette tips under UV light in a biosafety cabinet.

[0117] 2. Sample Preparation: Collect the cell suspension and transfer it to a 15 mL centrifuge tube. Centrifuge at 10,000 rpm for 10 min. Discard the supernatant and transfer the remaining liquid to a 1.5 mL EP tube. Add 1 mL of TRIzol and lyse the cells by pipetting until the lysate becomes clear.

[0118] 3. Separation: Add 200 μL of chloroform to the EP tube, shake vigorously for 15 seconds, let it sit for 3 minutes, and then centrifuge at 12,000 rpm at 4°C for 10 minutes. The sample should now separate into three layers: a pink organic phase, a middle layer, and an upper, colorless aqueous phase. The RNA is primarily in the colorless aqueous phase. Carefully aspirate the upper aqueous phase and transfer it to a new EP tube.

[0119] 4. RNA precipitation: Add an equal volume of isopropanol to the obtained aqueous phase, invert the liquid in the tube to mix, let it stand at room temperature for 10 minutes, then centrifuge at 12000r / min in a 4℃ centrifuge for 10 minutes, discard the supernatant, at this time RNA precipitate can be seen.

[0120] 5. RNA elution: Add 1 mL of 75% ethanol and gently invert to mix to elute the RNA precipitate. Then centrifuge at 10,000 rpm at 4°C for 5 minutes and discard the supernatant (be careful not to pour out the precipitate. The remaining small amount of liquid can be aspirated with a pipette after brief centrifugation. Be careful not to aspirate the precipitate).

[0121] 6. RNA Redissolution: Place at room temperature for 5-10 minutes to dry thoroughly. After drying, add an appropriate amount of DNase / RNase-free water to dissolve the RNA. Mix by pipetting repeatedly to fully dissolve the RNA. Alternatively, place the RNA in a high-performance dry bath (70°C) for 10-15 minutes to promote dissolution.

[0122] 7. RNA concentration and purity determination: Take 1 μL of sample and measure the OD value on a nuclear protein analyzer. After the measurement, the sample is stored in a -80℃ refrigerator to prevent degradation.

[0123] (2) cDNA synthesis

[0124] Using the extracted RNA as a template, prepare the reverse transcription reaction solution according to the following components (Table 8):

[0125] Table 8 Reverse transcription system

[0126]

[0127] *: A 10μL reaction system can reverse transcribe a maximum of 500ng of total RNA. The reaction volume can be expanded as needed depending on the reaction system.

[0128] Vortex the prepared reaction system and centrifuge briefly. Turn on the PCR instrument, set the reverse transcription program (Table 9), and then place the reverse transcription system into the PCR instrument for reverse transcription.

[0129] Table 9 Reverse transcription procedure

[0130] temperature time 37℃ 15min 85℃ 5s 4℃ ∞

[0131] After the reaction is completed, the cDNA can be stored in a -20°C refrigerator.

[0132] (3) qRT-PCR

[0133] 1. Preparation: Dilute the primers to 10 μM in DNase / RNase-free water on ice.

[0134] 2. Add cDNA to the PCR plate according to the following system (Table 10).

[0135] Table 10 RT-qPCR reaction system

[0136]

[0137] 3. After sample loading, attach the membrane to the PCR plate and place it in a horizontal centrifuge for a brief centrifugation.

[0138] 4. After centrifugation, amplify using a real-time fluorescence quantitative PCR instrument. The amplification procedure is as follows (Table 11):

[0139] Table 11 RT-qPCR reaction procedure

[0140] temperature time Number of cycles 95℃ 30s 40 95℃ 5s 40 40℃ 30s 40

[0141] 5. Using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal reference, the amplification signal of each target gene was obtained. The relative mRNA expression level of each target gene was calculated by the 2-ΔΔCt method, which is its transcription level.

[0142] Part IV: Study the effects of type I ROP16 truncations on MH-S cell polarization;

[0143] Total RNA, total protein and cell culture supernatant were extracted from the normal group, negative control group, △1-180, △181-360, △361-540 and full-length ROP16 groups. The expressions of INOS, ARG-1, CD86, CD206, NLRP3, IL-1β, IL-6, IL-10, TNF-α and TGF-β in macrophages of each group were detected by qPCR-PCR, Western blot and ELISA. The effect of type I ROP16 truncation on the polarization of MH-S cells was analyzed.

