Optimized construction method and application of Spodoptera frugiperda protein interaction verification system BiFC-Venus

Abstract

The invention discloses an optimized construction method of a protein interaction verification system BiFC-Venus for spodoptera frugiperda and application of the optimized construction method of the protein interaction verification system BiFC-Venus for spodoptera frugiperda. The design principle of the Spodoptera frugiperda protein interaction verification system BiFC-Venus provided by the invention is as follows: plasmids of a pIZ-to-be-tested gene A-VN173-His and a pIZ-to-be-tested gene B-VC155-His are co-transfected into an Sf9 cell through the method, so as to judge whether the two proteins have interaction or not. The protein researched by the invention is eukaryotic expression and has natural conformation; consumed time is short, and data processing is simple; the background is clean, the fluorescence has high brightness and long service life, and the detection is sensitive; the temperature tolerance is high, and observation can be carried out under the physiological temperature condition. Therefore, the system can verify the interaction between the proteins, and a convenient and rapid research method is provided for the research of the insect protein function.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to an optimized construction method and application of a protein interaction verification system BiFC-Venus for Spodoptera frugiperda. Background Art

[0002] Spodoptera frugiperda is an agricultural pest native to the tropical and subtropical regions of the Americas and belongs to the family Noctuidae of the order Lepidoptera. In recent years, Spodoptera frugiperda has rapidly spread to parts of Asia, Africa, and Europe, posing a serious threat to various crops such as corn, wheat, and rice, and has become one of the major migratory pests attracting global attention. Studying the molecular mechanisms of its survival and immunity helps to screen biomarkers for disease diagnosis and determine the targets for disease treatment / intervention, which is of great significance for the prevention and control of Spodoptera frugiperda-related diseases and the breeding of Spodoptera frugiperda-resistant varieties. Existing verification methods such as pull down and dual luciferase reporter system have the disadvantages of long time consumption, high requirements for experimental instruments, being able to detect only strong protein interactions, and being unable to quickly and intuitively observe weak interactions or transient interactions.

[0003] Bimolecular fluorescence complementation (BiFC) is a special tool used in a technical platform for detecting protein-protein interactions in molecular biology and cell biology research. The BiFC technique is designed based on the characteristics of fluorescent proteins. Usually, two halves (N-terminal and C-terminal) of a fluorescent protein are used. When these two halves are fused and expressed with two proteins of interest respectively, if these two proteins interact with each other in living cells, the N-terminal and C-terminal will approach and spontaneously recombine to form a complete fluorescent protein, thereby causing the cells to emit a fluorescent signal, indicating the interaction between proteins. The advantages of this technique are its real-time monitoring, non-destructive detection, and the ability to observe protein interactions under physiological conditions, which are very suitable for studying dynamic biological processes such as signal transduction, protein localization changes, and complex assembly. Since BiFC does not rely on the addition of chemical substances, it avoids the possibility of false positive results, and thus has broad application prospects in fields such as drug screening and disease mechanism exploration. However, the BiFC technique also has certain limitations. For example, larger fluorescent protein fragments may interfere with the normal functions of proteins, or in some cases, weak interactions between two proteins may not be sufficient to result in a strong enough fluorescent signal. Summary of the Invention

[0004] The present invention aims to solve the technical problems to be solved, overcome the deficiencies of the prior art, and provide an optimized construction method and application of a protein interaction verification system for Spodoptera frugiperda.

