CN111117970B - Monoclonal antibody for recognizing N6 subtype avian influenza virus neuraminidase protein and application thereof - Google Patents

Abstract

The invention discloses a monoclonal antibody for recognizing the neuraminidase protein of N6 subtype avian influenza virus and its application. The monoclonal antibody is secreted and produced by the hybridoma cell line 3H7 with the deposit number of CGMCC No. 18900. Experiments show that the monoclonal antibody 3H7 obtained by the present invention can specifically bind to N6 subtype avian influenza virus, and can bind to 15 strains of N6 subtype avian influenza viruses of different evolution branches, indicating that 3H7 has a good broad spectrum. The invention utilizes the better broad-spectrum and specificity of the monoclonal antibody to establish an ELISA detection kit and a detection method for N6 subtype avian influenza virus neuraminidase protein antibody blocking. Experiments show that the method has high sensitivity and good repeatability, and can be used to detect the antibody level of N6 subtype avian influenza virus neuraminidase protein in clinical serum samples, and has a good development prospect.

Description

A monoclonal antibody that recognizes the neuraminidase protein of N6 subtype avian influenza virus and its application

technical field

The present invention relates to a hybridoma cell line capable of secreting a monoclonal antibody against the neuraminidase protein of N6 subtype avian influenza virus, as well as the monoclonal antibody secreted by the hybridoma cell line and its application. The present invention belongs to the field of biotechnology.

Background technique

Avian influenza (AI) is an acute infectious disease of poultry caused by influenza A virus of the family Orthomyxoviridae, and is classified as a class A severe infectious disease by the World Organization for Animal Health. Avian influenza virus (AIV) is a single-stranded negative-stranded segmented RNA virus with numerous subtypes, which can be divided into 16 types according to the antigenic differences of its surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). In addition, H17N10 and H18N11 influenza viruses were isolated from bats.

NA is a type II glycoprotein encoded by fragment 6. The NA protein forms a heterotetramer in structure. Each NA protein monomer is composed of a short amino-terminal cytoplasmic region and a hydrophobic transmembrane region. , the stem region and the bulbous head region. The NA protein has neuraminidase activity. In the late stage of virus infection, it can cleave sialic acid on the surface of host cells and the surface of newly formed virus particles, thereby promoting the release and diffusion of newly formed virus particles and preventing the accumulation of new virus particles on the cell surface.

N6 is one of the main NA subtypes of avian influenza viruses currently circulating in China, among which the combination of H5N6 and H6N6 subtypes is the most common. The H5N6 subtype avian influenza virus is not only harmful to the poultry industry, but can also infect humans across the interspecific barrier. As of August 8, 2019, 22 cases of human infection with the H5N6 subtype avian influenza virus have been reported. In addition, there are also reports of H6N6 subtype avian influenza virus crossing the interspecies barrier to infect pigs. Therefore, strengthening the surveillance of N6 subtype avian influenza virus has important public health significance. At present, the NA subtype detection method recommended by WHO is the neuraminidase inhibition test, but this method has a long experimental period and the experimental reagent sodium arsenite is a highly toxic reagent, so it is difficult to popularize and use this method in clinical practice.

SUMMARY OF THE INVENTION

One of the objects of the present invention is to provide a hybridoma cell line capable of stably secreting a monoclonal antibody against the neuraminidase protein of N6 subtype avian influenza virus.

The second object of the present invention is to provide a monoclonal antibody with broad-spectrum anti-N6 subtype avian influenza virus neuraminidase protein secreted by the hybridoma cell line.

The third object of the present invention is to apply the above-mentioned monoclonal antibody to the diagnosis and treatment of N6 subtype avian influenza.

The present invention achieves the above object and is achieved through the following technical solutions:

The present invention first selects 3 strains of N6 subtype avian influenza viruses located in different evolutionary branches, immunizes 6-week-old BALB/c female mice with the inactivated viruses at intervals of 14 days, and fuses the spleen of the immunized mice with SP2/0 cells . By indirect immunofluorescence method, a hybridoma cell line that can stably secrete monoclonal antibody with broad-spectrum anti-N6 subtype avian influenza virus neuraminidase protein was screened, named 3H7, and classified as hybridoma cell. The strain is deposited in the General Microbiology Center of the China Microorganism Culture Collection Management Committee, and its address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The preservation number is CGMCC No. 18900, and the preservation time is December 4, 2019. day.

