CN108586586A - With the HIV P10 albumen of HGV RNA E2 protein-interactings - Google Patents

Abstract

The invention discloses an HIV P10 protein interacting with hepatitis G virus, the amino acid sequence of which is shown in SEQ ID NO: 1, and the nucleotide sequence of the gene encoding the HIV P10 protein is shown in SEQ ID NO: 2; It is to use the fourth-generation yeast two-hybrid method to screen the HIV cDNA library through the hepatitis G virus E2 protein to obtain the HIV P10 protein interacting with the hepatitis G virus E2 protein, and to identify the interacting protein by co-immunoprecipitation. Carry out positive verification; the present invention can provide a certain theoretical basis for studying the mechanism of action between hepatitis G virus and HIV-1 virus.

Description

HIV P10 Protein Interacting with Hepatitis G Virus E2 Protein

technical field

The invention relates to a research method and the field of genetic engineering of interacting proteins in the inhibitory effect of hepatitis G virus on HIV-1, in particular to an HIV P10 protein interacting with E2 protein of hepatitis G virus.

Background technique

Given that hepatitis G virus (GB Virus C, GBV-C) itself is not pathogenic, but co-infection with HIV-1 is beneficial to delay the course of HIV-1 patients, elucidating the mechanism of GBV-C's inhibition of HIV-1 will It is a key scientific problem to be solved urgently in the application of GBV-C in the treatment of HIV-1 disease. Based on the research progress at home and abroad, the inhibitory effect of GBV-C on HIV-1 mainly includes the following aspects: (1) The envelope glycoprotein E2 of GBV-C hinders the entry of HIV through the combination with HIV-1 Gp41. (2) The short peptides in the NS5A region of GBV-C may prevent HIV-1 from entering cells by inhibiting the expression of HIV-1 co-receptors CCR5 and CXCR4 in T cells. (3) By reducing the expression of FAs protein in T cells, GBV-C affected the FAs-induced apoptosis pathway, increased the number of CD4 ⁺ T lymphocytes, and delayed the course of HIV-1 patients. (4) GBV-C inhibits the expression of Th2 cytokines IL-4, IL-13 and IL-10 by enhancing the expression of Th1 cytokines IL-2 and IL-12 in T cells, and finally hinders HIV infection. -1 for duplication. (5) GBV-C decreases the activity of T cells by reducing the expression of LCK protein in the T cell signaling pathway, resulting in a significant decrease in the expression of CD38, CD69, CD25 and CCR5 molecules, affecting the replication of HIV-1. (6) At present, studies have found that GBV-C E2 protein can also inhibit the replication of HIV-1 by hindering the assembly and release of HIV-1 GAG protein. The above studies laid the foundation for elucidating the interaction mechanism between GBV-C and HIV-1. However, these studies are somewhat one-sided. Therefore, the mechanism by which GBV-C inhibits HIV-1 remains unclear.

Determining the proteins and their functions that directly interact between viruses is a key link in the study of the interaction mechanism between two viruses. At present, the interaction mechanism between GBV-C and HIV can be divided into two categories. The first category is that GBV-C inhibits the replication or entry of HIV through the influence of host cell activity; the second category is that GBV-C directly interacts with HIV proteins. The effect is to inhibit HIV from entering host cells or inhibit its growth. There are parts that can interact between proteins. Only by understanding the interaction network between proteins can we understand the principles of the organism's life process and the mechanism of disease occurrence, because many diseases occur because of the interaction between proteins. Interactions between proteins are disrupted, so the interaction between proteins plays a very important role. Therefore, the research on protein interaction is of great significance to reveal the interaction mechanism between GBV-C and HIV-1.

Contents of the invention

The object of the present invention is to provide an HIV P10 protein interacting with the hepatitis G virus E2 protein.

In order to achieve the purpose of the present invention, the present invention provides an HIV P10 protein that interacts with the hepatitis G virus E2 protein, the amino acid sequence of the HIV P10 protein is shown in SEQ ID NO:1.

