A human G9 rotavirus HN-28 strain and its application

Abstract

The present invention discloses a human G9 rotavirus HN‑28 strain, which is Reoviridae / Rotavirus A / Human rotavirus (G9P8) HN‑28 Strain, and has been deposited in the China Center for Type Culture Collection on January 13, 2022, with a deposit number of CCTCC NO: V202203. RNA‑PAGE shows that the electrophoresis type of the virus strain is long, and the 11 bands present a unique electrophoresis pattern of group A rotavirus in a 4:2:3:2 arrangement; the whole genome second-generation sequencing results show that this strain is a G9P[8] rotavirus. The present invention successfully isolates a G9 rotavirus wild strain that can be stably propagated through the isolation, culture and cloning and purification of rotavirus-positive fecal samples, named HN‑28 strain, which can be used to prepare rotavirus vaccines.

Description

Technical Field

[0001] The present invention relates to the field of biological preparations, and in particular to a human G9 rotavirus HN-28 strain and an application thereof. Background Art

[0002] Human rotavirus group A (RVA) is the main pathogen of gastroenteritis in infants and young children worldwide. A large number of studies have shown that rotavirus infection is the main cause of diarrhea in children under 5 years old (20% to 60%), and approximately 450,000 to 600,000 children die from diarrhea caused by rotavirus infection each year. Rotavirus belongs to the genus Rotavirus of the family Reoviridae. The viral genome has 11 gene segments, encoding 6 structural proteins (VP1-4, VP6, VP7) and 6 non-structural proteins (NSP1-6), including 3 inner core proteins (VP1-VP3), 1 inner capsid protein (VP6), and 2 outer capsid proteins (VP4 and VP7). According to the antigenic specificity of VP6, RV can be divided into 9 groups (AD and FJ), among which group A rotavirus (RVA) infection is the most common. RVA typing is based on the specificity and diversity of the viral coat proteins VP7 and VP4. It is divided into G serotypes according to the VP7 sequence and P genotypes according to the VP4 sequence. G1-G4 and G9 serotypes are the main types of human rotavirus infection (>80%). G9 HRV infection has now spread throughout the world and has become the dominant epidemic strain in some countries and / or regions, and is likely to become the fifth most common epidemic dominant strain in the world.

[0003] There is no specific treatment for rotavirus infection yet. Vaccination is the only way to effectively prevent rotavirus diarrhea and reduce mortality. Vaccines currently being promoted and used abroad include the attenuated live vaccine Rotarix produced by GlaxoSmithKline, with the vaccine strain G1P[8], the pentavalent reassortant live vaccine RotaTeq produced by Merck, which contains the main human epidemic strains G1, G2, G3, G4 and P1A[8], and the oral rotavirus live vaccine produced by Lanzhou Institute of Biological Products Co., Ltd., with the vaccine strain LLR strain G10P

[12] . Summary of the invention

[0004] The purpose of the present invention is to provide a human G9 rotavirus HN-28 strain and a separation and identification method and application thereof.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] A human G9 rotavirus HN-28 strain, Reoviridae / Rotavirus A / Human rotavirus (G9P8) HN-28 Strain, was deposited in the China Center for Type Culture Collection on January 13, 2022, with the deposit number: CCTCC NO: V202203. RNA-PAGE showed that the electrophoretic type of the virus strain was long, and the 11 bands showed a unique electrophoretic pattern of group A rotavirus in a 4:2:3:2 arrangement; the whole genome second-generation sequencing results showed that this strain was a G9P[8] rotavirus; CCID 50 The virus titer was determined to be 10 5.5 ~10 6.5 ; SDS-PAGE and Western-blot results showed that protein bands of the expected sizes of VP7, VP6 and VP4 were formed at molecular weights of approximately 34KD, 45KD and 88KD, respectively; electron microscopy detected typical rotavirus particles with a diameter of approximately 75nm; indirect immunofluorescence test showed that the virus proliferating on MA-104 cells was rotavirus.