Further, the present invention also provides a monoclonal antibody 3H7 which is secreted by the above hybridoma cell line and has broad-spectrum anti-N6 subtype avian influenza virus neuraminidase protein. The heavy chain subclass of the antibody was identified as IgG2b, and the light chain subclass was κ chain. Take the monoclonal antibody 3H7 and N6 subtype avian influenza virus for Western Blot detection, the results show that 3H7 can specifically bind to the neuraminidase protein of the denatured N6 subtype avian influenza virus, indicating that 3H7 recognizes the linear expression of the N6 protein. bit. The ELISA titer of hybridoma 3H7 supernatant was 1:6400, and the ELISA titer of mouse ascites was 1:409600. 15 strains of N6 subtype AIV with different evolutionary branches were selected to infect DF-1 cells and tested by IFA. The results showed that 3H7 could bind to these 15 viruses, indicating that 3H7 has a good broad spectrum. The N1-N9 subtype AIV and Newcastle disease virus were used as the coating antigen to carry out indirect ELISA test. The results showed that 3H7 can only bind to N6 subtype AIV, indicating that 3H7 has good specificity.

Further, the present invention also proposes the use of the monoclonal antibody in preparing a reagent for detecting N6 subtype avian influenza virus. And the application of the monoclonal antibody in the preparation of medicine for treating N6 subtype avian influenza.

A pharmaceutical combination comprising the monoclonal antibody of the present invention.

Furthermore, the present invention establishes a blocking ELISA detection kit and a detection method for detecting N6 subtype avian influenza virus neuraminidase protein antibody by utilizing the better broad-spectrum and specificity of 3H7.

The described blocking ELISA detection kit for detecting N6 subtype avian influenza virus neuraminidase protein antibody contains the monoclonal antibody of the present invention.

Wherein, preferably, the kit also contains inactivated antigen A/chicken/Hunan/S3003/2009 (H6N6), ELISA plate, coating solution, PBST solution, HRP-labeled goat anti-mouse secondary antibody, TMB Chromogenic solution and stop solution.

When the kit is used to detect N6 subtype avian influenza virus antibodies, follow the steps below:

(1) Dilute the inactivated antigen A/chicken/Hunan/S3003/2009 (H6N6) with an HA titer of 2 ⁸ in a 1:80-fold dilution with the coating solution and coat the ELISA plate, 100 μL per well, overnight at 4°C ;

(2) Discard the supernatant, wash the ELISA plate with PBST solution, 200 μl per well, add 5% skim milk solution for blocking after washing 3 times, 200 μL per well, incubate at 37°C for 1 h;

(3) Discard the supernatant, wash the ELISA plate with PBST solution, 200 μl per well, add the serum to be tested, negative serum and positive serum after washing 3 times, 100 μL per well, incubate at 37°C for 1 h;

(4) Discard the supernatant, wash the ELISA plate with PBST solution, 200 μl per well, add 1:12800-fold diluted monoclonal antibody ascites 3H7 after washing 3 times, 100 μL per well, incubate at 37°C for 1 h;

(5) Discard the supernatant, wash the ELISA plate with PBST solution, 200 μl per well, add 1:3000-fold diluted HRP-labeled goat anti-mouse secondary antibody after washing 3 times, 100 μL per well, incubate at 37°C for 1 h;

(6) Discard the supernatant, wash the ELISA plate with PBST solution, 200 μl per well, wash 3 times, and add TMB color developing solution in the dark, 100 μL per well, develop color for 15 minutes, add 50 μL of 2M sulfuric acid to stop color development, And read the OD ₄₅₀ nm value on a microplate reader.

Experiments show that the method has high sensitivity and good repeatability, and can be used to detect the antibody level of N6 subtype avian influenza virus neuraminidase protein in clinical serum samples, and has a good development prospect.

