Method for detecting activity of HPV (human papillomavirus) trivalent therapeutic vaccine

Abstract

The invention relates to the technical field of vaccines, in particular to an activity detection method of an HPV (human papilloma virus) trivalent therapeutic vaccine. The method comprises the following steps: inoculating and infecting BEAS-2B cells by adopting constructed HPV16 / 18 / 52 type E6 / E7 antigen-loaded non-replicating type 5 adenovirus, culturing, extracting total protein, sequentially performing SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis), western blot detection, immunoreaction and developing imaging, and detecting the fusion protein; the transfection complex number MOI of the constructed adenovirus is 15-25, and the convergence degree of BEAS-2B cells is greater than 80%; the membrane transfer current of the western blot method is 220-280 mA, and the time is 50-80 minutes. The method avoids the problem of inaccurate detection results caused by obvious difference of biological activities in different animal species, has good accuracy, stability and durability, is simple in steps and low in cost, and can be effectively popularized and used.

Description

Technical Field

[0001] The present invention relates to the field of vaccine technology, and in particular to a method for detecting the activity of a HPV trivalent therapeutic vaccine. Background Art

[0002] At present, the HPV trivalent therapeutic vaccine with genetically modified human adenovirus type 5 as a carrier has gradually been put into clinical use. The design principle of this vaccine is to infect host cells by intramuscular injection into the human body. The host cells express HPV16 / 18 / 52 type E7 / E6 fusion protein, which is cut into small antigenic peptide fragments. The antigenic peptides are presented to T cells through two different pathways via antigen presenting cells (APC), specifically killing cervical epithelial cells infected with the virus, blocking the progression of the disease, and achieving the purpose of treatment.

[0003] In therapeutic vaccines, antigen presentation depends on MHC molecules. Different MHC typing will lead to obvious differences in antigen peptide binding sequences. Laboratory mice are all inbred mice, and the H-2 locus is homozygous. All offspring express the same MHC haplotype. In order to detect the biological activity of the six antigens in the fusion protein of HPV trivalent therapeutic vaccine (such as KDTV001), the current method is to test in multiple animal species including C57BL / 6J mice, babl / c mice, golden hamsters and SD rats. However, after many studies, it was found that the biological activity of KDTV001 in different animal species is significantly different.

[0004] Biological activity is a key quality control item for the KDTV001 product, and the robustness of its detection method should be ensured. Due to the limitation of a single animal species, it is impossible to fully verify the biological activity of the six antigens in the fusion protein of the KDTV001 product, and the large batch-to-batch variability in animal experiments, it is difficult to effectively and stably evaluate the activity of the HPV trivalent therapeutic vaccine. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for detecting the activity of a trivalent HPV therapeutic vaccine.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] The present invention provides a method for detecting HPV16 / 18 / 52 type E7 / E6 fusion protein in a host cell, wherein the host cell is a BEAS-2B cell transfected with a constructed adenovirus, and the detection method comprises the steps of extracting total protein in the host cell, SDS-PAGE electrophoresis, Western blotting detection, immune response and color imaging;

[0008] The constructed adenovirus is an adenovirus carrying HPV16 / 18 / 52 type E6 / E7 antigens, the transfection multiplicity MOI of the constructed adenovirus is 15-25, and before transfection, the confluence of the BEAS-2B cells is greater than 80%; in the protein blotting method, the transmembrane current is 220-280mA, and the time is 50-80min.

[0009] Based on the above technical solution, the present invention can also be improved as follows.

[0010] Furthermore, the transfection multiplicity MOI of the constructed adenovirus was 25, and before transfection, the confluence of the BEAS-2B cells was 90%; in the protein blotting method, the transfer current was a constant current of 220 mA, and the time was 80 min.

[0011] Furthermore, before transfection, the secondary counted cell number of the BEAS-2B cells was 1.21×10 6 cells / well.

[0012] Furthermore, in the SDS-PAGE electrophoresis, the loading amount in each loading well is greater than or equal to 30 μg.

[0013] Furthermore, in the immune reaction, a diluted primary antibody solution and a diluted secondary antibody solution are sequentially used for incubation;

[0014] The primary antibody solution is one of an HPV 16E7 antibody solution, an HPV 18E7 antibody solution, an HPV 16E6 antibody solution, an HPV 18E6 antibody solution, and an HPV 52E7 antibody solution, wherein the dilution ratio of the HPV 16E7 antibody solution is 1:50, the dilution ratio of the HPV 18E7 antibody solution is 1:1000, the dilution ratio of the HPV 16E6 antibody solution is 1:250, the dilution ratio of the HPV 18E6 antibody solution is 1:250, and the dilution ratio of the HPV 52E7 antibody solution is 1:100;

[0015] The secondary antibody solution is goat anti-mouse IgG H&L antibody solution or goat anti-rabbit IgG H&L antibody solution, and the dilution ratio of the secondary antibody solution is 1:2000.

[0016] Furthermore, the primary antibody solution is an HPV18E7 antibody solution, and the secondary antibody solution is a goat anti-mouse IgG H&L antibody solution.

[0017] Furthermore, the amino acid sequence of the fusion protein is shown in SEQ ID NO.1.

[0018] Furthermore, the constructed adenovirus vector is a non-replicating type 5 adenovirus.

[0019] Further, when a specific band appears at the position of 100 KDa in the obtained electrophoretogram, the sample to be tested contains the fusion protein, and when a specific band does not appear at the position of 100 KDa in the obtained electrophoretogram, the sample to be tested does not contain the fusion protein.

[0020] The present invention also provides a method for detecting the activity of a trivalent HPV therapeutic vaccine, comprising the step of detecting HPV16 / 18 / 52 E7 / E6 fusion protein; the step of detecting the HPV16 / 18 / 52 E7 / E6 fusion protein is to infect host cells with an HPV trivalent therapeutic vaccine containing a constructed non-replicating type 5 adenovirus carrying HPV16 / 18 / 52 E6 / E7 antigens, and then detecting the HPV16 / 18 / 52 E7 / E6 fusion protein in the host cells using the method as described above, and determining the activity of the trivalent HPV therapeutic vaccine based on the detection results.

[0021] The beneficial effects of the present invention are:

[0022] (1) The detection method of HPV16 / 18 / 52 type E7 / E6 fusion protein of the present invention has good robustness and can accurately detect the content of HPV16 / 18 / 52 type E7 / E6 fusion protein, so that its detection structure can be used as an effective basis for judging the activity of the constructed virus;

[0023] (2) The detection method of HPV16 / 18 / 52 type E7 / E6 fusion protein of the present invention has suitable membrane transfer conditions, which avoids the problem of low accuracy caused by interference in the detection results;

[0024] (3) The detection method of HPV16 / 18 / 52 type E7 / E6 fusion protein of the present invention is based on normal human cell lines, making the detection method more practical;

[0025] (4) The HPV trivalent therapeutic vaccine activity detection method of the present invention uses the detection results of HPV16 / 18 / 52 type E7 / E6 fusion protein as an indicator for evaluating vaccine activity, thereby avoiding the problem of inaccurate detection results caused by obvious differences in biological activity in different animal species;

[0026] (5) The HPV trivalent therapeutic vaccine activity detection method of the present invention has good accuracy, stability and durability, and has simple steps and low cost, and can be effectively promoted and used. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1The detection method of HPV16 / 18 / 52 type E7 / E6 fusion protein of the present invention, the electrophoresis diagram of Example 1, Figure 1 The primary antibody used in a is 18E7 antibody. Figure 1 The primary antibody used in b is 52E7 antibody. Figure 1 The primary antibody used in c is 16E6 antibody. Figure 1 The primary antibody used in d is 16E7 antibody. Figure 1 The primary antibody used in e is 52E6 antibody. Figure 1 The primary antibody used in f is 18E6 antibody;