[0007] The application of the HN-28 strain of the present invention can be used to prepare a rotavirus vaccine, and the preparation steps are as follows:

[0008] S1. Freeze and thaw the cell culture inoculated with HN-28 virus at -20°C once, centrifuge at 8000rpm, 4°C for 30min, collect the supernatant, and collect the virus particles by ultracentrifugation. Add 30mL of the supernatant to a 50mL special centrifuge tube, gently push 20mL of 50% sucrose solution from the bottom with an 8cm long needle to pad the bottom, keep the interface clear, place in an ultracentrifuge, ultracentrifuge at 35000rpm, 4°C for 4h, discard the supernatant, obtain the purified rotavirus particle precipitate, resuspend the precipitate with RPMI-1640, and obtain the purified virus liquid.

[0009] S2. The purified virus solution was sterilized by filtering with a 0.22μm syringe filter, and then dispensed into 100mL sterile Erlenmeyer flasks. A 40% formaldehyde solution was added, and the solution was shaken while adding to a final concentration of 0.1%. After mixing evenly, the solution was placed in a 60°C water bath for 30 minutes, and then placed in a 37°C water bath for 72 hours for inactivation. After inactivation, sodium bisulfite was added to the virus solution to neutralize the residual formaldehyde. The protein concentration was determined by the BCA method, and the protein concentration was diluted to 200μg / mL with RPMI-1640 culture medium to prepare the G9 type HN-28 rotavirus vaccine stock solution, and samples were taken for virus inactivation verification testing; three consecutive generations of blind subculture were performed on MA-104 cells to observe the cytopathic condition, and each generation was harvested after 6 days of culture, and finally tested by CCID 50The virus inactivation effect was detected by the method. When immunizing animals, the first dose was added with inactivated virus stock solution and Freund's complete adjuvant at a ratio of 1:1, and the second and third doses were added with inactivated virus stock solution and Freund's incomplete adjuvant at a ratio of 1:1, so that the final protein concentration of the vaccine was 100 μg / mL, and the mixture was mixed with an oscillator to prepare a G9 type HN-28 rotavirus vaccine sample containing adjuvant.

[0010] The present invention successfully separated a G9 rotavirus wild strain capable of stable propagation through separation, culture and cloning and purification of rotavirus-positive fecal samples, named as HN-28 strain, which can be used to prepare rotavirus vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Cytopathic effects at different times in the fifth passage virus culture of HN-28 strain;

[0012] In the figure: (a) 1 day after infection; (b) 2 days after infection; (c) 3 days after infection; (d) 4 days after infection; (e) 5 days after infection; (f) 6 days after infection; (g) control cells 6 days.

[0013] Figure 2 Nucleic acid banding pattern of the HN-28 strain RNA-PAGE genome.

[0014] Figure 3 The HN-28 strain forms plaques on MA-104 cells.

[0015] Figure 4 Agarose gel electrophoresis of segment9 PCR products (1 and 2 are gene segment9, M is marker).

[0016] Figure 5 Phylogenetic analysis of 11 gene fragments of HN-28 strain

[0017] In the figure: (a) Segment1-VP1; (b) Segment2-VP2; (c) Segment3-VP3; (d) Segment4-VP4; (e) Segment5 NSP1; (f) Segment6 VP6; (g) Segment7 NSP3; (h) Segment8 NSP2; (i) Segment9 VP7; (j) Segment10 NSP4; (k) Segment11 NSP5 / NSP6.

[0018] Figure 6 Mycoplasma test results;

[0019] In the figure: 1: HN-28 virus suspension; 2: negative control; M: DNA marker; 3: oral mycoplasma; 4: mycoplasma pneumoniae.

[0020] Figure 7 Hemodsorption virus test results;

[0021] In the figure: (a) HN-28 strain virus liquid; (b) negative control; (c) positive control.

[0022] Figure 8 Proliferation curve of HN-28 strain virus.

[0023] Fig. 9 Indirect immunofluorescence assay was used to detect the infection of MA-104 cells with different dilutions of HN-28 strain;

[0024] In the figure: (a) Negative; (b) Dilution: 1X10 -6 ; (c) Dilution: 1X 10 -5 ; (d) Dilution: 1X10 -4 (e) Dilution: 1X 10 -3 ; (f) Dilution: 1X 10 -2 .