Description of drawings

Fig. 1 is the indirect immunofluorescence detection of N6 subtype avian influenza virus neuraminidase protein monoclonal antibody 3H7;

A: 3H7; B: negative control

Fig. 2 is the Western Blot detection of N6 subtype avian influenza virus neuraminidase protein monoclonal antibody 3H7;

1:3H7:2: negative control

Fig. 3 is the broad-spectrum detection of N6 subtype avian influenza virus neuraminidase protein monoclonal antibody 3H7;

A:DK/HuB/SP143/2014(H5N6);B:DK/HuN/SE226/2017(H5N6);

C:CK/HuN/S3003/2009(H6N6); D:DK/HuN/S4270/2010(H6N6);

E:DK/GX/S4111/2010(H6N6);F:DK/HuN/S1351/2009(H6N6);

G:DK/ZJ/S4354/2016(H11N6);H:DK/GD/S4180/2017(H6N6);

I: CK/GD/S1031/2018(H6N6); J: DK/JX/S40199/2017(H6N6);

K:DK/JX/S1129/2018(H6N6); L:DK/JX/S1200/2018(H6N6);

M:DK/GX/S40437/2017(H6N6); N:DK/GX/S31293/2017(H6N6);

O: CK/HuB/S1246/2018 (H4N6); P: negative control.

Detailed ways

In order to explain the present invention more clearly, the present invention will be described in detail below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

Example 1 Preparation and identification of monoclonal antibody against neuraminidase protein of N6 subtype avian influenza virus

1.1 Preparation of immunogens and animal immunization

Three strains of avian influenza viruses of N6 subtype in different evolutionary branches (CK/HuN/S3003/2009(H6N6), referred to as HuN3003; DK/HuB/SP143/2014(H5N6), referred to as HuB143; DK/HuN/SE226/2017 (H5N6), referred to as HuN226) as an immunogen, the amino acid homology between HuN226 and HuN3003 is 95%, the amino acid homology between HuN3003 and HuB143 is 94.1%, and the amino acid homology between HuN226 and HuB143 is 91.7%. First, the viruses HuN226, HuN3003 and HuB143 were diluted 10-fold, inoculated with 9-11-day-old SPF chicken embryos, placed in a 37°C incubator for 48 hours, collected allantoic fluid, and added β- Propiolactone is used to inactivate the virus. After the chicken embryo is inoculated, it is tested whether the virus inactivation is sufficient, and after the sterility test, it is stored at -70 °C for future use.

Eight 6-week-old female BALB/c mice were selected and immunized with inactivated HuN226, HuN3003 and HuB143 in sequence. Three days before fusion, HuN3003 was used for booster immunization. The specific immunization program is as follows (Table 1):

Table 1 The immunization procedure for preparing the monoclonal antibody against neuraminidase protein of N6 subtype avian influenza virus

1.2 Preparation of hybridoma cell lines

The mouse spleen was collected aseptically, and the spleen was triturated with the plunger of a 2.5 mL syringe to disperse the splenocytes evenly, and then transferred to a sterile 50 mL centrifuge tube. The SP2/0 cells were blown down with serum-free DMEM and placed in a centrifuge tube containing splenocytes, mixed gently, centrifuged at 800 rpm for 10 min, and the supernatant was discarded. The centrifuge tube was then placed in a 37°C water bath, and 1 mL of pre-warmed PEG was slowly added dropwise within 45 s while the centrifuge tube was shaken to make it evenly mixed. After standing for 1 min, slowly add serum-free DMEM to 40 mL, centrifuge at 800 rpm for 10 min, and discard the supernatant. The confluent cells were resuspended in DMEM containing 1% HAT and 20% FBS pre-warmed at 37°C into 96-well plates, 100 μL per well, and then placed in a 37°C cell incubator with 5% CO ₂ . cultivated in.