[0028] Figure 2 The electrophoresis diagram of Example 2 is a method for detecting HPV16 / 18 / 52 type E7 / E6 fusion protein of the present invention;

[0029] Figure 3 The detection method of HPV16 / 18 / 52 type E7 / E6 fusion protein of the present invention, the electrophoresis diagram of the MRC-5 cell line used in Example 3;

[0030] Figure 4 The detection method of HPV16 / 18 / 52 type E7 / E6 fusion protein of the present invention, the electrophoresis diagram of BEAS-2B cell line used in Example 3;

[0031] Figure 5 The electrophoresis diagram of Example 4 is a method for detecting HPV16 / 18 / 52 type E7 / E6 fusion protein of the present invention;

[0032] Figure 6 The electrophoresis diagram of Example 5 is the detection method of HPV16 / 18 / 52 type E7 / E6 fusion protein of the present invention;

[0033] Figure 7 The electrophoresis diagram of Example 6 is the detection method of HPV16 / 18 / 52 type E7 / E6 fusion protein of the present invention;

[0034] Figure 8 The detection method of HPV16 / 18 / 52 type E7 / E6 fusion protein of the present invention is the detection flow chart of Example 6;

[0035] Fig. 9 The electrophoresis diagram of Example 7 is the detection method of HPV16 / 18 / 52 type E7 / E6 fusion protein of the present invention;

[0036] Fig.10 This is the electrophoresis diagram of Example 8 of the detection method of HPV16 / 18 / 52 type E7 / E6 fusion protein of the present invention. DETAILED DESCRIPTION

[0037] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0038] The present invention discloses a method for detecting HPV16 / 18 / 52 type E7 / E6 fusion protein in a host cell. The host cell is a BEAS-2B cell transfected with a constructed non-replicating type 5 adenovirus carrying HPV16 / 18 / 52 type E6 / E7 antigen. The detection method comprises the steps of extracting total protein, SDS-PAGE electrophoresis, protein blotting detection, immune response and color imaging. The transfection multiplicity MOI of the constructed adenovirus is 15-25, and before transfection, the confluence of the BEAS-2B cells is greater than 80%. In the protein blotting method, the transmembrane current is 220-280 mA, and the time is 50-80 min.

[0039] The detection method of the present invention is based on the HPV16 / 18 / 52 type E7 / E6 fusion protein and is detected according to its molecular weight. In the detection method, the total protein of the infected cells is first extracted, the protein is separated by SDS-PAGE electrophoresis and transferred to a solid carrier PVDF membrane, the primary antibody is used to specifically recognize and bind to the target protein, and then reacted with a labeled secondary antibody, and the substrate is developed and imaged. Whether the target gene correctly expresses the fusion protein is determined by whether a specific band appears in the theoretical area, thereby detecting the biological activity of the product. The method has good robustness and can accurately detect the content of the HPV16 / 18 / 52 type E7 / E6 fusion protein, so that its detection structure can be used as an effective basis for judging the activity of the constructed virus.

[0040] The detection method of the present invention detects an amino acid sequence of the fusion protein as shown in SEQ ID NO. 1. The sequence of the fusion protein is analyzed by ProtParam, and its molecular weight is 87038.30Da.

[0041] The constructed virus vector is human adenovirus type V.

[0042] Preferably, the constructed non-replicating type 5 adenovirus carrying HPV16 / 18 / 52 type E6 / E7 antigens is KDTV001, which is a product with a non-replicating type 5 adenovirus as a vector. Its main feature is that it is prepared from a non-replicating type 5 adenovirus with E1 and E3 regions deleted. For the specific sequence of KDTV001, please refer to patent CN202410543158 "A HPV16_18_52 therapeutic vaccine, preparation method and application".

[0043] Experimental verification shows that the transfer conditions in the detection method of the present invention are appropriate. When the transfer time is too long or the transfer current is too high, a Prestained Protein Ladder band will appear in the electrophoresis diagram, interfering with the detection results.

[0044] Since the constructed adenovirus is generally used as a therapeutic vaccine in actual use, and the therapeutic vaccine is specifically injected intramuscularly, the normal cells of the body are the main antigen production. The detection method based on human normal cell lines is more practical, therefore, the method of the present invention uses BEAS-2B (human normal lung epithelial cells) as transfected cells.

[0045] Preferably, the total protein is quantified after extraction. The protein quantification step can determine whether the protein extraction is successful. At the same time, based on the quantitative results, the total sample loading amount of subsequent immune response steps can be controlled, thereby reflecting the difference in the amount of target protein expressed in different samples.

[0046] Preferably, in the immune reaction, a diluted primary antibody solution and a diluted secondary antibody solution are used sequentially for incubation; the primary antibody solution is one of an HPV 16E7 antibody solution, an HPV 18E7 antibody solution, an HPV 16E6 antibody solution, an HPV 18E6 antibody solution, and an HPV 52E7 antibody solution, the dilution ratio of the HPV 16E7 antibody solution is 1:50, the dilution ratio of the HPV 18E7 antibody solution is 1:1000, the dilution ratio of the HPV 16E6 antibody solution is 1:250, the dilution ratio of the HPV 18E6 antibody solution is 1:250, and the dilution ratio of the HPV 52E7 antibody solution is 1:100; the secondary antibody solution is a goat anti-mouse IgG H&L antibody solution or a goat anti-rabbit IgG H&L antibody solution, and the dilution ratio of the secondary antibody solution is 1:2000. It has been experimentally verified that when the concentration of the primary antibody is too high, it is difficult to detect the fusion protein.

[0047] More preferably, the primary antibody solution is an HPV18E7 antibody solution, and the secondary antibody solution is a goat anti-mouse IgG H&L antibody solution.

[0048] The detection method of the present invention comprises the following specific steps:

[0049] 1. Virus transfection: Culture cells first and then inoculate the virus. After inoculation, continue to culture the transfected cells.

[0050] Preferably, the cell culture before inoculation is carried out by plating, specifically: prepare BEAS-2B cells with a confluence of more than 80%, discard the culture medium therein, add 3 ml of trypsin to the culture flask for rinsing, add 3 ml of trypsin again, digest for 13 minutes, and then prepare 2.8×105 Cell suspension of 10 cells / ml was inoculated into 6-well plates at 2 ml / well and cultured overnight.

[0051] Preferably, the specific steps of virus inoculation are to observe the cell growth status and cell confluence, ensure that the cell confluence is about 80-90%, and the secondary counting cell volume is: 1.21×10 6 cells / well; BEAS-2B cells proliferate rapidly, and secondary counting can ensure the accuracy of the experiment.

[0052] According to the inoculation volume of virus transfection multiplicity MOI of 25, use DMEM medium containing 10% FBS to dilute the test sample to 10MOI / ml. Take out the 6-well plate from the incubator, carefully discard the culture supernatant in the biosafety cabinet, rinse once with PBS or culture medium, and add 2.5ml / well of the sample diluted in the previous step. The same volume of fresh culture medium is used as a negative control, and placed at 37°C and 5% CO 2 Continue culturing in the incubator for 48 h ± 2 h.

[0053] 2. Extract total cell protein: Take out the cell culture plate after the culture is completed, observe the cell morphology under a microscope, collect the cell culture supernatant of the test sample and negative control into centrifuge tubes in the biosafety cabinet, centrifuge at 1000rpm for 5min, and discard the supernatant. After 200ul of cells in each well of the 6-well plate are digested with trypsin for 10 minutes, add 2ml of 10% FBSDMEM to each well to terminate the digestion and collect them into the corresponding centrifuge tube in the previous step. Centrifuge at 1000rpm for 10min at room temperature and discard the supernatant. Add 1ml PBS to the centrifuge tube, resuspend and transfer to a 1.5ml EP tube, and centrifuge at 4000rpm for 5min. Discard the supernatant and repeat the previous step twice. The collected cell sediment is placed at -80℃ for use.