[0005] One of the purposes of the present invention is to provide a construction method of a protein interaction verification system for Spodoptera frugiperda, which is characterized by including the following steps: Step 1: Design specific primers to split the yellow fluorescent protein into two fragments, Venus-VN173 and Venus-VC155. The Venus-VN173 fragment is within the amino acids (a.a.) 1 - 173 of the yellow fluorescent protein. The forward primer sequence of the Venus-VN173 fragment is as shown in SEQ ID NO:1, i.e., 5’-AGCACAGTGGCGGCCGCTCGAGTATGGTGAGCAAGGGCGA-3’, and the reverse primer sequence of Venus-VN173 is as shown in SEQ ID NO:2, i.e., 5’-GATGATGACCGGTACGCGTCTCGATGTTGTGGCGGATCTT-3’. The Venus-VC155 fragment is within the amino acids (a.a.) 156 - 239 of the yellow fluorescent protein. The forward primer sequence of the Venus-VC155 fragment is as shown in SEQ ID NO:3, i.e., 5’-AGCACAGTGGCGGCCGCTCGAGTGACAAGCAGAAGAACGG-3’, and the reverse primer sequence of Venus-VC155 is as shown in SEQ ID NO:4, i.e., 5’-GATGATGACCGGTACGCGTCTTGTACAGCTCGTCCATG-3’; Step 2: Homologously recombine the Venus-VN173 and Venus-VC155 fragments cut in Step 1 into the insect expression vector pIZ-V5 / His respectively to obtain the empty vectors pIZ-VN173-His and pIZ-VC155-His; Step 3: Connect the mouse protein p53 protein (a.a. 72 - 390) and SV40 large T antigen (a.a. 87 - 708) fragments to the N-terminals of the empty vectors pIZ-VN173-His and pIZ-VC155-His constructed in Step 2 respectively; Step 4: Inoculate Sf9 cells into a cell plate with an inoculation density of about 0.5 - 2*105 cell / well and culture the cells for 24 h; Step 5: Transfect the two groups of plasmids constructed in Step 3 into the cells cultured in Step 4 and continuously culture at 28 °C for 48 h; Step 6: Place the cells in Step 5 under an inverted fluorescence microscope, observe the distribution of yellow fluorescence (i.e., the intensity of yellow fluorescence), and judge the construction result of the BiFC-YFP system.

[0006] The present invention uses molecular biology and cell biology means to explore the molecular mechanism of protein-protein interaction in Spodoptera frugiperda, constructs and optimizes a verification system BiFC-Venus for protein-protein interaction in Spodoptera frugiperda, and applies this system to verify whether there is an interaction between two target proteins in vivo or in vitro in Spodoptera frugiperda.

[0007] The further optimized technical solution of the present invention is as follows: In the step 1, the cleavage positions of the yellow fluorescent protein are 155 / 156 and 173 / 174 aa.

[0008] In the step 2, the restriction enzyme sites are XhoⅠ and MluⅠ.

[0009] In the step 3, the C-terminal restriction enzyme site of mouse protein p53 and SV40 large T antigen is XhoⅠ.

[0010] In the step 4, the density of Sf9 cells in the culture medium during transfection is 70-90%l.

[0011] The second object of the present invention is to provide a Spodoptera frugiperda protein-protein interaction verification system BiFC-Venus constructed by the above method.

[0012] The third object of the present invention is to provide the application of the Spodoptera frugiperda protein-protein interaction verification system BiFC-Venus in verifying the existence of interaction between two target proteins in vivo or in vitro in Spodoptera frugiperda.

[0013] In the above application, the criterion for judging the existence of interaction between two target proteins is: under an inverted fluorescence microscope, the cells show yellow fluorescence.

[0014] The present invention constructs a vector containing the fusion of the protein to be tested and the fluorescent protein fragment, and observes whether there is yellow fluorescence after co-expression to judge whether there is an interaction between the two proteins to be tested.

[0015] The above application is characterized in that the protein-protein interaction in Spodoptera frugiperda includes strong interaction, weak interaction and transient interaction between proteins.

[0016] The Spodoptera frugiperda protein-protein interaction verification system BiFC-Venus of the present invention co-transfects pIZ-test gene A-VN173-His and pIZ-test gene B-VC155-His plasmids into Sf9 cells to judge whether two proteins have an interaction. The proteins studied in the present invention are eukaryotic expressed and have natural conformations; they take less time and the data processing is simple; the background is clean, the fluorescence has high brightness and long lifespan, and the detection is sensitive; they have high temperature tolerance and can be observed under physiological temperature conditions. Therefore, this system can verify the interaction between proteins and provide a convenient and fast research method for the study of insect protein functions.

[0017] The beneficial effects of the present invention are as follows: simple operation, capable of quickly obtaining experimental results and presenting clear and visible experimental phenomena; high sensitivity, capable of accurately capturing the interaction between proteins; the selected yellow fluorescence of the present invention has high temperature tolerance; the result verification of the protein interaction relationship conforms to the actual situation, with intuitive phenomena and high result credibility; convenient cell culture of model insects and strong representativeness. Brief Description of the Drawings

[0018] Figure 1 It is the schematic diagram of the principle of the present invention.