P10 is a sequence starting from the overlapping region of the pol and gal genes, and is a protein encoded by the PR gene. It is an aspartic acid protease that acts as a dimer and functions to cleave Gag protein and Gag-pol precursor protein during virus maturation.

Another object of the present invention is to provide the gene encoding the above HIV P10 protein, the nucleic acid sequence of which is shown in SEQ ID NO:2.

The present invention also provides a vector containing the above HIV P10 protein gene.

The invention also provides engineering bacteria containing the above HIV P10 protein gene.

The present invention further provides a method for screening the HIV P10 protein interacting with the hepatitis G virus E2 protein: using the fourth-generation yeast two-hybrid method to screen the HIV cDNA library through the hepatitis G virus E2 protein to obtain a protein that interacts with the hepatitis G virus The HIV P10 protein that E2 protein interacts, and carries out the positive verification of interaction by the method for co-immunoprecipitation, thus screens the HIV P10 protein that interacts with hepatitis G virus E2 protein, the amino acid sequence of said HIV P10 protein is as SEQ ID NO: 1, including the following steps:

(1) Cloning and identification of the gene encoding hepatitis G virus protein E2: RNA was extracted from the type 7 hepatitis G virus strain, and after the RNA was obtained, the cDNA was obtained by reverse transcription, and then passed ordinary PCR and agarose gel Electrophoresis and sequence determination analysis to identify the target fragment;

(2) Construction of the bait plasmid vector containing the target fragment of hepatitis G virus E2 protein: the target fragment was recovered, digested, ligated with pGBKT7, and transformed into Escherichia coli, positive clones were screened, and the BD-Bait fusion expressable was constructed Protein plasmid;

(3) Construction of HIV cDNA library;

(4) A plasmid capable of expressing AD-Library/prey fusion protein: HIV cDNA library constructed on pGADT7, purchased from Clontech;

(5) Use the fourth-generation yeast two-hybrid method to screen candidate proteins that interact with hepatitis G virus E2 protein in the HIV cDNA library;

(6) Co-immunoprecipitation method was used to further verify the positive interacting proteins, so as to screen the HIV P10 protein interacting with the hepatitis G virus E2 protein.

Among them, step (6) is specifically: constructing pHA-P10 and pflag-E2 vectors with the candidate protein obtained in step (5) and the E2 protein of hepatitis G virus, forming fusion proteins with different tag tags, and then using X- tremeGENE HPDNA Transfection Reagent liposome transfection method co-transfected HEK239 human embryonic kidney cells, lysed the cells after 48-72 hours, incubated with ANTI-FLAG M2 Affinity Gel to precipitate, and finally analyzed the positive interaction results by Western blot.

(7) The positive interacting proteins were further verified by laser confocal method, so as to screen the HIV P10 protein interacting with the hepatitis G virus E2 protein.

Specifically, a method for screening the HIV P10 protein interacting with hepatitis G virus of the present invention comprises the following steps:

1. Cloning and identification of hepatitis G virus E2 gene

The hepatitis G virus E2 protein extracts RNA from the type 7 hepatitis G virus strain, obtains the RNA, and then obtains cDNA by RT-PCR, and then through ordinary PCR, agarose gel electrophoresis and sequence determination, the sequence comparison To analyze and identify the target fragment;

2. Hepatitis G virus E2 protein constructs bait fusion protein

Separation and recovery of the above PCR products by agarose gel electrophoresis, double enzyme digestion of PCR products and plasmid vectors, and recovery and purification of linear digestion products; the vector and target fragments were connected at a molar ratio of 1:4, overnight at 16°C . The ligation product was transformed into Escherichia coli JM109 competent cells, and positive clones were identified on the resistant medium; after picking up the monoclonal culture medium and expanding the culture, the recombinant plasmid was extracted, and after identification by PCR, double enzyme digestion and sequencing, it was determined to be heptane The BD-bait fusion protein plasmid constructed from the hepatitis G virus E2 gene, and finally transformed into yeast to identify the self-activation and toxicity of the hepatitis G virus E2 protein recombinant, can be used for subsequent experiments;