[0054] Place the EP tube containing the cell sediment on ice, add 100ul / well of IP lysis buffer (WB(IP)Lysis Buffer:Protease Inhibitor (100×)=99:1) containing 1×COCKTAIL, and vortex. Lyse on ice for 30min, vortex every 10 minutes. Centrifuge the lysed sample at 4℃12000rpm for 30min, collect 80ul / well of the supernatant after centrifugation into a new EP tube, which is the total cell protein.

[0055] 3. Protein concentration quantification (BCA method):

[0056] First, prepare a plurality of protein standard working solutions with concentration gradients, perform BCA color development on each of the protein standard working solutions, and draw a standard curve of protein concentration and absorbance value. Prepare a protein solution to be tested containing the total cell protein, perform BCA color development on the protein solution to be tested, and obtain the content of the total cell protein according to the color development result and the standard curve.

[0057] Preferably, the specific calculation steps are: draw a standard curve with the gradient protein content (μg / ml) in the standard curve as the ordinate and the absorbance value as the abscissa. According to the absorbance value of the sample to be tested, the protein concentration (μg / ml) of the sample to be tested in the corresponding well can be found on the standard curve, and then multiplied by the sample dilution multiple to obtain the actual protein concentration of the sample to be tested.

[0058] 4. SDS-PAGE electrophoresis: After protein denaturation, load the protein sample and perform electrophoresis.

[0059] Preferably, the specific steps of protein denaturation are: add 0.25 times the sample volume of 5× loading buffer to each protein sample, mix well, seal tightly, and boil at 100° C. for 10 min.

[0060] Preferably, the specific steps of protein loading are: installing the precast gel on the vertical electrophoresis device, ensuring that the groove is aligned with the protrusion on the electrophoresis device, pouring the electrophoresis solution, checking to ensure that the device is leaking, vertically pulling the comb out of the gel, and adding 30 μg of the protein sample obtained in the previous step to the loading well.

[0061] Preferably, the specific steps of electrophoresis are: correctly connect the positive and negative power supplies, 60V, and start electrophoresis. After the Prestained Protein Ladder is clearly separated, adjust to 120V, and stop electrophoresis until the bromophenol blue is about 1 cm below.

[0062] 5. Western Blot: Place the transfer clip in the electrophoresis tank, pour in the transfer solution and transfer the membrane, and seal after transfer.

[0063] Preferably, the production process of the transfer sandwich is as follows: pour the transfer solution into the transfer plate, place the black part of the transfer clip on the plate, and half-suspend the white part, spread sponges on the black and white parts, and then spread 2 filter papers according to the thickness of the sponges, and all the sponge filter papers must be soaked.

[0064] Preferably, the membrane cutting step is to cut a 0.45 μm PVDF membrane of appropriate size, and to stick the glue and the membrane together according to the "black glue and white film", and to avoid bubbles in the middle. Before use, the membrane should be soaked in methanol for 1 minute, and then rinsed in water. The order of the transfer clip is: white part-sponge-filter paper-membrane-glue-filter paper-sponge-black part.

[0065] Preferably, the specific steps of transferring the membrane are: placing the clamp into the electrophoresis tank, pouring the transfer solution, and placing the entire electrophoresis tank on ice.

[0066] Preferably, the transfer current is 220 mA and the transfer time is 80 min.

[0067] Preferably, the specific step of blocking is to pick up the transferred membrane with tweezers and place it in a rapid blocking solution at room temperature on a shaking table for 30 minutes.

[0068] 6. Immune reaction: First use diluted primary antibody solution for incubation, and then use diluted secondary antibody solution to perform immune reaction on the blocked membrane.

[0069] Preferably, the specific steps of incubating the primary antibody are: using an antibody diluent to prepare the primary antibody to an appropriate concentration as shown in the following table, and using an appropriate amount of the primary antibody to fully immerse the PVDF membrane, and incubating at 2-8°C overnight.

[0070] Preferably, the primary antibody is Anti-HPV18 E7 antibody [8E2]

[0071] Preferably, the dilution ratio of the primary antibody solution is antibody volume: antibody diluent volume = 1:1000.

[0072] Wash the membrane: Take out the membrane solid phase carrier and place it in TBST for 10 min × 3 times, shaking on a shaker at room temperature.

[0073] Preferably, the secondary antibody is diluted with 5% BSA TBST, and an appropriate amount of the corresponding secondary antibody is used to fully immerse the PVDF membrane (the membrane can be fully shaken in the liquid), and incubated on a shaker at room temperature for 1 hour.

[0074] Preferably, the secondary antibody is goat anti-mouse IgG H&L, and the dilution ratio of the secondary antibody solution is antibody volume: antibody diluent volume = 1:2000.

[0075] Preferably, after incubation with the diluted primary antibody solution, the membrane is washed first. The specific steps of washing the membrane are to take out the membrane solid phase carrier and place it in TBST for 10 min×3 times and incubate on a shaker at room temperature.

[0076] 7. After the immune reaction, perform chemiluminescence and imaging.

[0077] When a specific band appears at the position of 100 KDa in the obtained electrophoretogram, the sample to be tested contains the fusion protein. When a specific band does not appear at the position of 100 KDa in the obtained electrophoretogram, the sample to be tested does not contain the fusion protein.

[0078] Preferably, a color developing solution is first used for chemiluminescence, and the color developing solution is a conventional color developing solution, which is prepared by a 1:1 ratio of solution A and solution B. The strip is placed in the color developing solution, soaked in the dark for 2 minutes, and then taken out for exposure.

[0079] Preferably, the specific steps of imaging are: using the chemiluminescence mode of the fully automatic chemiluminescence image analysis system, selecting continuous exposure after white light photography until specific bands appear, and superimposing the results with the previous white light image Prestained Protein Ladder and then analyzing the results.

[0080] The HPV trivalent therapeutic vaccine activity detection method of the present invention uses the above-mentioned method to detect the fusion protein in the vaccine, and determines the activity of the HPV trivalent therapeutic vaccine based on the detection results.

[0081] Specifically, when the test result shows that the fusion protein is contained, the activity of the HPV trivalent therapeutic vaccine is judged to meet the requirements; when the test result shows that the fusion protein is not contained, the activity of the HPV trivalent therapeutic vaccine is judged to not meet the requirements.

[0082] It should be noted that although there are many indicators for determining vaccine activity and it is not determined solely by the fusion protein content, before conducting complex in vivo and clinical trials, the method of the present invention is used to conduct qualitative detection of the fusion protein, which can play a role in preliminary judgment of the final result of the vaccine activity detection, which not only ensures the accuracy of the vaccine activity detection results, but also improves the detection efficiency.

[0083] The vaccine activity detection method of the present invention uses fusion protein as the detection target to judge the vaccine activity, thereby avoiding the problem of inaccurate detection results caused by obvious differences in biological activity in different animal species.

[0084] The present invention is described below by means of specific examples.