[0144] Part V: Study the effects of type I ROP16 truncations on MH-S cell proliferation, apoptosis and cell cycle

[0145] (1) Six groups of cells, including the normal group, negative control group, Δ1-180, Δ181-360, Δ361-540, and ROP16 full-length group, were plated and the proliferation of cells in each group was detected by CCK-8 assay.

[0146] (2) Flow cytometry was used to detect the effects of type I ROP16 truncations on the apoptosis and cell cycle of MH-S cells;

[0147] (3) Total RNA and total protein were extracted respectively, and the expression of BAX, BCL-2, Caspase3, Caspase9, CDK6, CyclinD1, P21 and P53 factors in macrophages of each group were detected by qPCR-PCR, Western-blot and flow cytometry, and the effects of type I ROP16 protein segment protein truncation on MH-S cell proliferation, apoptosis and cycle were analyzed.

[0148] Part VI: Studying the mechanism of regulation of type I ROP16 truncation on MH-S cell phenotype

[0149] The experiment was divided into blank group, empty vector group, Δ1-180, Δ1-360, Δ1-540, and full-length ROP16 groups. Cells in the logarithmic growth phase were collected for the next step. Protein was extracted from the normal group, negative control group, Δ1-180, Δ181-360, Δ361-540, and full-length ROP16 groups. Western blot was used to detect the protein expression of JAK, STAT3, and P-STAT3 pathway-related factors after overexpression of type I ROP16 protein truncations.

[0150] Part VII: Study the effect of siRNA-STAT3 on the phenotype of MH-S macrophages expressing type I ROP16 protein truncations

[0151] SiRNA-STAT3 (SiRNA-STAT3 is a method of silencing the STAT3 gene as a whole through small interfering technology) was transfected into each group of macrophages. The expression of polarization, apoptosis, cycle and pathway factors in the cells were detected by qPCR-PCR and Western-blot methods. The effects of various ROP16 protein segment protein fragments after siRNA-STAT3 on the polarization, apoptosis and cycle phenotypes and molecular mechanisms of each group of macrophages were analyzed.

[0152] result:

[0153] 1) Bioinformatics analysis results of type I ROP16 full-length protein

[0154] Bioinformatics analysis showed that type I ROP16 proteins are composed of 707 amino acids (amino acid sequence shown in SEQ ID No. 1) and are unstable alkaline proteins with hydrophilicity. The online software ProScale predicts the hydrophilicity of type I ROP16 proteins, such as Figure 1As shown, the type I ROP16 protein is a hydrophilic protein, and the 12th amino acid has the highest score of 2.911 points, while the 343rd amino acid has the lowest score of -3.544 points. The online software SignalP-4.1 and TMHMM-2.0 predicted the transmembrane structure and signal peptide sequence of ROP16. The type I protein has a signal peptide sequence located between amino acids 23 and 24, with D = 0.865 and D-cutoff = 0.450, indicating that it is a secreted protein without a transmembrane structure. Figure 2 As shown, the online software NetPhos-3.1 predicted the phosphorylation sites of the protein, and the results showed that the type I ROP16 protein had 83 phosphorylation sites (Ser:61, Tyr:8, Thr:18). Figure 3 As shown, the online software SOPMA predicted the secondary structure of the type I ROP16 protein, and the results showed that the Alphahelix accounted for 33.80%, the Extended strand accounted for 10.61%, the Betaturn accounted for 6.65%, and the Random coil accounted for 48.94%. The online software Phyre2 predicted the tertiary structure of the type I ROP16 protein, and the results showed that the type I ROP16 protein was modeled based on the template d1vzoa, and 278 amino acid residues (39% of the sequence) were modeled with 100% confidence using the single highest-scoring template.

[0155] 2) Validation of type I ROP16 truncated proteins in MH-S macrophages

[0156] After the type I ROP16 protein truncations act on MH-S macrophages, Figure 4 As shown in the figure, compared with the normal blank group, there was no difference in the expression levels of ROP16 gene and protein in the negative control group, while the expression levels of ROP16 gene and protein in the truncated group △1-180, △181-360, △361-540, and △ROP16 full-length group were significantly increased (all P < 0.05), which was statistically significant, indicating that ROP16 was successfully and stably expressed in each group of cells.