3. HIV cDNA library, constructed on the pGADT7 vector;

4. Co-transform the bait plasmid and the library plasmid into Y2H Gold yeast, and the reporter gene system is: -His, -Ade, AUR1-C, MEL1;

(1) Preparation of yeast competent cells

A. Take a small part of the frozen yeast cells and streak them on the YPDA agar plate (if it has been thawed before streaking, vortex to ensure the uniform distribution of yeast cells), invert the plate, and incubate at 30°C until colonies appear (about 3 days) );

B. Pick a single colony (≤4w, 2-3mm), inoculate a 5mL YPDA liquid culture based on a 15mL sterile centrifuge tube, and culture with shaking at 30°C for 12h;

C. Take 100 μl of the culture in a 250 mL flask containing 50 mL of YPDA, 30 ° C, 250 rpm shaking culture for 4-6 hours, OD600 should reach 0.6;

D. Centrifuge at room temperature at 3000rpm for 5-10min, discard the supernatant, and add 30mL sterile deionized water to resuspend the cell pellet;

E. Centrifuge at room temperature at 3000rpm for 5-10min, discard the supernatant, add 1.5mL 1.1×TE/LiAC solution to resuspend the cells, and centrifuge at 12000rpm at high speed for 15s;

F. Discard the supernatant, and resuspend the cells in 600 μL 1.1×TE/LiAC solution, which are competent cells;

(2) Transform baiT and prey into competent cells

A. Take 0.1 μg of baitT and prey plasmid DNA, 5 μL (0.1 mg) of Carrier DNA into a sterile 1.5mL EP tube, mix well, add 50 μL of yeast competent cells, and vortex to mix;

B. Add 0.5mL sterile 1×PEG/LiAC and mix well, incubate at 30°C for 30min, and mix every 10min;

C. Add 20 μL DMSO, gently invert and mix well, heat shock in 42°C water bath for 15 minutes and shake once every 5 minutes;

D. Centrifuge at 12000rpm for 15s at high speed, discard the supernatant; add 1mL YPDA, shake at 30°C, 250rpm for 1-2h;

E. Centrifuge at 12,000 rpm for 15 s at high speed, resuspend the cells with 0.9% NaCl, take 300 μL and spread it on the corresponding auxotrophic SD plate.

(3) Screening of positive clones

The yeast two-hybrid system used in the experiment has a total of four reporter genes, namely -His, -Ade, AUR1-C, and MEL1. Therefore, the final result of the screening finally determines that the positive clones should be able to activate the expression of these four reporter genes at the same time. The library screening process Firstly, the baiT and prey plasmid DNAs were respectively transformed and then hybridized in the double-deficiency culture with α-galactosidase (α-Galactosidase) chromogenic substrate X-α-Gal and antifungal aureobasidin Aba Based on the initial screening, the clones obtained in this step can either grow or turn blue. Theoretically, it is believed that the expression of the two reporter genes AUR1-C and MEL1 is activated due to the interaction between proteins; The positive clones obtained in the first round of screening were continued to be streak cultured on the four-deficient medium containing X-α-Gal and Aba (SD/-His/-Ade/-Trp/-Leu). The colonies that can still grow well on the four-deficiency medium are considered to have activated the expression of -His, -Ade genes, and these colonies are considered to be the positive results obtained by the preliminary screening.