[0025] Fig.10 SDS-PAGE (A) and Western-blot analysis of purified HN-28 strain;

[0026] In the figure: 1: purified HN-28 virus; 2: HN-28 virus and G9 RV antiserum reaction band; M1: Page Ruler Prestained Protein Ladder; M2: Magic Mark XP Western Standard.

[0027] Fig.11 Electron microscopic observation of HN-28 virus strain.

[0028] Fig.12 Nucleic acid banding patterns of viral genomes of different generations of HN-28 strain;

[0029] In the figure: Lanes 1 to 10: Virus harvest fluid from 1 to 10 consecutive passages.

[0030] Figure 13 Homology analysis of gene sequencing of HN-28 strain (5th generation) and each fragment of the virus continuously passed to the 10th and 15th generations.

[0031] Fig.14 Virus titers of HN-28 strain at different culture passages.

[0032] Fig.15 The growth status of cells at different times after the third generation of HN-28 inactivated stock solution infected cells;

[0033] In the figure: (a) inactivated HN-28 1d; (b) inactivated HN-28 2d; (c) inactivated HN-28 3d; (d) inactivated HN-28 4d; (e) control 1d; (f) control 2d; (g) control 3d; (h) control 4d; (i) inactivated HN-28 5d; (j) control 5d; (k) inactivated HN-28 6d; (l) control 6d.

[0034] Fig.16 Antibody responses in sera of mice infected with HN-28 strain.

[0035] Fig.17 Average body weight changes of mice in the vaccine group and control group over 28 days.

[0036] Fig.18 Changes in average body temperature of mice in the vaccine group and the control group 28 days after oral gavage.

[0037] Fig.19 RT-PCR was used to detect the detoxification results of the feces of mice in each group;

[0038] In the figure: lane1-10: corresponds to fecal samples of 1-10 mice, positive: HN-28 stock solution, M: marker. DETAILED DESCRIPTION

[0039] In order to make the purpose and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0040] Test data

[0041] 1 Materials and methods

[0042] 1.1 Cell lines and clinical specimens

[0043] Vero and MA-104 cells are preserved by the Second Research Laboratory of Lanzhou Institute of Biological Products Co., Ltd.; FL (amniotic membrane cells) cells are preserved by the Quality Inspection Laboratory of Lanzhou Institute of Biological Products Co., Ltd.; clinical samples are stool specimens from children with diarrhea collected during the Phase III clinical trial of oral trivalent rotavirus gene reassortment attenuated live vaccine (Vero cells), and the samples that were found to be positive for rotavirus by etiological identification were preserved at -80℃ by the Second Research Laboratory of Lanzhou Institute of Biological Products Co., Ltd.

[0044] 1.2 Main reagents and instruments

[0045] RPMI-1640 and trypsin were purchased from Thermo Fisher, USA; newborn calf serum was purchased from Lanzhou Rongye Technology Bioengineering Co., Ltd.; Plasmocin TM treatment was purchased from Thermo Fisher Scientific, USA; low melting point agarose (Agarose) was purchased from SIGMA; Mycoplasma pneumoniae: strain number ATCC15531, originated from China Food and Drug Inspection Institutes; Mycoplasma oralis: strain number ATCC23714, originated from China Food and Drug Inspection Institutes; Mycobacterium phlei: strain number CMCC95024, originated from China Food and Drug Inspection Institutes; secondary antibody: fluorescein goat anti-rabbit IgG (H+L) was purchased from Thermo Fisher Scientific; total RNA extraction kit RNeasyMini Kit and Agarose Gel DNA recovery kit were both purchased from QIGEN; One-Step PrimeScript RT-PCR Kit (Perfect Real Time) was purchased from TaKaRa; rotavirus ELISA kit was provided by the diagnostic supplies room of our company (National Medicine Approval Number: S10870016). The cell counter was purchased from Roche; the CO2 incubator was purchased from Memmert; the S1000 Thermal Cycler PCR amplifier was purchased from BIO-RAD; the NAN0DROP 2000 nucleic acid quantifier was purchased from Thermo Fisher, USA; and the SpectraMax M3 microplate reader was purchased from Molecular Devices.