[0085] The experimental materials used in each embodiment are shown in Table 1-4:

[0086] Table 1

[0087] cell source batch number BEAS-2B ATCC NA A549 Working cell bank (for testing) 20210501 MRC-5 Working cell bank (for testing) 20210502 HEK293 Working cell bank (for testing) 20210601

[0088] Table 2

[0089]

[0090]

[0091] Table 3

[0092]

[0093]

[0094] Table 4

[0095] sample batch number KDTV001 purified solution 20231121ALH03 KDTV001 original solution DS240822 KDTV001 original solution DS240921 KDTV001 original solution DS241008 KDTV001 finished product 20241001

[0096] Example 1

[0097] 1. Reagent preparation:

[0098] Preparation of electrophoresis buffer (1×): Measure 1L of purified water and mix it with a packet of electrophoresis solution powder to dissolve it into 1L solution before use.

[0099] Preparation of transfer buffer (1×): Measure 800 ml of purified water and mix it with 1 package of transfer buffer powder to dissolve. Before the transfer operation begins, measure 200 ml of methanol and add it to make a 1L solution for use.

[0100] Preparation of TBST solution: Measure 2L of purified water, take 1 packet of TBS powder and add it to the purified water, then add 2ml of TWEEN-20 at a ratio of 1:1000.

[0101] Preparation of secondary antibody diluent: Weigh 1.0 g BSA and add it to 20 ml of the above TBST to prepare 5% BSA TBST as the secondary antibody diluent.

[0102] 2. Virus transfection:

[0103] Cell plating: Take a bottle of T225 A549 cells with a confluence of 80%, discard the culture medium, add 3 ml of trypsin to the culture bottle to rinse once, add 3 ml of trypsin again, digest for 5 minutes, and then prepare 2.5×10 5 Cell suspension of 10 cells / ml was inoculated into 6-well plates at 2 ml / well and cultured for 24 h.

[0104] Virus inoculation: Observe the cell growth and cell confluence to ensure that the cell confluence is about 80-90%. According to the inoculation amount of virus transfection multiplicity MOI=10, dilute the test sample to 5MOI / ml using 10% FBS DMEM medium. Take out the 6-well plate from the incubator, carefully discard the culture supernatant in the biosafety cabinet, rinse once with PBS or culture medium, and add 2ml / well of the diluted sample in the previous step. The same volume of fresh culture medium is used as a negative control and placed at 37°C and 5% CO 2 Continue culturing in the incubator for 48 h ± 2 h.

[0105] 3. Extract total cell protein: Take out the cell culture plate after the culture is completed, observe the cell morphology under a microscope, collect the cell culture supernatant of the test sample and negative control into centrifuge tubes in the biosafety cabinet, centrifuge at 1000rpm for 5min, and discard the supernatant. After 200ul of cells in each well of the 6-well plate are digested with trypsin for 10 minutes, add 2ml of 0% FBSDMEM to each well to terminate the digestion and collect them into the corresponding centrifuge tube in the previous step. Centrifuge at 1000rpm for 10min at room temperature and discard the supernatant. Add 1ml PBS to the centrifuge tube, resuspend and transfer to a 1.5ml EP tube, and centrifuge at 4000rpm for 5min. Discard the supernatant, repeat the previous step twice, collect the cell sediment, and place it at -80℃ for use.

[0106] Place the EP tube containing the cell sediment on ice, add 100ul / well of IP lysis buffer (WB(IP)Lysis Buffer:Protease Inhibitor (100×)=99:1) containing 1×COCKTAI L, and vortex. Lyse on ice for 30min, vortex every 10 minutes. Centrifuge the lysed sample at 4℃12000rpm for 30min, collect 80ul / well of the supernatant after centrifugation into a new EP tube, which is the total cell protein.

[0107] 4. Protein concentration quantification (BCA method)

[0108] Prepare protein standard working solution: Take an appropriate amount of 25 mg / ml protein standard stock solution and dilute it 50 times with PBS to obtain a protein standard working solution with a final concentration of 0.5 mg / ml. Note that the dilution should be done in a 10-fold gradient to ensure accurate dilution.

[0109] Draw a standard curve (ELISA method): add 0, 1, 2, 4, 8, 12, 16, 20 μl of the protein standard working solution to a 96-well plate, then add 20, 19, 18, 16, 12, 8, 4, 0 μl of PBS or saline to make up the gradient working solution to 20 μl. Obtain a gradient curve with protein concentrations of 0, 25, 50, 100, 200, 300, 400, 500 μg / ml.

[0110] Prepare the samples to be tested: dilute the protein samples to be tested 20 times and add 20 μl of each sample to a 96-well plate. Dilute the samples to be tested and the protein standards with the same solution.

[0111] Prepare BCA colorimetric working solution: Mix BCA reagent and copper sulfate solution in a volume ratio of 50:1 to obtain BCA colorimetric working solution.

[0112] Detection: Add 200 μl of BCA colorimetric working solution to each of the standard curve sample wells and the sample wells to be tested, mix thoroughly (the 96-well plate can be placed on an oscillator for 30 seconds), react at 37°C for 30 minutes, use the standard curve No. 0 as a reference, perform colorimetric determination at a wavelength of 562 nm, and record the absorbance value of each well.

[0113] Calculation: Draw a standard curve with the gradient protein content (μg / ml) in the standard curve as the ordinate and the absorbance value as the abscissa. According to the absorbance value of the sample to be tested, the protein concentration (μg / ml) of the sample to be tested in the corresponding well can be found on the standard curve, and then multiplied by the sample dilution multiple to obtain the actual protein concentration of the sample to be tested (y=1767.5x-229.81, R 2 =0.9906), as shown in Table 5.

[0114] Table 5

[0115] Sample Information Dilution multiple OD-replicate 1 OD-Duplicate 2 OD-mean Actual concentration ug / ul 20231121ALH03 10 0.609 0.605 0.6070 8.4306 Negative control 10 0.413 0.42 0.4165 5.0635

[0116] 5. SDS-PAGE electrophoresis

[0117] Protein denaturation: Add 0.25 times the sample volume of 5× loading buffer to each protein sample, mix well, seal tightly, and boil at 100℃ for 10 minutes.

[0118] Protein loading: Install the precast gel on the vertical electrophoresis device, make sure that the groove is aligned with the protrusion on the electrophoresis device, pour the electrophoresis solution, check to make sure there is no leakage in the device, pull the comb teeth vertically upward from the gel, as shown in Table 6, and add 25 μg of the protein solution obtained in the previous step to the loading well.

[0119] Table 6

[0120] Sample Information Sample volume ug Sample volume ul 20231121ALH03 25 2.97 Negative control 25 4.94

[0121] Electrophoresis: Connect the positive and negative power supply correctly, start electrophoresis at 60V. After the Prestained Protein Ladder is clearly separated, adjust to 120V and stop electrophoresis until the bromophenol blue is about 1cm below.

[0122] 6. Western Blot

[0123] Transfer sandwich: Pour transfer solution into the transfer plate, place the black part of the transfer sandwich on the plate, with the white part half suspended in the air, place sponges on the black and white parts, and then place 2 filter papers on each part according to the thickness of the sponges (all sponge filter papers must be soaked).

[0124] Cut the membrane: Cut a 0.45μm PVDF membrane of appropriate size, and stick the glue and membrane together according to the "black glue and white film" order, and be careful to avoid bubbles in the middle. Before using the membrane, soak it in methanol for 1 minute, and then rinse it in water. The order of transferring the membrane clip is: white part-sponge-filter paper-membrane-glue-filter paper-sponge-black part.

[0125] Transfer: Place the clamp into the electrophoresis tank, pour in the transfer solution, place the entire electrophoresis tank on ice, and transfer at 280 mA for 60 minutes.

[0126] Blocking: Pick up the transferred membrane with tweezers and place it in rapid blocking solution at room temperature on a shaker for 30 minutes.