(4) Sequencing analysis of insert fragments of positive clones

Extract the plasmids of all positive cloned yeast strains, and transform Escherichia coli JM109 competent cells by electric shock method. After transformation, the bacterial solution is spread on the plate of LB+Amp to screen the AD plasmid of the library. Since the plasmid expressing the bait protein is kana resistant, and The plasmid expressing the prey protein is Amp-resistant, and the bait plasmid can be screened out, while only the prey plasmid is retained. Pick out at least 10 single clones on each plate, extract plasmids after colony PCR detection to remove duplication, sequence and perform sequence analysis;

(5) Verification of positive clone results

The positive proteins obtained from the above sequence analysis were co-transformed with the hepatitis G virus E2 protein into the Y2H Gold yeast strain, and yeast two-hybrid was performed to confirm their interaction;

(6) Further verification of positive interacting proteins by co-immunoprecipitation experiments

Construct the obtained positive protein and hepatitis G virus E2 protein on the p-HA vector and pflag vector respectively, form fusion proteins with different tag tags, and then co-transfect with X-tremeGENE HP DNA Transfection Reagent liposome transfection method Human embryonic kidney cells HEK239 were lysed after 48-72 hours, incubated with ANTI-FLAG M2 Affinity Gel to precipitate, and finally analyzed the results of positive interactions by Western blot.

(7) Positive interacting proteins were further verified by laser confocal experiments

The cell colocalization of P10 and E2 was detected by laser confocal method. pFlag-P10 and pCDNA3.1-myc-E2 were respectively transfected into HEK293 and untreated HEK293 cells as a control, and pFlag-P10 and pCDNA3.1-myc-E2 were co-transfected into Huh7.5.1 cells. After 48 hours, the primary antibody and For the secondary antibody, rabbit antibody was selected for Flag tag antibody, and Rhodamine with red light was used as the fluorescent secondary antibody; mouse antibody was selected for Myc tag antibody, and FITC with green light was used as the fluorescent secondary antibody. Finally, the results of positive interactions were detected and analyzed by confocal laser microscopy.

The invention provides an HIV P10 protein interacting with the hepatitis G virus E2 protein, which provides a new research method for the study of the interaction mechanism between the hepatitis G virus and HIV, and provides a new research method for the study of the hepatitis G virus and HIV. The mechanism of action between them has laid the foundation.

The present invention has the following advantages:

1. Certainty: Aiming at the biggest problem in the traditional research on the mechanism of action between hepatitis G virus and HIV, that is, the uncertainty caused by the "black box" of each pathway chain between cause and effect, the present invention directly analyzes the phenomenon Start with the most fundamental reason and get a certain conclusion;

2. Efficiency: Some positive interacting proteins can be obtained through library screening and verification by yeast two-hybrid, and the results are clear and easy to analyze;

3. Practicality: Yeast two-hybrid method is more mature, simpler and easier to operate than comparative analysis method and omics technology;

4. Wide applicability: the method of the present invention can be used for the research on the interaction between any virus and viral protein.

Description of drawings

Figure 1 is the results of the present invention to verify whether the target protein can use the yeast two-hybrid screening system and self-activation, that is, pGBKT7 empty vector and pGBKT7-E2 plasmid are respectively transformed into yeast in SD/-Trp and SD/-His/-Ade/- Growth results on the Trp/-Leu plate; in the figure A is the pGBKT7 empty vector transformed into yeast on the SD/-His/-Ade/-Trp/-Leu plate without growing colonies, B is the pGBKT7 empty vector transformed into yeast in SD Colonies grown on /-Trp plate, C: pGBKT7-E2 empty vector transformed into yeast on SD/-Trp plate, D: pGBKT7-E2 empty vector transformed into yeast on SD/-His/-Ade/- No colonies grew on the Trp/-Leu plate;

Figure 2 is the result of the positive protein screened by the yeast two-hybrid verification library of the present invention, in which BD-P53/AD-T is a positive control; BD-Lam/AD-T is a negative control; BD /AD-P10 is pGBKT7 empty vector Figure of yeast colonies co-transformed with pGADT7-P10; BD-E2/AD-P10 is a picture of yeast colonies co-transformed with pGBKT7-E2 and pGADT7-P10; SD/-2 is SD/-Trp/-Leu two auxotrophic media ; SD/-4 is SD/-His/-Ade/-Trp/-Leu four auxotrophic medium; SD/-4/X-gal is SD/-His/-Ade/-Trp/-Leu four An auxotrophic medium was added with antifungal aureobasidin Aba and 4X-α-gal;