[0046] 1.3 Experimental animals

[0047] 20 female BALB / c mice, 7 weeks old, weighing 16-18 g; 10 suckling mice, within 24 hours after birth; 10 Kunming mice, weighing 15-20 g; 10 SPF chicken embryos, 9-11 days old and 5-7 days old.

[0048] 1.4 Cell culture

[0049] The MA-104 cell cryopreservation tube was taken out of liquid nitrogen and revived in a 37°C water bath. The MA-104 cells were cultured in RPMI-1640 (containing 10% newborn calf serum) for 24 h and then replaced with the corresponding culture medium (containing 5% newborn calf serum) for another 72 h. After the cells grew to a monolayer, the growth medium was discarded and the cells were digested with 0.25% Trypsin-0.01% EDTA. 5×10 4T25-FLASK was inoculated with 10 mL cell / mL concentration in each bottle and cultured at 37°C for 96 h. The cells were observed to be confluent as a monolayer under a microscope. The MA-104 cells were washed twice with RPMI-1640 and set aside.

[0050] 1.5 Virus isolation and culture

[0051] Resuspend the rotavirus-positive fecal samples with PBS, shake gently on a vortex oscillator until the feces are dispersed, centrifuge, aspirate the supernatant, filter and sterilize with a 0.22μm filter, and dispense 400μL / tube. Add 300μg / mL pancreatin to the filtrate to a final concentration of 15μg / mL, and bathe at 37℃ for 60min. Then inoculate in MA-104 cells that have grown into a monolayer, inoculate 1 bottle of T25-FLASK for each sample, inoculate 0.8mL per bottle, adsorb at 37℃ for 1h, discard the sample filtrate, and add a solution containing pancreatin and Plasmocin TM RPMI-1640 medium maintenance solution for treatment 10mL / bottle (final concentration of trypsin is 0.5μg / mL, Plasmocin TM The cells were cultured in a 36.5°C, 5% CO2 incubator, and the cell status and cytopathic effects were observed daily. The cells were harvested after 5 to 7 days and the harvest fluid was propagated for 5 generations using the same method. The 5th generation was propagated to T75-FLASK, and the 5th generation culture harvest fluid was used for plaque cloning.

[0052] 1.6 RNA-PAGE assay

[0053] Take 400 μL of the 5th generation virus harvest fluid of HN-28 strain, extract the viral genomic RNA by phenol-chloroform extraction method, perform RNA-PAGE electrophoresis under 20 mA constant current conditions, and detect the nucleic acid banding type of the virus.

[0054] 1.7 Plaque cloning and purification of HN-28 strain The fifth generation virus harvest of HN-28 strain was serially diluted 10-fold, with the dilution factor ranging from 10 -1 Up to 10 -8 times, 10 -3 Up to 10 -8The diluted virus solution was inoculated into a 6-well plate with MA-104 cells. Each dilution was inoculated with 2 replicates, and 0.9 mL was inoculated in each well. After inoculation, the cells were adsorbed in a cell culture incubator at 37°C and 5% CO2 for 4 hours. The supernatant was discarded, and 2 mL / well of 1% low-melting-point agarose prepared with RPMI-1640 was added. After solidification at room temperature, the cells were placed in a cell culture incubator at 37°C and 5% CO2 for 5 days. 2 mL / well of 1% low-melting-point agarose prepared with RPMI-1640 containing neutral red was added. The cells were solidified at room temperature and placed in a cell culture incubator at 37°C and 5% CO2 for 4 to 24 hours to observe the plaques. A single plaque was picked and inoculated into a 24-well plate containing MA-104 cells. The cells were cultured at 37°C and 5% CO2 for 6 days. The positive wells were cloned again, and cloned 5 times in a row. The HN-28 strain with the highest virus titer was selected for gene sequencing and virus purity inspection.