1.3 Screening of positive hybridoma cells

When the hybridoma cells in the cell wells grow to about half, aspirate the cell supernatant for IFA detection to determine the positive wells. The day before screening, 293T cells were transfected with eukaryotic expression plasmid pCAGGS-N6 (HuN3003). After 24 hours of culture, the cell supernatant was discarded, washed three times with PBST solution, and then formalin fixative was added, 100 μL per well, and fixed at room temperature. 30min. After fixation, wash three times with PBST solution, then add 5% skim milk solution for blocking, 200 μL per well, block at 37°C for 2h or 4°C overnight, and wash three times as above after blocking. Pipette 100 μL of the supernatant of the fused hybridoma cells into the 293T cell plate, and the mouse negative and positive serum diluted 1:400 was used as the negative and positive control, incubated at 37°C for 1 h, and washed three times as above. Then, 100 μL of a 1:1000-fold diluted FITC-labeled goat anti-mouse fluorescent secondary antibody was added to each well, incubated at 37° C. for 1 h, and washed three times as above. The process was performed in a dark environment. Then, the results were observed by fluorescence microscope, and finally a hybridoma cell that could recognize N6 protein was screened and named as 3H7. The cell line is deposited in the General Microbiology Center of the China Microorganism Culture Collection Management Committee, and its address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. The preservation number is CGMCC No. 18900, and the preservation time is December 2019. 4th of May.

1.4 Subcloning of positive hybridoma cells 3H7

First, the cells in the 3H7 well of the positive hybridoma cells were gently pipetted and mixed, and the cells were counted. The cells were diluted with DMEM containing 20% FBS to contain about 100 cells in 10 mL, and then added to a 96-well plate after mixing. 100 μL per well, and then each well was supplemented with 100 μL of DMEM medium containing 20% FBS, and cultured in a 37°C cell incubator containing 5% CO ₂ . The cells were observed every day until cell colonies appeared, and the single-cell wells were marked, and the positive hybridoma cells were screened when the cells had grown to about half. This subcloning was repeated 3 times.

1.5 Preparation of monoclonal antibody 3H7

The mice were intraperitoneally injected with incomplete Freund's adjuvant, each with 0.5 mL. 3d after injection, the hybridoma cells with good cell condition 3H7 were blown down, centrifuged at 800 rpm for 10 min, the supernatant was discarded, the cells were resuspended with sterile PBS, centrifuged again, and the supernatant was discarded. Add sterile PBS to resuspend the cells and perform a cell count, diluting the cells to contain 5 x 10 ⁵ cells per 500 μL. The cell dilutions were mixed evenly, and then injected into the abdominal cavity of mice, 500 μL per injection. Within 7-10 days after cell injection, the state of the mice was observed every day. When the abdomen of the mice was swollen, the ascites fluid of the mice was drawn with a syringe, and after overnight at 4°C, centrifuged at 4500 rpm for 10 minutes, and the supernatant of the ascites was collected and placed in a -70°C refrigerator. spare.

1.6 Subclass identification of monoclonal antibody 3H7

The 3H7 hybridoma cells were cultured with serum-free medium, and the cell supernatant was collected, and the 3H7 subclass was identified according to the instructions of the antibody subclass identification kit. The heavy chain subclass was determined to be IgG2b, and the light chain subclass was kappa chain.

1.7 Titer determination of monoclonal antibody 3H7

The N6 subtype AIV was formulated into 8 units of antigen and coated on an ELISA plate, overnight at 4°C. After washing three times with PBST, 200 μL of 5% skim milk solution was added for blocking, and incubated at 37°C for 1 h. The 3H7 hybridoma cell supernatant or ascites was serially diluted as the primary antibody, and incubated at 37°C for 1 h. After washing, add 1:3000-fold diluted HRP-labeled goat anti-mouse secondary antibody, 100 μL per well, and incubate at 37°C for 1 h. After washing, add TMB chromogenic solution for color development in the dark, 100 μL per well, add 50 μL of 2M H ₂ SO ₄ to each well after 15 min of color development to stop color development, and read the OD ₄₅₀ nm value on a microplate reader. Finally, the ELISA titer of the hybridoma supernatant was determined to be 1:6400, and the ELISA titer of ascites was 1:409600.

1.8 IFA detection of monoclonal antibody 3H7

The supernatant of 3H7 cells was detected by IFA using the method in 1.3, and the plasmid used for transfection was the constructed pCAGGS-NA (HuN226) eukaryotic expression recombinant plasmid. The primary antibody was cell supernatant, and the secondary antibody was FITC-labeled goat anti-mouse fluorescent secondary antibody diluted 1:1000 times, and the results were recorded under a fluorescence microscope. It was found that 3H7 could react with the N6 protein of HuN226 to emit green fluorescence (Figure 1).