Figure 3 is the in vivo interaction between hepatitis G virus E2 protein and HIV P10 protein verified by co-immunoprecipitation experiments in the embodiment of the present invention; Input is the whole cell lysate control group, and the other two groups are the interaction between HIV P10 protein and E2 protein Verify the experimental group; Flag-E2 + is the addition of the E2 plasmid, and the HA-P10 + position is the addition of the P10 plasmid; E2 and P10 in the right column are detected with the tag antibody carried by E2 or P10; when co-transfecting E2 and P10, the In the control group, E2 and P10 can be detected at the same time, and E2 and P10 can also be detected by the co-precipitation method, so it can be explained that the hepatitis G virus E2 protein and HIV P10 protein can interact;

Fig. 4 shows the cell co-localization of E2 and P10 detected by laser confocal method in the present invention; P10 has green light, E2 has red light, and the nucleus is blue light.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention; If not specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available items.

Example 1: Screening for HIV P10 protein interacting with hepatitis G virus E2 protein

1. Cloning and identification of hepatitis G virus E2 gene

Analyze the target gene fragment sequence and the multiple cloning site information of the connected vector, and design primers: 5' end primer pGBKT7-E25'- CCG GAATTC GGCGCCCCGGCCTCGGTGCTAG, the underlined part is the EcoRI restriction site;

3' end primer pGBKT7-E2 3' - ACG CGTCGA CCCTGCCCGAGGAGAGCCATGCGAAC, the underlined part is the Sal Ⅰ restriction site;

The cDNA of hepatitis G virus type 7 was amplified by PCR to obtain gene fragments. 50 μl PCR system: 1 μl upstream primer, 1 μl downstream primer, 25 μl rTaq mixture, 21 μl dd H ₂ O, 2 μl template; PCR conditions: pre-denaturation at 94°C 5min, 35 cycles; 94°C for 30s, 50°C for 30s, 72°C for 2min, and finally extension at 72°C for 7min; the PCR product was electrophoresed on a 1% agarose gel to identify the size of the fragment.

2. Construction of bait plasmid vector

The above-mentioned PCR products were recovered after agarose gel electrophoresis, and EcoRI and SalI double enzymes were used to digest the target fragment and the plasmid vector pflag respectively, and the linear digestion products were recovered and purified. The vector and the target gene were ligated at a molar ratio of 1:4 at 16°C overnight. The ligation product is transformed into Escherichia coli JM109 competent cells, and the ligation product is transformed into Escherichia coli JM109 competent cells, and positive clones are identified on the resistant medium. After picking out the monoclonal culture medium and expanding the culture, the recombinant plasmid was extracted, and after identification by PCR, double enzyme digestion and sequencing, it was determined to be the BD-bait fusion protein plasmid constructed by the E2 gene of hepatitis G virus, and finally transformed into yeast to identify hepatitis G The self-activation and toxicity of the viral E2 protein recombinant can be used for subsequent experiments.

3. Library screening

Use yeast two-hybrid method to screen candidate proteins in HIV cDNA library that interact with hepatitis G virus E2 protein; in the present invention, E2 protein is used as Bait to connect to vector pGBKT7 to form BD-Bait, and the library protein is used as prey to construct AD -prey/Library. Transformed into yeast strain Y2H Gold yeast strain and Y187 yeast strain respectively, the reporter gene system is: -His, -Ade, AUR1-C and MEL1.