[0019] Figure 2 It is the schematic diagram of the binding situation of double fluorescence fragments observed under the inverted fluorescence microscope of the present invention. Detailed Embodiments

[0020] The technical solution of the present invention will be further described in detail below in conjunction with the embodiments: This embodiment is implemented on the premise of the technical solution of the present invention, and the detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0021] The materials and reagents mentioned in the present invention can be purchased from domestic and foreign commercial channels or obtained through free channels by the public, and will not be described one by one here. Example 1

[0022] As Figure 1 shown, an optimized construction method and application of a protein interaction verification system BiFC-Venus for Spodoptera frugiperda include the following steps: 1) Construct empty vectors pIZ-VN173-His and pIZ-VC155-His by PCR amplification and ligation.

[0023] The amino acid sequence of the fluorescent protein Venus fragment is shown in SEQ ID NO: 7. Using the fluorescent protein Venus fragment as a template, two fragments, Venus-VN173 (a.a. 1-173) and Venus-VC155 (a.a. 156-239), are amplified and homologous arm sequences are added. The cleavage sites are 155 / 156 and 173 / 174 aa respectively.

[0024] Design specific primers. The forward primer sequence of Venus-VN173 is 5’-AGCACAGTGGCGGCCGCTCGAGTATGGTGAGCAAGGGCGA-3’ (SEQ ID NO:1), and the reverse primer sequence of Venus-VN173 is 5’-GATGATGACCGGTACGCGTCTCGATGTTGTGGCGGATCTT-3’ (SEQ ID NO:2). The forward primer sequence of Venus-VC155 is 5’-AGCACAGTGGCGGCCGCTCGAGTGACAAGCAGAAGAACGG-3’ (SEQ ID NO:3), and the reverse primer sequence of Venus-VC155 is 5’-GATGATGACCGGTACGCGTCTTGTACAGCTCGTCCATG-3’ (SEQ ID NO:4); The PCR reaction conditions are as follows: pre-denaturation at 94°C for 3 minutes, then denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 60 seconds, for a total of 35 cycles, and finally extension at 72°C for 10 minutes. The PCR products were identified by agarose gel electrophoresis and recovered, ligated overnight at 16°C with the pMD18T vector (Takara), transformed into DH5α competent cells (TransGen Biotech), and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing after obtaining positive clones. The sequencing results showed that the Venus-VN173 gene with a full length of 519 bp and the Venus-VC155 gene with a full length of 252 bp were successfully cloned. The sequence of the Venus-VN173 gene is shown as SEQ ID NO:5, and the sequence of the Venus-VC155 gene is shown as SEQ ID NO:6. Then, the two fragments were respectively ligated to the pIZ-V5 / His vector (V5 tag removed, THERMO FISHER) digested with two enzymes (the restriction sites are XhoⅠ and MluⅠ) using 2xHieff Clone*UniversalⅡ homologous recombination enzyme.

[0025] 2) Construct an overexpression plasmid carrying a gene fragment of the fusion target protein and a reporter gene through single enzyme digestion and ligation.

[0026] The empty vectors pIZ-VN173-His and pIZ-VC155-His were digested with the restriction endonuclease XhoⅠ, and then ligated at 50°C for 1 hour using 2xHieff Clone*UniversalⅡ homologous recombination enzyme. The vector fragments pIZ-VN173-His and pIZ-VC155-His were ligated with the gene fragments of the target proteins mouse p53 protein and SV40 large T antigen protein respectively, and then transformed into DH5α competent cells. The plasmids were extracted and their concentrations were measured, and then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing to ensure that the plasmids were correct and suitable for cell transfection.

[0027] 3) Cell transfection and subsequent culture.

[0028] Sf9 cells with a density of about 60%-70% were seeded into a 24-well cell culture plate, 500 μL of op ti-MEM medium per well. 24 hours after cell seeding, the constructed plasmids were co-transfected into the cells. The empty transfection of pIZ-VN173-His and pIZ-VC155-His was used as the control group, and the co-transfection of pIZ-target protein gene-VN173-His and pIZ-target protein gene pIZ-target protein gene-VC155-His plasmids was used as the experimental group. 0.5 μg of the overexpression plasmid and the transfection reagent were configured into a Lipormaster3000Reagent / DNA / T3000 Enhancer Reagent complex mixture for transfection. The cells were continuously cultured in an incubator at 28°C for 48 hours.

[0029] 4) Fluorescence observation to verify whether the target proteins interact.