1.9 Western Blot detection of monoclonal antibody 3H7

The whole virus HuN3003 was mixed with 5×SDS-PAGE loading buffer, heated and denatured, electrophoresed, and transferred to membrane. After transfer, the membrane was blocked with 5% skim milk solution and incubated at 37°C for 2h. Wash with PBST solution, 5min each time. The 3H7 cell supernatant was used as the primary antibody, placed on a shaker at 37 °C for 1 h, washed with the same method as above, and then added with a 1:1000-fold dilution of infrared-labeled anti-mouse secondary antibody, and incubated at 37 °C for 1 h in the dark. After washing the membrane in the same way, place it on an infrared scanner to scan the results. The results showed that 3H7 could react specifically with the N6 protein of HuN3003 (Figure 2).

1.10 Specific detection of monoclonal antibody 3H7

N1-N9 subtype AIV and Newcastle disease virus were selected as antigens, and these viruses were prepared into an 8-unit antigen-coated ELISA plate, overnight at 4°C. After washing three times with PBST, 200 μL of 5% skim milk solution was added for blocking, and incubated at 37°C for 1 h. The 3H7 cell supernatant was used as the primary antibody and incubated at 37°C for 1 h. After washing, add 1:3000-fold diluted HRP-labeled goat anti-mouse secondary antibody, 100 μL per well, and incubate at 37°C for 1 h. After washing, add TMB chromogenic solution for color development in the dark, 100 μL per well, add 50 μL of 2M H ₂ SO ₄ to each well after 15 min of color development to stop color development, and read the OD ₄₅₀ nm value on a microplate reader. The results showed that 3H7 can only bind to N6 subtype AIV (Table 2), indicating that 3H7 has good specificity.

Table 2 Specificity detection of N6 subtype avian influenza virus neuraminidase protein monoclonal antibody 3H7

1.11 Broad-spectrum detection of monoclonal antibody 3H7

First, DF-1 cells were cultured in a 96-well plate. When the cells grew to about 80%, 15 strains of N6 subtype avian influenza viruses in different evolutionary branches were infected with DF-1 cells. After culturing in a 37°C cell incubator for 48 hours Remove and discard the supernatant and wash 3 times with PBST. 100 μL of formalin fixative solution was added to each well, and the supernatant was discarded after fixation at room temperature for 30 min, and washed three times with PBST. The primary antibody was the culture supernatant of hybridoma cell 3H7, 100 μL per well, incubated at 37°C for 1 h, and washed three times with PBST. The secondary antibody was FITC-labeled goat anti-mouse fluorescent secondary antibody diluted 1:1000 times, 100 μL per well, and the process was performed in the dark. After incubation at 37°C for 1 h, washed three times with PBST and recorded the results under a fluorescence microscope. The cells infected with these 15 strains of viruses were observed to emit green fluorescence under a fluorescence microscope (Fig. 3), indicating that 3H7 has a good broad spectrum.

Example 2 Establishment of ELISA detection method for N6 subtype avian influenza virus neuraminidase protein antibody blocking

2.1 N6 subtype avian influenza virus neuraminidase protein antibody blocking ELISA operation method

(1) Dilute the inactivated antigen A/chicken/Hunan/S3003/2009 (H6N6) with an HA titer of ²⁸ with a coating solution (50mmol/L carbonate buffer (pH 9.6)) 1:80 times After coating the microtiter plate, 100 μL per well, overnight at 4°C.

(2) Discard the supernatant, wash the ELISA plate with PBST solution (50 mmol/L pH 7.2 phosphate buffer containing 0.05% Tween-20), 200 μl per well, wash 3 times and add 5% w/w The PBST solution of skim milk was used for blocking, 200 μL per well, and incubated at 37°C for 1 h.

(3) Discard the supernatant, wash the ELISA plate with PBST solution, 200 μl per well, add the serum to be tested, negative serum and positive serum after washing 3 times, 100 μL per well, incubate at 37°C for 1 h.

(4) Discard the supernatant, wash the ELISA plate with PBST solution, 200 μl per well, add 1:12800-fold diluted monoclonal antibody ascites 3H7 after washing 3 times, 100 μL per well, incubate at 37°C for 1 h.