(1) Preparation of yeast competent cells

A. Take a small portion of frozen yeast cells and streak them on YPDA agar plates (if they have been thawed before streaking, vortex to ensure the uniform distribution of yeast cells). Invert the plate and incubate at 30°C until colonies appear (about 3 days);

B. Pick a single colony (≤4w, 2-3mm), inoculate a 5mL YPDA liquid culture based on a 15mL sterile centrifuge tube, and culture with shaking at 30°C for 12h;

C. Take 100 μl of the culture in a 250 mL flask containing 50 mL of YPDA, 30 ° C, 250 rpm shaking culture for 4-6 hours, OD600 should reach 0.6;

D. Centrifuge at room temperature at 3000rpm for 5-10min, discard the supernatant, and add 30mL sterile deionized water to resuspend the cell pellet;

E. Centrifuge at room temperature at 3000rpm for 5-10min, discard the supernatant, add 1.5mL 1.1×TE/LiAC solution to resuspend the cells, and centrifuge at 12000rpm at high speed for 15s;

F. Discard the supernatant, and resuspend the cells in 600 μL 1.1×TE/LiAC solution, which are competent cells;

(2) Transform baiT and prey into competent cells

A. Take 0.1 μg of baitT and prey plasmid DNA, 5 μL (0.1 mg) of Carrier DNA into a sterile 1.5mL EP tube, mix well, add 50 μL of yeast competent cells, and vortex to mix;

B. Add 0.5mL sterile 1×PEG/LiAC and mix well, incubate at 30°C for 30min, and mix every 10min;

C. Add 20 μL DMSO, gently invert and mix well, heat shock in 42°C water bath for 15 minutes and shake once every 5 minutes;

D. Centrifuge at 12000rpm for 15s at high speed, discard the supernatant; add 1mL YPDA, shake at 30°C, 250rpm for 1-2h;

E. Centrifuge at 12,000 rpm for 15 s at high speed, resuspend the cells with 0.9% NaCl, take 300 μL and spread it on the corresponding auxotrophic SD plate.

(3) Screening of positive clones

The yeast two-hybrid system used in the experiment has a total of four reporter genes, namely -His, -Ade, AUR1-C, and MEL1, so the final result of the screening finally determined that the positive clones should be able to activate the expression of these four reporter genes at the same time. In the library screening process, firstly, the bacterium liquid transformed with bait and prey plasmid DNA and then hybridized was mixed with α-galactosidase (α-Galactosidase) chromogenic substrate X-α-Gal and antifungal aureobasidin Aba The initial screening on the double-deficient medium, the clones obtained in this step can grow and turn blue colonies, it is theoretically believed that the two reporter genes AUR1-C and MEL1 are activated due to the interaction between proteins Express. The positive clones obtained after the first round of screening were continued to be streak cultured on the four-deficient medium (ie SD/-His/-Ade/-Trp/-Leu) containing X-α-Gal and Aba. The colonies that can still grow well on the four-deficiency medium are considered to have activated the expression of -His, -Ade genes, and these colonies are considered to be the positive results obtained by the preliminary screening. The positive results of screening on the four-deficient medium are shown in Figures 1 and 2. In Figure 1, the pGBKT7 empty vector and pGBKT7-E2 plasmid were transformed into yeast respectively; in Figure 2, the AD of the pGBKT7-E2 and pGBKT7 empty plasmid and the positive protein were respectively Co-transfer into yeast strains, pGBKT7-LAM and pGBKT7-P53 were co-transfected with pGADT7-T into yeast as negative and positive controls, respectively, pGBKT7 empty vector and pGBKT7-E2 plasmids can grow on SD/-Trp plates, It shows that the vectors have been transferred into the bacteria, and the pGBKT7-E2 plasmid cannot grow on the SD/-Trp/X-α-Gal plate, which shows that neither the pGBKT7 empty vector nor the pGBKT7-E2 plasmid has self-activation.

The experimental results in Figure 2 show that the two fusion protein vectors of AD-P10+BD-E2 can grow on both double-deficient medium and four-deficient medium after co-transformation into yeast, and when X-α-Gal and Aba are added It can still grow on the four-deficiency medium and the plaques turn blue. At the same time, co-transfection experiments with pGBKT7 empty plasmid and AD-P10 show that the protein we screened has no self-activation.

Yeast two-hybrid results show that HIV P10 protein and hepatitis G virus E2 protein interact, and bind together through this interaction, thereby causing some subsequent effects; the above results show that P10 protein and hepatitis G virus E2 protein interact effect.