[0127] 7. Immune response

[0128] Incubate with primary antibody: Use antibody diluent to prepare the primary antibody to the appropriate concentration as shown in Table 7, and use an appropriate amount of primary antibody to fully immerse the PVDF membrane, and incubate overnight at 2-8°C.

[0129] Table 7

[0130]

[0131] Wash the membrane: Take out the membrane solid phase carrier and place it in TBST for 10 min × 3 times, shaking on a shaker at room temperature.

[0132] Incubation with secondary antibody: dilute the secondary antibody with 5% BSA TBST, the dilution ratio is shown in Table 8, and use an appropriate amount of the corresponding secondary antibody to fully immerse the PVDF membrane (the PVDF membrane can be fully shaken in the liquid), and incubate on a shaker at room temperature for 1 hour.

[0133] Table 8

[0134]

[0135] Wash the membrane: Take out the membrane solid phase carrier and place it in TBST, incubate at room temperature on a shaker for 10 min × 3 times.

[0136] 8. Chemiluminescence and imaging

[0137] Color development: Prepare color developing solution, prepare solution A and solution B in a ratio of 1:1, place the strip in the color developing solution, soak it in the dark for 2 minutes and then take it out for exposure.

[0138] Imaging: Select the chemiluminescence mode of the fully automatic chemiluminescence image analysis system. After taking white light photos, select continuous exposure until specific bands appear. Superimpose the results with the previous white light image Prestained Protein Ladder and analyze them. For detailed operations, see "4800Multi Fully Automatic Chemiluminescence Image Analysis System (Tianneng) SOP"

[0139] SOP(RD)-QC-005.

[0140] 9. Experimental results and analysis

[0141] like Figure 1 As shown, some Prestained Protein Ladder bands can be seen on the filter paper, indicating that the transfer conditions in this example are not suitable. The transfer conditions should be properly optimized, the transfer time should be reduced, or the transfer current should be lowered.

[0142] Anti-HPV18 E7 antibody [8E2] and Anti-HPV52 E7 / Protein E7 Polyclonal Antibody showed specific bands at the lower part of 100KDa, such as Figure 1 The red box in a and b shows that there is no specific band in the negative control in the same area. The samples treated with the other four antibodies did not show specific bands in the target area, such as Figure 1 As shown in the blue box in cf.

[0143] In view of the above results of this example, it is considered to appropriately adjust the target protein amount and the antibody dilution ratio.

[0144] Example 2

[0145] This example optimizes the method of Example 1, and the specific optimization conditions are: increasing the amount of protein loaded to 30ug; adjusting the transfer conditions to 280mA, 50min; no obvious target protein bands were observed for the four antibodies HPV 16E6, HPV 18E6, HPV 52E7, and HPV 52E6, the antibody dilution ratio was reduced, and the concentration of the primary antibody incubation was increased.

[0146] Specifically, in the SDS-PAGE electrophoresis step of this embodiment, the sample loading amount and sample loading volume of the experimental group and the negative control group are shown in Table 9:

[0147] Table 9

[0148] Sample Information Sample volume ug Sample volume ul 20231121ALH03 30 2.97 Negative control 30 4.94

[0149] The Western Blot transfer conditions in this example are 280 mA and 50 min.

[0150] The specific steps of the immune response are as follows:

[0151] Incubate with primary antibody: Use antibody diluent to prepare the primary antibody to an appropriate concentration as shown in Table 10, and use an appropriate amount of primary antibody to fully immerse the PVDF membrane, and incubate overnight at 2-8°C.

[0152] Table 10

[0153]

[0154] Wash the membrane: Take out the membrane solid phase carrier and place it in TBST for 10 min × 3 times, shaking on a shaker at room temperature.

[0155] Incubation with secondary antibody: dilute the secondary antibody with 5% BSA TBST, the dilution ratio is shown in Table 11, and use an appropriate amount of the corresponding secondary antibody to fully immerse the PVDF membrane (the PVDF membrane can be fully shaken in the liquid), and incubate on a shaker at room temperature for 1 hour.

[0156] Table 11

[0157]

[0158] Wash the membrane: Take out the membrane solid phase carrier and place it in TBST, incubate at room temperature on a shaker for 10 min × 3 times.

[0159] The specific operations of chemiluminescence and imaging in this embodiment are the same as those in embodiment 1.

[0160] Experimental results and analysis:

[0161] like Figure 2 As shown in the figure, after the transfer, some Prestained Protein Ladder bands can be seen remaining on the gel surface. The transfer current can be further lowered to extend the transfer time.

[0162] The test results of Anti-HPV18 E7 antibody [8E2], Human Papillomavirus type 18 E6 antibody, Human Papilloma virus type 16 E6 antibody, and Anti-HPV52 E7 / Protein E7 Polyclonal Antibody showed obvious target bands below 100KDa, such as Figure 3 The red box marks the middle area, and there is no band in the negative control area. The results suggest that this antibody dilution is more appropriate, and the primary antibody dilution of the above 4 antibodies will be basically determined later.

[0163] HPV Type 16 E7 Monoclonal Antibody (8C9) and Anti-HPV52 E6 / ProteinE6Polyclonal Antibody showed no specific bands in the target protein region. Figure 3 Marked by the blue box in the middle, consider optimizing the antibody dilution conditions again or consider changing the antibody, or increase the total amount of protein loaded again.

[0164] Although 4 antigens were detected in this experiment, Figure 2It can be seen that non-specific protein coloring is more common, which seriously interferes with the judgment of experimental results. Considering that KDTV001 product is used for intramuscular injection, normal cells in the body are the main force of antigen production. The use of human normal cell lines is more representative than the use of cancer cells to identify the biological activity of the drug.

[0165] Example 3

[0166] According to the experimental results of Example 1 and Example 2, Anti-HPV18 E7 antibody [8E2] has strong specificity and high sensitivity. Therefore, in the optimization experiment of this example, in order to reduce costs and ensure stability, Anti-HPV18 E7 antibody [8E2] was used for further research.

[0167] This embodiment makes adjustments in the following four directions based on Embodiment 2.

[0168] 1) Change the cell line: MRC-5 (human embryonic lung fibroblasts), BEAS-2B (human normal lung epithelial cells).

[0169] 2) MOI screening: Different cells have different tolerance to viruses and target protein production. In order to further increase the expression of target proteins, the cell density and MOI of different cell lines when infected are screened to further improve the detection throughput of WB.

[0170] 3) Adjustment of transfer conditions: Considering that too high current and voltage will lead to high system temperature, and large proteins may be degraded into small molecular fragments if the temperature is too high, and Prestained Protein Ladder is also easily degraded, resulting in a decrease in the light color indication effect, so it is adjusted to a constant current of 220mA. In addition, there are differences in the number of membranes transferred at a time (when two membranes are in the same system, the increase in resistance affects the efficiency of membrane transfer), and a small amount of concentrated hydrochloric acid can be used to adjust the resistance and control the voltage. After debugging, when the constant current is 220mA (the voltage is adjusted to 100-120V) and the membrane is transferred for 80 minutes, the electrotransfer results are relatively ideal (judged by the retention of Prestained Protein Ladder on the gel / membrane / filter paper).

[0171] The transfer conditions of this embodiment are 220 mA, 80 min (voltage: 100-120 V).

[0172] The specific steps for cell line and MOI screening are as follows:

[0173] Cell plating: Take one bottle of T225-sized MRC-5 and BEAS-2B cells with a confluence of more than 80%, discard the culture medium, add 3 ml of trypsin to the culture bottle to rinse once, add 3 ml of trypsin again, and prepare cell suspension with 10% FBSDMEM after digestion and detachment, inoculate in a 6-well plate at 2 ml / well, and culture for 24 hours.