[0055] 1.8 Viral gene identification

[0056] 1.8.1 RT-PCR

[0057] According to the RV-VP7 gene sequence in GenBank, RV G9-type specific primers were designed and synthesized using Primer5.0 software, upstream: 5'-GGCTTTAAAAGAGAGAATTTCCG -3', downstream: 5'-GGT CACATCATACAATTCTAATC-3', and the amplification size was 1062bp. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the expected amplification fragment length was 1062bp. Take 120μL of the 5th generation virus harvest fluid of HN-28 strain, and extract total RNA according to the instructions of the total RNA extraction kit. According to the instructions of the reverse transcription kit, a 25 μL system was used: 10 μL of RNA template was taken, and 12.50 μL of one-step buffer (2×) was added in sequence, 1 μL of upstream primer and downstream primer, 0.5 μL of PrimeScript one-step Enzyme Mix were added, and a total of 25 μL was used in a PCR amplification instrument for the following procedures: 48°C reverse transcription for 30 min, 94°C pre-denaturation for 2 min, 94°C denaturation for 30 s, 52°C annealing for 30 s, 68°C for 2 min, and 68°C extension for 5 min after 40 cycles. After the reaction was completed, 5.00 μL of the PCR amplification product was taken, and 1% agarose gel electrophoresis was performed for 30 min, and the pattern was observed and saved.

[0058] 1.8.2 Determination and analysis of the whole gene nucleotide sequence

[0059] The HN-28 strain was sequenced and identified using the second-generation gene sequencing method (the specific process was completed by Shanghai Berger Medical Technology Co., Ltd.).

[0060] 1.9 Virus purity check

[0061] The fifth generation harvest fluid of the HN-28 strain was subjected to sterility inspection, mycoplasma inspection, exogenous viral factor inspection and mycobacterium inspection according to the current edition of the "Chinese Pharmacopoeia".

[0062] 1.9.1 Sterility test (membrane filtration method)

[0063] Take 30mL of the 5th generation virus harvest fluid of HN-28 strain, of which 20mL of the virus fluid was filtered and loaded into two filter cartridges respectively, and 100mL of thioglycollate medium was added to each. After the remaining 10mL of the virus fluid was filtered, 100mL of tryptic soytone liquid medium was added to the filter cartridge. One of the filter cartridges containing thioglycollate medium was placed at 30-35℃ for incubation, and the other filter cartridges containing thioglycollate medium and tryptic soytone liquid medium were placed at 20-25℃ for incubation; negative control: take RPMI-1640 liquid and operate in the same way. Culture for 14 days and observe the negative control at the same time.

[0064] 1.9.2 Nested-PCR method for mycoplasma detection

[0065] Nested-PCR was used to detect mycoplasma in the fifth generation harvest fluid, negative control and positive standard fluid of HN-28 strain. The detection primers were designed by Lanzhou Institute of Biological Products Co., Ltd. and verified by mycoplasma positive standard. The primer sequences are shown in Table 1.

[0066] Table 1 Mycoplasma primer names and sequences

[0067]

[0068] 1.9.3 Exogenous factor examination

[0069] 1.9.3.1 Cell culture method

[0070] 1.9.3.1.1 Non-hematosorbent virus test Take 2.5 mL of HN-28 virus suspension neutralized with anti-RV serum and inoculate it into amniotic cells, Vero cells and MA-104 cells respectively. Make 6 parallel replicates for each cell type, place them in culture at 36±1℃ and observe for 14 days.

[0071] 1.9.3.1.2 Blood adsorption virus test

[0072] On the 6th to 8th day and the 14th day of culture, take the above virus-inoculated cell cultures (2 bottles of each) for blood adsorption virus inspection. Pour out the cell culture medium, cover the cell surface with 3mL of 0.5% guinea pig and chicken red blood cell mixed suspension, place one bottle at 2-8℃ for 30min, and place the other bottle at 20-25℃ for 30min. After absorbing and discarding the excess red blood cells, perform microscopic examination to observe the red blood cell adsorption.