(4) Sequencing analysis of insert fragments of positive clones

Extract the plasmids of all positive cloned yeast strains, transform Escherichia coli JM109 competent cells by electric shock method, and spread the transformed bacteria solution on the LB+Amp plate to screen the AD plasmids of the library. Since the plasmid expressing the bait protein is kana-resistant, and the plasmid expressing the prey protein is Amp-resistant, the bait plasmid can be screened out, and only the prey plasmid is kept; pick at least 10 single clones on each plate, and colony PCR detection Plasmids were extracted after deduplication, sequenced and sequenced.

(5) Verification of positive clone results

The positive proteins obtained from the above sequence analysis were co-transformed with the hepatitis G virus E2 protein into the Y2H Gold yeast strain, and yeast two-hybrid was performed to confirm their interaction. Finally, the HIV protein interacting with the hepatitis G virus E2 protein was obtained through screening: the HIV P10 protein is a sequence starting from the overlapping region of the pol and gal genes, and is a protein encoded by the PR gene. It is an aspartic acid protease that acts as a dimer and functions to cleave Gag protein and Gag-pol precursor protein during virus maturation.

(6) Further verification of positive interacting proteins by co-immunoprecipitation experiments

HEK293 cells were cultured in high-glucose DMEM medium with 10% fetal bovine serum at 37°C in a 5% carbon dioxide incubator. The cells were seeded in a 10cm culture dish and could be used for transfection after 18-24h when they grew to 1×10 ⁶ . The obtained positive protein and hepatitis G virus E2 protein were respectively constructed on p-HA and pflag to form fusion proteins with different tag tags, and then co-transfected human embryos with X-tremeGENE HP DNA Transfection Reagent liposome transfection method Kidney cells HEK239, after 48-72h, lyse the cells with 1mL lysate, lyse the cells at 4°C or in an ice bath for 30min, centrifuge at 14000rpm for 10min, and use the supernatant for co-immunoprecipitation and western blot analysis.

Co-immunoprecipitation: Incubate the supernatant protein solution collected in the previous step with ANTI-FLAG M2 Affinity Gel at 4°C overnight, centrifuge at 5000g for 30sec to recover the column, wash with cleaning solution three times, add 0.1 M glycine HCl, pH 3.5, shake at room temperature for 5min, 5000g Centrifuge for 30 sec to take the supernatant, add 10 μL 0.5 M Tris-HCl (pH 7.4, add 1.5 M NaCl) to obtain a co-precipitated protein sample.

Western blot detection: The protein sample obtained in the previous step was subjected to polyacrylamide gel electrophoresis (gel concentration 12%), transferred to the membrane, blocked (5% calf serum), and rabbits with anti-FlAG and anti-HA for western blotting The source antibody was incubated overnight at 4°C. After washing with TBST, it was incubated with the secondary antibody (rabbit-derived antibody) for 1 hour at room temperature and developed for detection. The results showed that HIV P10 protein had a significant interaction with E2 protein (Figure 3). In Figure 3, Input is the whole cell lysate control group, and the other two groups are the HIV P10 protein and E2 protein experimental groups; when detecting IP results, use E2 fusion label FlAG to incubate with FlAG Beeds, and after elution, western blot, Western Using P10 protein fusion tag HA to develop, the results show that E2 can immunoprecipitate P10 protein, and the two have an interaction.

(7) Further verification of positive interacting proteins through laser confocal experiments

A. Take several 18mm×18mm coverslips, clean them with 75% ethanol, put them in gauze, and sterilize at 121°C for 30 minutes.

B. Use tweezers to pick up clean slides and place them in a six-well plate, add rat tail collagen dropwise on the slides until the slides are just covered by the liquid, coat for 30 minutes, discard the coating solution, dry naturally and use Rinse with PBS, spread about 3×105 Hun7.5.1 cells in each well, and transfect when the cell adhesion rate reaches 60%-70%.