[0030] The cultured cells were placed under an inverted fluorescence microscope for observation. In the control group, since the N-terminal and C-terminal fragments of the Venus fluorescent protein could not approach each other, no yellow fluorescence was presented. In the experimental group, if the target proteins interacted, the N-terminal and C-terminal fragments of the fluorescent protein could approach each other, and then form a complete Venus fluorescent protein chromophore to emit a yellow fluorescence signal. If there was no interaction, no fluorescence signal appeared.

[0031] The availability of this BiFC system was reversely proved by the protein interaction verification of the known interacting proteins mouse p53 protein (a.a. 72-390) and SV40 large T antigen (a.a. 87-708). Yellow fluorescence could be observed under the fluorescence microscope in the cells transfected with pIZ-p53-VN173-His and pIZ-T-VC155-His, while no fluorescence signal was observed in the control group transfected with the empty vector, proving that this system had the function of verifying protein interaction (see Figure 2 )). After transfection and Figure 2 the same brightness was strong interaction, there was fluorescence but no Figure 2Weak interaction is indicated by low fluorescence, and transient interaction can be observed by fluorescence a few hours after transfection.

[0032] As described above, only the specific embodiments of the present invention are provided herein, but the protection scope of the present invention is not limited thereto. Any variations or substitutions that can be understood and conceived by those skilled in the art within the technical scope disclosed by the present invention should be covered within the scope of the present invention. Therefore, the protection scope of the present invention should be determined by the protection scope of the claims.

Claims

1. An optimized construction method and application of BiFC-Venus, a protein interaction verification system for fall armyworm, characterized in that: The following steps are involved: Step 1, designing specific primers to split the yellow fluorescent protein into two fragments, Venus-VN173 and Venus-VC155, wherein the amino acid of the Venus-VN173 fragment is within the range of 1-173 of the yellow fluorescent protein, the forward primer sequence of the Venus-VN173 fragment is shown in SEQ ID NO: 1, and the reverse primer sequence is shown in SEQ ID NO: 2; the amino acid of the Venus-VC155 fragment is within the range of 156-239 of the yellow fluorescent protein, the forward primer sequence of the Venus-VC155 fragment is shown in SEQ ID NO: 3, and the reverse primer sequence is shown in SEQ ID NO: 4; Step 2, homologously recombining the Venus-VN173 and Venus-VC155 fragments cut in step 1 into the insect expression vector pIZ-V5 / His to obtain empty vectors pIZ-VN173-His and pIZ-VC155-His; Step 3, mouse protein p53 protein and SV40 large T antigen fragment were connected to the N-terminus of the empty vectors pIZ-VN173-His and pIZ-VC155-His constructed in step 2, respectively; Step 4, inoculate Sf9 cells into the cell plate at a density of about 0.5-2*105 cell / well, and culture the cells for 24 hours; Step 5, transfect the two sets of plasmids constructed in step 3 into the cells cultured in step 4, and culture them continuously at 28° C. for 48 h; Step 6: Place the cells from step 5 under an inverted fluorescence microscope, observe the distribution of yellow fluorescence, and determine the results of the BiFC-YFP system construction.

2. The method for constructing the fall armyworm protein interaction verification system according to claim 1, characterized in that: In step 1, the cleavage positions of the yellow fluorescent protein are 155 / 156 and 173 / 174 aa.

3. The method for constructing the fall armyworm protein interaction verification system according to claim 1, characterized in that: In the step 2, the restriction sites are XhoⅠ and MluⅠ.

4. The method for constructing the fall armyworm protein interaction verification system according to claim 1, characterized in that: In step 3, the C-terminal restriction site of mouse protein p53 protein and SV40 large T antigen is XhoⅠ.

5. The method for constructing the fall armyworm protein interaction verification system according to claim 1, characterized in that: In step 4, the density of Sf9 cells in the culture medium during transfection is 70-90%.

6. A fall armyworm protein interaction verification system constructed according to the method described in any one of claims 1 to 5.

7. The fall armyworm protein interaction verification system as described in claim 6 is used to verify the existence of interaction between two target proteins in vivo or in vitro in fall armyworm.

8. The use according to claim 7, characterized in that: The criterion for judging the existence of interaction between two target proteins is that the cells show yellow fluorescence under an inverted fluorescence microscope.

9. The use according to claim 8, characterized in that: The fall armyworm protein interactions include strong interactions, weak interactions and transient interactions between proteins.

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