[0174] Virus inoculation: Observe cell growth and cell confluence, and count twice. Dilute the test samples in 10% FBS DMEM medium according to the virus transfection multiplicity shown in Table 12.

[0175] Table 12

[0176]

[0177]

[0178] Inoculation of test sample: Take out the 6-well plate from the incubator, carefully discard the culture supernatant in the biosafety cabinet, rinse once with PBS or culture medium, add the diluted sample in the previous step at a volume of 2 ml / well, and the same volume of fresh culture medium as a negative control, and place at 37°C, 5% CO 2 Continue culturing in the incubator for 48 h ± 2 h.

[0179] The steps of extracting total cell protein in this example are the same as those in Example 1.

[0180] The steps of protein concentration quantification (BCA method) in this example are the same as those in Example 1. The standard curve in this example is y=1132.4x-121.56, R 2 =0.9983, as shown in Table 13.

[0181] Table 13

[0182]

[0183] The specific operation of SDS-PAGE electrophoresis in this example is the same as that in Example 1, and the sample loading amount and loading volume are shown in Table 14.

[0184] Table 14

[0185]

[0186]

[0187] The Western Blot experimental steps of this embodiment are the same as those of Example 1, and the transfer conditions are: 220 mA, 80 min (voltage 100-120 V). The immune reaction steps of this embodiment are:

[0188] Incubation with primary antibody: Prepare the primary antibody to an appropriate concentration using antibody diluent, and fully immerse the PVDF membrane with an appropriate amount of primary antibody. Incubate overnight at 2-8°C (Anti-HPV18 E7 antibody [8E2], 1:1000 dilution).

[0189] Wash the membrane: Take out the membrane solid phase carrier and place it in TBST for 10 min × 3 times, and shake horizontally on a shaker at room temperature.

[0190] Incubation with secondary antibody: prepare secondary antibody of appropriate concentration with 5% BSA TBST, and fully immerse the PVDF membrane with an appropriate amount of corresponding secondary antibody (goat anti-mouse IgG H&L (HRP), 1:2000 dilution), and fully immerse the PVDF membrane with an appropriate amount of secondary antibody (the membrane can be fully shaken in the liquid), and incubate on a shaker at room temperature for 1 hour.

[0191] Wash the membrane: take out the membrane solid phase carrier and place it in TBST for 10 min×3 times, shake horizontally on a shaker at room temperature. The steps of chemiluminescence and imaging in this example are the same as those in Example 1.

[0192] Experimental results and analysis:

[0193] like Figure 3 and 4 As shown in the figure, compared with the negative control, a weak target protein band can be detected in the MRC-5 cell line when inoculated at 15 MOI; when the confluence of the BEAS-2B cell line was 90%, the expression of the target protein with higher specificity could be detected at MOI=15-25. Therefore, the test cell line was initially determined to be BEAS-2B, with a confluence of 80-90% and MOI=15-25.

[0194] Example 4

[0195] According to the test results of Example 2, HPV Type 16 E7 Monoclonal Antibody (8C9) and Anti-HPV52 E6 / Protein E6Polyclonal Antibody still did not show specific bands, and the specific bands of Human Papillomavirus type 18 E6 antibody, Human Papilloma virus type 16 E6 antibody, and Anti-HPV52E7 / Protein E7 Polyclonal Antibody were unclear, and the antibody dilution conditions needed to be optimized and adjusted.

[0196] Based on the optimized cell lines, MOI and transfection conditions in Example 3, the dilution ratio of the antibody was further adjusted in this example.

[0197] Specifically, the SDS-PAGE electrophoresis steps of this example are the same as those of Example 1. When the confluence of the BEAS-2B cell line was 90%, the virus was inoculated at 25 MOI.

[0198] The Western Blot experimental steps of this example are the same as those of Example 1, and the transfer conditions are: 220 mA, 80 min (voltage 100-120 V).

[0199] The immune response steps of this embodiment are as follows:

[0200] Incubate with primary antibody: Use antibody diluent to prepare the primary antibody to an appropriate concentration as shown in Table 15, and use an appropriate amount of primary antibody to fully immerse the PVDF membrane, and incubate overnight at 2-8°C.

[0201] Table 15

[0202]

[0203]

[0204] Wash the membrane: Take out the membrane solid phase carrier and place it in TBST for 10 min × 3 times, shaking on a shaker at room temperature.

[0205] Incubation with secondary antibody: dilute the secondary antibody with 5% BSA TBST, the dilution ratio is shown in Table 16, and use an appropriate amount of the corresponding secondary antibody to fully immerse the PVDF membrane (the membrane can be fully shaken in the liquid), and incubate on a shaker at room temperature for 1 hour.

[0206] Table 16

[0207]

[0208] Wash the membrane: Take out the membrane solid phase carrier and place it in TBST, incubate at room temperature on a shaker for 10 min × 3 times.

[0209] The steps of chemiluminescence and imaging in this embodiment are the same as those in embodiment 1.

[0210] Experimental results and analysis:

[0211] like Figure 5 As shown in the figure, after adjusting the dilution ratio of the antibodies, five antibodies, namely HPV Type 16 E7 Monoclonal Antibody (8C9), Anti-HPV18 E7 Antibody [8E2], Human Papilloma virus type 16 E6 Antibody, Human Papillomavirus type 18 E6, and Anti-HPV52 E7 / Protein E7 Polyclonal Antibody, can meet the experimental requirements, such as Figure 5 As shown in the red box.

[0212] Anti-HPV52 E6 / Protein E6Polyclonal Antibody has poor specificity, and its ability to bind to the expressed target protein has no direct relationship with its concentration. It is impossible to obtain ideal results by adjusting the antibody concentration (the concentration of the primary antibody is too high, the membrane background is darker, and the background is complex). We tried to change the antibody supplier, but there are very few supply channels for this antibody in the global antibody market, and we also failed to achieve ideal results by trying to make our own antibodies.

[0213] According to the mechanism of action of KDTV001 product, the protein expressed by the target gene is a fusion protein. Detection of any one antigen epitope can prove that the target gene is successfully and correctly expressed. Therefore, no further testing will be conducted on the 52E6 antibody.

[0214] Example 5

[0215] The antibody brand used in Example 4 is GeneTex's 18E6 antibody. In actual use, this antibody has been out of stock for a long time in the global market, and the shortage of antibody reagents has made the test impossible. Therefore, in order to prevent the situation of untimely supply of antibodies, this example investigated other brands of HPV16\18\52 E6 / E7 antibodies, recommended alternative products through technical support from relevant suppliers, and obtained Abcam HPV18E6 and Saint-Luc HPV16E6 / 18E6 antibodies through online searches, and these two antibodies were screened for trial.

[0216] The SDS-PAGE electrophoresis step, Western Blot step, chemiluminescence, and imaging of this embodiment are exactly the same as those of Example 4.

[0217] The specific steps of the immune response are:

[0218] Incubate with primary antibody: Use antibody diluent to prepare the primary antibody to an appropriate concentration as shown in Table 17, and use an appropriate amount of primary antibody to fully immerse the PVDF membrane, and incubate overnight at 2-8°C.

[0219] Table 17

[0220]

[0221] Wash the membrane: Take out the membrane solid phase carrier and place it in TBST for 10 min × 3 times, shaking on a shaker at room temperature.

[0222] Incubation with secondary antibody: dilute the secondary antibody (goat anti-mouse IgG H&L (HRP) 1:2000) with 5% BSA TBST, and fully immerse the PVDF membrane with an appropriate amount of the corresponding secondary antibody (the membrane can be fully shaken in the liquid), and incubate on a shaker at room temperature for 1 hour.