[0073] 1.9.3.2 Animal testing methods

[0074] Take 10 suckling mice within 24 hours after birth, inoculate 0.01mL of HN-28 virus suspension neutralized with antiserum into the brain, inject 0.1mL into the abdomen, and observe for at least 14 days. Take 10 Kunming mice weighing 15-20g, inoculate 0.03mL of virus suspension neutralized with antiserum into the brain of each mouse, and inject 0.5mL into the abdomen, and observe for at least 21 days.

[0075] 1.9.3.3 Chicken embryo test

[0076] Two groups of SPF chicken embryos, 9-11 days old and 5-7 days old, were selected, with 10 in each group, and the virus suspension neutralized with RV antiserum was inoculated into the allantoic cavity and yolk sac, 0.5 mL per embryo. After 7 days of incubation, the survival of the chicken embryos was observed, and the allantoic fluid was taken for hemagglutination test with 0.2%-0.5% chicken and guinea pig red blood cell mixed suspension.

[0077] 1.9.4 Mycobacterium testing

[0078] Use a sterile syringe to draw 5 mL of HN-28 virus suspension, connect it to a Millipore express syringe filter (0.22 μm), filter the bacteria using a membrane filtration method, and then place the filter membrane in a solid modified Roche culture medium. Make three parallel cultures and place them in a 37°C incubator for 56 days.

[0079] Positive control: Inoculate a tube of solid modified Roche medium with Mycobacterium phlei strain CMCC 95024 and culture in an incubator at 37°C for 56 days.

[0080] Negative control: Take 1 tube of uninoculated solid modified Roche medium and culture it in a 37°C incubator for 56 days.

[0081] 1.10 Virulence testing of the virus

[0082] The efficacy test was carried out in accordance with the 2000 edition of the "Regulations on Biological Products in China", supplemented in 2002. The virus titer was determined by microlesion titration (CCID50) and enzyme-linked immunosorbent assay (ELISA). The specific operation was as follows: MA-104 cells in good growth state were digested with 0.25% trypsin and inoculated into a 96-well culture plate, placed in a 37°C, 5% CO2 incubator and cultured until they grew into a monolayer. The HN-28 virus solution to be tested was diluted 10 times in series with RPMI-1640 maintenance solution, and 10 -2 ~10 -6 Samples with 5 dilutions were inoculated in 4 wells at each dilution in parallel, with 100 μL in each well. RPMI-1640 maintenance solution was used as a blank control. The cells were then placed in a 37°C, 5% CO2 incubator for 6 days and observed daily until the cells showed pathological changes. When harvesting, the 96-well cell plate was frozen and thawed twice, 50 μL of supernatant was aspirated, and the rotavirus ELISA kit was used to detect the cells. A 450 Value, Karber method calculates the corresponding lgCCID 50 / mL.

[0083] 1.11 Determination of HN-28 virus proliferation curve

[0084] The 5th generation harvest fluid of HN-28 strain that passed the exogenous factor inspection was taken and inoculated with 0.01 MOI of MA-104 cells in good growth state. The cells were cultured at 35°C and 5% CO2. Samples were taken once a day. The culture was terminated on the 8th day because most cells showed obvious CPE shedding. The virus titer of the samples harvested every day was measured, and the Karber method was used to calculate 1gCCID 50 / mL value, and draw a growth curve.

[0085] 1.12 Indirect immunoinfluscent assay (IFA)

[0086] The HN-28 virus strain was diluted 10-fold in serum-free RPMI-1640 culture medium. -2 -10 -6MA-104 cells were inoculated in 96-well plates at 5 dilutions, and 8 replicates were made for each dilution. At the same time, blank controls and positive controls were set up. After inoculation of the virus, the cells were placed in a 37°C, 5% CO2 incubator for 18 hours; fixed with pre-cooled 80% acetone solution for 20 minutes, dried at room temperature, added with 1mL / L Tween 20, pH7.4 PBS 200μL / well, soaked for 2 minutes, shaken off, patted dry, added rabbit anti-RV G9 antiserum (primary antibody, diluted 1:200 with pH7.4 PBS), 100μL / well, incubated at 37°C for 1 hour, aspirated and discarded, washed 6 times with PBST, patted dry, added FITC-labeled goat anti-rabbit IgG (secondary antibody, 4 replicates per dilution), incubated at 37°C in the dark for 1 hour, washed in the same way, patted dry, sealed with 60% glycerol, observed and photographed with a fluorescent inverted microscope.