C. Transfect 2ug of the plasmid (co-transfected with Flag-tagged and Myc-tagged plasmids, and set up the control of transfection of Flag alone and Myc-tagged plasmid alone) into Huh7.5.1 cells.

D. Discard the medium after 48 hours, and wash the cells 3 times with pre-cooled PBS, 5 minutes each time. Add 1 mL of cell fixation solution (70% methanol + 30% acetone) to each well and fix for 5 min.

E. Discard the fixative, wash the cells 3 times with PBS, and place for 5 minutes each time. Add 800uL of 10% FBS to each well and block at 37°C for 2h. Repeat step 6.

F. Mark the primary antibody, dilute the primary antibody with blocking solution according to the instruction manual, wash the liquid around the slide, drop the antibody directly onto the slide, add 50-80 μL to each slide; Flag is rabbit antibody, Myc is For mouse antibody, dilute after the antibody is mixed; you can choose to label at 37°C for 2 hours, or choose to label at 4°C overnight; repeat step 6.

G. Label the fluorescent secondary antibody, choose the fluorescent secondary antibody from the corresponding source (Rhodamine for rabbit source, FITC for mouse source), label at 37°C for 2 hours, and operate in the dark. Repeat step 6.

H. Label DAPI, dilute DAPI with PBS according to the instructions, the labeling method is the same as step 9, after the addition, use tinfoil to protect from light, and label at room temperature for 20 minutes. Repeat step 6.

I. Take a piece of clean glass slide, drop a little PBS in the center, carefully pick up the cover glass with tweezers, put the cell side down, and slowly put it down from one side to the other to avoid air bubbles, and immediately Put it in a dark box and place it roughly off.

J. Observed and photographed under a confocal microscope. P10 has red light, E2 has green light, and the nucleus is blue light. Colocalization of P10 with E2 was detected using confocal microscopy. The results showed that P10 and E2 could be expressed in the cytoplasm, and the co-localized fluorescence superimposed was orange-yellow (Figure 4). The results showed that the interaction region of the two proteins was in the cytoplasm.

Finally, the protein interacting with hepatitis G virus E2 protein was obtained through screening: HIV P10 protein, which is a protease. P10 is a sequence starting from the overlapping region of the pol and gal genes, and is a protein encoded by the PR gene. It is an aspartic acid protease that acts as a dimer and functions to cleave Gag protein and Gag-pol precursor protein during virus maturation. This can lay the foundation for the follow-up research on the mechanism of interaction between hepatitis G virus and AIDS.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications and improvements can be made on the basis of the present invention. Therefore, the modifications and improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

sequence listing

<110> Kunming University of Science and Technology

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gaggcaggaa ccgaaagaca gggaagcctt aactgccctc agatcactct ttggcagcga 180

ccccttgtct caataaaagt aggaggccag ataaaagagg ctctcttaga tacaggagca 240

gatgatacag tattagaaga aatagatttg ccaggaaaat ggaaaccaaa aatgatagga 300

ggaattggag gttttatcaa agtaagacaa tatgaacaaa tatctataga aatttgtgga 360

aaaaaggcta taggtacagt attagtggga cccacacctg tcaacataat tggaagaaat 420

atgttgaccc agcttggatg cacactaaat tttccaatca gtccc 465

<210> 3

<211> 31

<212>DNA

<213> Artificial sequence (Artificial)

<400> 3

ccggaattcg gcgccccggc ctcggtgcta g 31

<210> 4

<211> 35

<212>DNA

<213> Artificial sequence (Artificial)

<400> 4

acgcgtcgac cctgcccgag gagagccatg cgaac 35

Claims (3)

1. the HIV P10 albumen with HGV RNA E2 protein-interactings, the amino acid sequence of the HIV P10 albumen is such as SEQ ID NO：Shown in 1.

2. encoding the gene of HIV P10 albumen described in claim 1.

3. gene according to claim 2, it is characterised in that：Its nucleotide sequence SEQ ID NO：Shown in 2.