(5) Discard the supernatant, wash the ELISA plate with PBST solution, 200 μl per well, add 1:3000-fold diluted HRP-labeled goat anti-mouse secondary antibody after washing 3 times, 100 μL per well, incubate at 37°C for 1 h.

(6) Discard the supernatant, wash the ELISA plate with PBST solution, 200 μl per well, wash 3 times, and add TMB color developing solution in the dark, 100 μL per well, develop color for 15 minutes, add 50 μL of 2M sulfuric acid to stop color development, And read the OD ₄₅₀ nm value on a microplate reader.

2.2 Determination of critical value

Select 60 negative sera to do 1:160 dilution, and use SPF chicken serum as negative control, use the established blocking ELISA method to measure the inhibition rate of these 60 sera (Table 3), and obtain an average inhibition rate of 9.04% , with a standard deviation of 4.36%. According to the critical value criterion, the positive critical value was 22.12%, and the negative critical value was 17.76%. If it is in the middle of the two, it is judged as suspected, and if it is still less than the positive threshold after re-measurement, it is judged as negative.

Table 3 Determination of critical value of N6 subtype avian influenza virus neuraminidase protein antibody blocking ELISA detection method

2.3 Specific detection of blocking ELISA method

The N1-N9 subtype AIV serum and the Newcastle disease virus serum were selected for blocking ELISA detection, and the SPF chicken serum was used as a negative control. 4), indicating that this method has good specificity.

Table 4 Specificity detection of N6 subtype avian influenza virus neuraminidase protein antibody blocking ELISA detection method

2.4 Repeated detection of blocking ELISA method

Five serum samples were selected and repeated 4 times on one ELISA plate and 4 ELISA plates respectively, and the average inhibition rate X and standard deviation SD were obtained, and the coefficient of variation CV(%)=SD×100%/X. The calculated intra-plate variation coefficient was between 1.14% and 6.57%; the inter-plate variation coefficient was between 2.22% and 4.98% (Table 5), all of which were less than 10%, indicating that the method had good repeatability.

Table 5 Repeated detection of N6 subtype avian influenza virus neuraminidase protein antibody blocking ELISA detection method

2.5 Sensitivity detection of blocking ELISA method

30 positive sera with clear background were selected for detection by blocking ELISA, and the inhibition rate was calculated to be between 27.83% and 75.31% (Table 6), and the coincidence rate was 100%, indicating that this method has good sensitivity.

Table 6 Sensitivity detection of N6 subtype avian influenza virus neuraminidase protein antibody blocking ELISA detection method

The above results show that the blocking ELISA method established in the present invention has good specificity, repeatability and sensitivity, and can be used to detect the antibody level of N6 subtype avian influenza virus neuraminidase protein in clinical serum samples.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the examples, and any other changes, modifications, combinations, substitutions, and simplifications made without departing from the spirit and principle of the present invention All should be equivalent alternatives, and all are included within the protection scope of the present invention.

Claims (6)

1. A monoclonal antibody hybridoma cell strain capable of stably secreting N6 subtype avian influenza virus resistant neuraminidase protein is named as 3H7, the cell strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the microbial research institute of China academy of sciences No. 3, West Lu No.1, North Chen, Xilu, in the area of the morning of Beijing, the preservation number is CGMCC No.18900, and the preservation time is 12 months and 4 days in 2019.

2. A monoclonal antibody secreted by the hybridoma cell line of claim 1 and having broad-spectrum resistance to N6 subtype avian influenza virus neuraminidase protein.

3. Use of the monoclonal antibody of claim 2 in the preparation of a reagent for detecting avian influenza virus subtype N6.

4. A blocking ELISA detection kit for detecting N6 subtype avian influenza virus neuraminidase protein antibody, characterized in that the kit contains the monoclonal antibody of claim 2.

5. The ELISA detection kit of claim 4 further comprising inactivated antigen A/chicken/Hunan/S3003/2009(H6N6), an ELISA plate, a coating solution, a PBST solution, a goat anti-mouse secondary antibody labeled with HRP, a TMB color developing solution and a stop solution.

6. A pharmaceutical combination comprising the monoclonal antibody of claim 2.

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