[0157] 3) Effect of type I ROP16 protein on MH-S macrophage polarization

[0158] After the type I ROP16 protein truncations act on MH-S macrophages, Figure 5 As shown in the figure, compared with the normal blank group, the expression levels of M2 polarization factors ARG-1 and CD206 genes and proteins in the negative control group, truncated groups △1-180, and △181-360 were not different (all P>0.05), while the expression levels of ARG-1 and CD206 in △361-540 and △ROP16 full-length groups were significantly increased (all P<0.05); Figure 6As shown in the results, compared with the normal blank group, the expression levels of NLRP3, IL-10, and TGF-β genes and proteins in the negative control group were not different (all P>0.05), while the expression levels of NLRP3, IL-10, and TGF-β genes and proteins in the △361-540 and △ROP16 full-length groups were significantly increased (all P<0.05).

[0159] 4) Effects of type I ROP16 protein truncations on the proliferation of MH-S macrophages

[0160] The experiment used CCK-8 method to analyze the effect of type I ROP16 protein truncations on the proliferation ability of MH-S macrophages. Figure 7 As shown, compared with the normal blank group, the negative control group showed no significant changes in cell proliferation and viability at any time point (all P>0.05), with no statistically significant differences. Compared with the normal blank group, the cell proliferation and viability of all groups did not change significantly at 6 hours (all P>0.05), with no statistically significant differences. At 24 hours, the △1-180 group showed no significant changes in cell proliferation and viability (P>0.05), while the △181-360, △361-540, and full-length △ROP16 groups showed significant increases in cell proliferation and viability (all P<0.001). At 48 hours, the cell proliferation and viability of all groups were significantly increased (all P<0.001), and at 72 hours, the cell proliferation and viability of all groups were significantly increased (all P<0.001). These results indicate that type I ROP16 protein truncations can significantly enhance the proliferation of MH-S cells in a time-dependent manner.

[0161] 5) Effects of type I ROP16 protein truncations on apoptosis of MH-S macrophages

[0162] Flow cytometry was used to detect apoptosis. Figure 8 As shown, compared with the normal blank group, the negative control group,

[0163] There was no significant difference in the degree of apoptosis between the △1-180 and △181-360 groups, which was not statistically significant (both P>0.05); the degree of apoptosis in the △361-540 and △ROP16 full-length groups was significantly inhibited, which was statistically significant (both P<0.001). Western-blot and qRT-PCR results showed that Figure 9As shown, compared with the normal blank group, there were no significant differences in the protein and mRNA levels of apoptotic factors BAX, BCL-2, Caspase3, and Caspase9 in the negative control group (all P>0.05). However, in the △361-540 and △ROP16 full-length groups, BAX, Caspase3, and Caspase9 protein and mRNA levels were significantly decreased (all P<0.01), while BCL-2 protein and mRNA levels were significantly increased (all P<0.001). This indicates that the type I ROP16 protein truncation △361-540 and the △ROP16 full-length protein significantly inhibited cell apoptosis, with more significant differences.

[0164] 6) Effects of type I ROP16 protein truncations on the cell cycle of MH-S macrophages

[0165] Flow cytometry detection cycle, such as Figure 10 As shown in the figure, compared with the normal blank group, there were no significant differences in the G0 / G1, G2, and S phases of the negative control group, △1-180, and △181-360 groups, which were not statistically significant (all P>0.05); the G0 / G1, G2, and S phases of the △361-540 and △ROP16 full-length groups were significantly decreased, which was statistically significant (all P<0.05). Western-blot and qRT-PCR results showed that Figure 11 As shown in the figure, compared with the normal blank group, there were no significant differences in the protein and mRNA expressions of cycle factors CDK6, CyclinD1, P21, and P53 in the negative control group, with no statistical significance (all P>0.05); the protein and mRNA expressions of CDK6 and CyclinD1 in the △361-540 and △ROP16 full-length groups were significantly increased (all P<0.01), and the protein and mRNA expressions of P21 and P53 were significantly decreased (all P<0.01), indicating that the type I ROP16 protein truncation △361-540 and the full-length △ROP16 protein induced S / G2 phase cell cycle arrest in MH-S cells, and the differences were more significant.