[0223] Wash the membrane: Take out the membrane solid phase carrier and place it in TBST, incubate at room temperature on a shaker for 10 min × 3 times.

[0224] Experimental results and analysis:

[0225] like Figure 6 As shown, the results of the two screenings showed that the antibodies of both companies could not meet the experimental requirements.

[0226] As there are relatively few relevant antibody categories on the market, some antibodies are not specific enough and are out of stock from time to time, HPV18E7 was selected as the subsequent detection reagent after comprehensive consideration, and the remaining antibodies were selected as alternatives.

[0227] By investigating and analyzing the supply market of five antibodies, HPV 16E7, HPV 18E7, HPV 16E6, HPV 18E6, and HPV 52E7, combined with the application of antibodies in the early method development process of our company, (HPV 16E7, HPV 16E6, HPV 18E6, HPV52E7) antibodies all have varying degrees of instability. As a key quality control indicator of the KDTV001 product, this analysis method must ensure that it can be carried out stably and sustainably. In the conclusion description of the aforementioned embodiment, it is also analyzed that "according to the mechanism of action of the KDTV001 product, the protein expressed by the target gene is a fusion protein, and the detection of any one of the antigenic epitopes can prove that the target gene is successfully and correctly expressed", so among the five antibodies of HPV 16E7, HPV 18E7, HPV 16E6, HPV 18E6, and HPV 52E7, the HPV18E7 type antibody with the strongest specificity, the most stable market supply, and the most reliable test results is selected for this method.

[0228] Example 6

[0229] This embodiment uses the optimized method to detect different samples, and the specific steps are as follows:

[0230] 1. Viral transfection

[0231] Cell plating: Take a bottle of T225 BEAS-2B cells with a confluence of more than 80%, discard the culture medium, add 3 ml of trypsin to the culture bottle to rinse once, add 3 ml of trypsin again, digest for 13 minutes, and then prepare 2.8×10 5 Cell suspension of 10 cells / ml was inoculated into 6-well plates at 2 ml / well and cultured overnight.

[0232] Virus inoculation: Observe the cell growth and cell confluence to ensure that the cell confluence is about 80-90%. The secondary cell count is: 1.21×10 6cells / well (BEAS-2B cells proliferate quickly, so count twice to ensure the accuracy of the experiment). According to the inoculation amount of virus transfection multiplicity MOI=25, use DMEM medium containing 10% FBS to dilute the test sample to 10MOI / ml. Take out the 6-well plate from the incubator, carefully discard the culture supernatant in the biosafety cabinet, rinse once with PBS or culture medium, and add 2.5ml / well of the sample diluted in the previous step. The same volume of fresh culture medium is used as a negative control, and placed at 37°C and 5% CO 2 Continue to culture in the incubator for 48h±2h. The inoculation conditions of each test product are shown in Table 18.

[0233] Table 18

[0234]

[0235]

[0236] 2. Extraction of total cell protein

[0237] Take out the cell culture plate at the end of the culture, observe the cell morphology under a microscope, collect the cell culture supernatant of the test sample and negative control into centrifuge tubes in the biosafety cabinet, centrifuge at 1000rpm for 5min, and discard the supernatant. After 200ul of cells in each well of the 6-well plate are digested with trypsin for 10 minutes, add 2ml of 10% FBSDMEM to each well to terminate the digestion and collect them into the corresponding centrifuge tube in the previous step. Centrifuge at 1000rpm for 10min at room temperature and discard the supernatant. Add 1ml PBS to the centrifuge tube, resuspend and transfer to a 1.5ml EP tube, and centrifuge at 4000rpm for 5min. Discard the supernatant and repeat the previous step twice. The collected cell sediment is placed at -80℃ for use.

[0238] Place the EP tube containing the cell sediment on ice, add 100ul / well of IP lysis buffer (WB(IP)Lysis Buffer:Protease Inhibitor (100×)=99:1) containing 1×COCKTAIL, and vortex. Lyse on ice for 30min, vortex every 10 minutes. Centrifuge the lysed sample at 4℃12000rpm for 30min, collect 80ul / well of the supernatant after centrifugation into a new EP tube, which is the total cell protein.

[0239] 3. Protein concentration quantification (BCA method)

[0240] Prepare protein standard working solution: Take an appropriate amount of 25 mg / ml protein standard stock solution and dilute it 50 times with PBS to obtain a protein standard working solution with a final concentration of 0.5 mg / ml. Note that the dilution should be diluted according to the 10-fold gradient method to ensure accurate dilution.

[0241] Draw a standard curve (ELISA method): add 0, 1, 2, 4, 8, 12, 16, 20 μl of the protein standard working solution to a 96-well plate, then add 20, 19, 18, 16, 12, 8, 4, 0 μl of PBS or saline to make up the gradient working solution to 20 μl. Obtain a gradient curve with protein concentrations of 0, 25, 50, 100, 200, 300, 400, 500 μg / ml.

[0242] Prepare the samples to be tested: dilute the protein samples to be tested 20 times and add 20 μl of each sample to a 96-well plate. Dilute the samples to be tested and the protein standards with the same solution.

[0243] Prepare BCA colorimetric working solution: Mix BCA reagent and copper sulfate solution in a volume ratio of 50:1 to obtain BCA colorimetric working solution.

[0244] Detection: Add 200 μl of BCA colorimetric working solution to each of the standard curve sample wells and the sample wells to be tested, mix thoroughly (the 96-well plate can be placed on an oscillator for 30 seconds), react at 37°C for 30 minutes, use the standard curve No. 0 as a reference, perform colorimetric determination at a wavelength of 562 nm, and record the absorbance value of each well.

[0245] Calculation: Draw a standard curve with the gradient protein content (μg / ml) in the standard curve as the ordinate and the absorbance value as the abscissa. According to the absorbance value of the sample to be tested, the protein concentration (μg / ml) of the sample to be tested in the corresponding well can be found on the standard curve, and then multiplied by the sample dilution multiple to obtain the actual protein concentration of the sample to be tested (y=1179.6x-136.73, R 2 =0.9981), as shown in Table 19.

[0246] Table 19

[0247]

[0248]

[0249] 4. SDS-PAGE electrophoresis

[0250] Protein denaturation: Add 0.25 times the sample volume of 5× loading buffer to each protein sample, mix well, seal tightly, and boil at 100℃ for 10 minutes.

[0251] Protein loading: Install the precast gel on the vertical electrophoresis device, make sure that the groove is aligned with the protrusion on the electrophoresis device, pour in the electrophoresis solution, check to make sure there is no leakage in the device, pull the comb out of the gel vertically, and add 30 μg of the protein sample obtained in the previous step to the loading well, as shown in Table 20.

[0252] Table 20

[0253] Test product information Sample volume ug Sample volume ul KDTV001 original solution DS240822 30 9 KDTV001 stock solution DS240921 30 10 KDTV001 stock solution DS241008 30 11 KDTV001 finished product 20241001 30 8 Negative control 30 12

[0254] Electrophoresis: Connect the positive and negative power supply correctly, start electrophoresis at 60V. After the Prestained Protein Ladder is clearly separated, adjust to 120V and stop electrophoresis until the bromophenol blue is about 1cm below.

[0255] 5. Western Blot:

[0256] Transfer sandwich: Pour transfer solution into the transfer plate, place the black part of the transfer sandwich on the plate, with the white part half suspended in the air, place sponges on the black and white parts, and then place 2 filter papers on each part according to the thickness of the sponges (all sponge filter papers must be soaked).

[0257] Cut the membrane: Cut a 0.45μm PVDF membrane of appropriate size, and stick the glue and membrane together according to the "black glue and white film" order, and be careful to avoid bubbles in the middle. Before using the membrane, soak it in methanol for 1 minute, and then rinse it in water. The order of transferring the membrane clip is: white part-sponge-filter paper-membrane-glue-filter paper-sponge-black part.

[0258] Transfer: Place the clamp into the electrophoresis tank, pour in the transfer solution, place the entire electrophoresis tank on ice, and transfer at 220 mA for 80 minutes.

[0259] Blocking: Pick up the transferred membrane with tweezers and place it in a rapid blocking solution at room temperature on a shaker for 30 minutes.

[0260] 6. Immune response:

[0261] Incubate with primary antibody: Use antibody diluent to prepare the primary antibody to an appropriate concentration as shown in Table 21, and use an appropriate amount of primary antibody to fully immerse the PVDF membrane, and incubate overnight at 2-8°C.

[0262] Table 21

[0263]

[0264]

[0265] Wash the membrane: Take out the membrane solid phase carrier and place it in TBST for 10 min × 3 times, shaking on a shaker at room temperature.

[0266] Incubation with secondary antibody: As shown in Table 22, dilute the secondary antibody with 5% BSA TBST, and fully immerse the PVDF membrane with an appropriate amount of the corresponding secondary antibody (the membrane can be fully shaken in the liquid), and incubate on a shaker at room temperature for 1 hour.

[0267] Table 22

[0268]

[0269] Wash the membrane: Take out the membrane solid phase carrier and place it in TBST, incubate at room temperature on a shaker for 10 min × 3 times.

[0270] 7. Chemiluminescence and imaging:

[0271] Color development: Prepare color developing solution, prepare solution A and solution B in a ratio of 1:1, place the strip in the color developing solution, soak it in the dark for 2 minutes and then take it out for exposure.

[0272] Imaging: Select the chemiluminescence mode of the fully automatic chemiluminescence image analysis system. After taking white light photos, select continuous exposure until specific bands appear. Superimpose the results with the previous white light image Prestained Protein Ladder and analyze them. For detailed operations, see "4800Multi Fully Automatic Chemiluminescence Image Analysis System (Tianneng) SOP"

[0273] SOP(RD)-QC-005.

[0274] 8. Experimental results and analysis:

[0275] The experimental results showed that all four groups of test samples had obvious specific bands at the lower part of 100KDa, while the negative control had no bands in the same area. Figure 7 As shown, the experiment was established. The developed analytical method can accurately identify whether the target gene can be successfully expressed in the cell and synthesize active intermediates, thereby confirming the viral biological activity of the KDTV001 product. Analysis process Figure 8 shown.

[0276] Example 7

[0277] This example verifies the specificity of the detection method of the present invention. Specifically, the source of cells (BEAS-2B) was changed in this example, specifically derived from the cell line of ATCC, and the specific experimental steps were the same as those of Example 6.

[0278] The samples and test results of this example are shown in Table 23 and Fig. 9 shown.

[0279] Table 23

[0280]

[0281] It can be seen from the above test results that the method of the present invention is specific for BEAS-2B cells.

[0282] Example 8

[0283] This example verifies the durability of the detection method of the present invention.

[0284] Specifically, this example uses the method of Example 6 to test different batches of test samples, and uses two common brands of protease inhibitors. The samples and test results of this example are shown in Tables 24 and Fig.10 shown.

[0285] Table 24

[0286]

[0287] According to the above test results, it can be seen that the method of the present invention can effectively detect different batches of test samples, and the test results are not affected by the brand of protease inhibitors, which shows that the method of the present invention has good durability.

[0288] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting HPV16 / 18 / 52 type E7 / E6 fusion protein in a host cell, characterized in that: The host cell is a BEAS-2B cell transfected with a constructed adenovirus, and the detection method comprises the steps of extracting total protein from the host cell, SDS-PAGE electrophoresis, Western blotting detection, immune response and color imaging; The constructed adenovirus is an adenovirus carrying HPV16 / 18 / 52 type E6 / E7 antigens, the transfection multiplicity MOI of the constructed adenovirus is 15-25, and before transfection, the confluence of the BEAS-2B cells is greater than 80%; in the protein blotting method, the transmembrane current is 220-280mA, and the time is 50-80min.

2. The method for detecting HPV16 / 18 / 52 type E7 / E6 fusion protein in a host cell according to claim 1, characterized in that: The transfection multiplicity MOI of the constructed adenovirus is 25, and before transfection, the confluence of the BEAS-2B cells is 90%; in the Western blotting method, the transfer current is a constant current of 220 mA, and the time is 80 minutes.

3. The method for detecting HPV16 / 18 / 52 type E7 / E6 fusion protein in a host cell according to claim 2, characterized in that: Before transfection, the secondary count of the BEAS-2B cells was 1.21×10 6 cells / holes.

4. The method for detecting HPV16 / 18 / 52 type E7 / E6 fusion protein in a host cell according to claim 2, characterized in that: In the SDS-PAGE electrophoresis, the loading amount in each loading well is greater than or equal to 30 μg.

5. The method for detecting HPV16 / 18 / 52 type E7 / E6 fusion protein in a host cell according to claim 1, characterized in that: In the immune reaction, a diluted primary antibody solution and a diluted secondary antibody solution are sequentially used for incubation; The primary antibody solution is one of an HPV 16E7 antibody solution, an HPV 18E7 antibody solution, an HPV 16E6 antibody solution, an HPV 18E6 antibody solution, and an HPV 52E7 antibody solution, wherein the dilution ratio of the HPV 16E7 antibody solution is 1:50, the dilution ratio of the HPV 18E7 antibody solution is 1:1000, the dilution ratio of the HPV 16E6 antibody solution is 1:250, the dilution ratio of the HPV 18E6 antibody solution is 1:250, and the dilution ratio of the HPV 52E7 antibody solution is 1:100; The secondary antibody solution is goat anti-mouse IgG H&L antibody solution or goat anti-rabbit IgG H&L antibody solution, and the dilution ratio of the secondary antibody solution is 1:2000.

6. The method for detecting HPV16 / 18 / 52 type E7 / E6 fusion protein in a host cell according to claim 5, characterized in that: The primary antibody solution is an HPV18E7 antibody solution, and the secondary antibody solution is a goat anti-mouse IgG H&L antibody solution.

7. A method for detecting HPV16 / 18 / 52 type E7 / E6 fusion protein in a host cell according to any one of claims 1 to 6, characterized in that: The amino acid sequence of the fusion protein is shown in SEQ ID NO.

1.

8. The method for detecting HPV16 / 18 / 52 type E7 / E6 fusion protein in a host cell according to claim 7, characterized in that: The constructed adenovirus vector is a non-replicating type 5 adenovirus.

9. The method for detecting HPV16 / 18 / 52 type E7 / E6 fusion protein in a host cell according to claim 8, characterized in that: When a specific band appears at the position of 100 KDa in the obtained electrophoretogram, the sample to be tested contains the fusion protein. When a specific band does not appear at the position of 100 KDa in the obtained electrophoretogram, the sample to be tested does not contain the fusion protein.

10. A method for detecting the activity of a trivalent HPV therapeutic vaccine, characterized in that: The method comprises the step of detecting HPV16 / 18 / 52 E7 / E6 fusion protein; the step of detecting HPV16 / 18 / 52 E7 / E6 fusion protein is to transfect a HPV trivalent therapeutic vaccine containing a constructed non-replicating type 5 adenovirus carrying HPV16 / 18 / 52 E6 / E7 antigens into a host cell, and then detecting the HPV16 / 18 / 52 E7 / E6 fusion protein in the host cell by using the method described in any one of claims 1 to 9, and determining the activity of the HPV trivalent therapeutic vaccine according to the detection result.

Images