[0166] 7) Molecular mechanism of type I ROP16 protein truncations regulating MH-S macrophages

[0167] Western-blot and qRT-PCR were used to detect the mechanism of type I ROP16 protein truncations in regulating MH-S macrophages. The results of Western-blot showed that Figure 12As shown, compared with the normal blank group, the negative control group showed no significant differences in the expression of pathway-related factors JAK, STAT3, and P-STAT3 proteins (all P>0.05). However, the expression of JAK and P-STAT3 proteins in the Δ361-540 and ΔROP16 full-length groups was significantly increased (all P<0.01). This suggests that type I ROP16 protein truncations regulate the polarization, apoptosis, and phenotypic changes of MH-S macrophages through the JAK-STAT3 signaling pathway.

[0168] 8) Effect of siRNA-STAT3 on the polarization of MH-S macrophages expressing type I ROP16 protein truncations

[0169] This study used siRNA to silence STAT3 in MH-S cells overexpressing ROP16. Three siRNAs (siRNA-1393, siRNA-368, and siRNA-644) were constructed and synthesized. After transfection into the cells, the silencing effect was verified by qRT-PCR and Western blot. The results showed that compared with the blank group, the STAT3 protein level in the cells transfected with siRNA-368 was significantly downregulated (P < 0.001, Figure 13 Middle A, Figure 13 Middle B), the mRNA expression level was significantly down-regulated (P < 0.0001, Figure 13 (C). Figure 14 、 Figure 15 As shown in the results, after transfection with siRNA-STAT3, the expression levels of polarization factors ARG-1, CD206, NLRP3, IL-10, and TGF-β in the △361-540 and △ROP16 full-length groups were significantly decreased (all P<0.05).

[0170] 9) Effect of siRNA-STAT3 on apoptosis of MH-S macrophages expressing type I ROP16 protein truncations

[0171] Western-blot and qRT-PCR results showed that Figure 16 As shown in the results, after transfection with siRNA-STAT3, the BAX, Caspase3, and Caspase9 proteins and mRNA levels in the △361-540 and △ROP16 full-length groups were significantly increased (all P<0.01), while the BCL-2 protein and mRNA levels were significantly decreased (both P<0.001).

[0172] 10) Effects of siRNA-STAT3 on the phenotype of MH-S macrophages expressing type I ROP16 protein truncations

[0173] Western-blot and qRT-PCR results showed that Figure 17As shown in the figure, after transfection with siRNA-STAT3, the protein and mRNA levels of P21 and P53 in the △361-540 and △ROP16 full-length groups were significantly increased (all P<0.01), while the protein and mRNA levels of CDK6 and CyclinD1 were significantly decreased (all P<0.001).

[0174] 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 truncated form of the rhoptry protein ROP16 of type I Toxoplasma gondii, characterized in that: The amino acid sequence of the type I Toxoplasma gondii rhoptry protein ROP16 truncation is shown in SEQ ID No.

1.

2. The type I Toxoplasma gondii rhoptry protein ROP16 truncation according to claim 1, characterized in that The nucleotide sequence corresponding to the type I Toxoplasma gondii rhoptry protein ROP16 truncation is shown in SEQ ID No.

2.

3. Use of the type I Toxoplasma gondii rhoptry protein ROP16 truncation according to claim 1 or 2 in the preparation of a drug for treating pulmonary toxoplasmosis by regulating macrophage phenotype.

4. The use according to claim 3, characterized in that The type 1 Toxoplasma gondii rhoptry protein ROP16 truncations promote macrophage polarization.

5. The use according to claim 3, characterized in that The type I Toxoplasma gondii rhoptry protein ROP16 truncated form promotes the proliferation of macrophages.

6. The use according to claim 3, characterized in that The type I Toxoplasma gondii rhoptry protein ROP16 truncated form inhibits macrophage apoptosis.

7. The use according to claim 3, characterized in that The type I Toxoplasma gondii rhoptry protein ROP16 truncation induces macrophage S / G2 phase cell cycle arrest.

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