[0087] 1.13 Virus purification

[0088] The cell culture inoculated with HN-28 virus was placed at -20°C for one freeze-thaw, centrifuged at 8000rpm, 4°C for 30min, and the supernatant was collected. The supernatant was taken for sucrose-based ultracentrifugation to collect virus particles. The specific steps are as follows: 30mL of supernatant was added to a 50mL special centrifuge tube, and 20mL of 50% sucrose solution was gently pushed in from the bottom with an 8cm long needle to keep the interface clear. The tube was placed in an ultracentrifuge (model: Hitachi CP 70MX), and ultracentrifuged at 35000rpm at 4°C for 4h. The supernatant was discarded to obtain a purified rotavirus particle precipitate, and the precipitate was resuspended in RPMI-1640 to obtain a purified virus solution.

[0089] 1.14 Viral protein detection

[0090] Protein content determination: Use NanoDrop to detect the protein concentration of the virus suspension, take an appropriate amount of suspension and dilute it to 1 mg / mL with RPMI-1640 medium. SDS-PAGE electrophoresis: Take 40uL of the dilution, add SDS loading buffer, boil at 100℃ for 10min, use 12% gel for SDS-PAGE electrophoresis, stain with Coomassie brilliant blue dye, decolorize, and observe the molecular weight of the protein. Western blot detection: After SDS-PAGE electrophoresis, take the protein sample and transfer it to PVDF membrane, wash 3 times with PBST, and block it with PBST buffer containing 1% BSA at room temperature for 1h; add RV G9 rabbit serum (1:1000 dilution) and incubate for 1h; wash 3 times with PBST, add enzyme (HRP) labeled goat anti-rabbit IgG (1:1000 dilution) and incubate for 1h; wash 3 times with PBST, and color it with DAB colorimetric solution, and perform Western-blot analysis.

[0091] 1.15 Electron microscopy observation

[0092] Electron microscopic observation of virus particles in concentrated samples from ultracentrifugation was performed by the Electron Microscopy Center of Lanzhou University.

[0093] 1.16 Study on the stability of HN-28 strain

[0094] The HN-28 strain virus was continuously passaged on MA-104 cells at a fixed MOI (0.01) for 10 generations and subjected to RNA-PAGE detection; the virus seed, 5th and 10th generation culture harvest fluids were subjected to 2nd generation sequencing to complete homology analysis; and the virus titer of single harvest fluids for 10 consecutive generations was determined.

[0095] 1.17 Preparation of inactivated vaccines

[0096] The sample prepared in 1.13 was sterilized by filtering with a 0.22μm syringe filter, and then dispensed into 100mL sterile Erlenmeyer flasks. A 40% formaldehyde solution was added and shaken to a final concentration of 0.1%. After mixing evenly, it was placed in a 60°C water bath for 30 minutes, and then replaced in a 37°C water bath for 72 hours for inactivation. After inactivation, sodium bisulfite was added to the virus solution to neutralize the residual formaldehyde. The protein concentration was determined by the BCA method, and the protein concentration was diluted to 200μg / mL with RPMI-1640 culture medium to prepare the G9 type HN-28 rotavirus vaccine stock solution, and samples were taken for virus inactivation verification testing; three consecutive generations of blind passages were carried out on MA-104 cells to observe the cytopathic condition, and each generation was harvested after 6 days of culture, and finally tested by CCID 50 The virus inactivation effect was detected by the method. When immunizing animals, the first dose was added with inactivated virus stock solution and Freund's complete adjuvant at a ratio of 1:1, and the second and third doses were mixed with inactivated virus stock solution and Freund's incomplete adjuvant at a ratio of 1:1, so that the final protein concentration of the vaccine was 100 μg / mL, and the mixture was mixed on an oscillator to prepare a G9 type HN-28 rotavirus vaccine sample containing adjuvant.

[0097] 1.18 Immunogenicity test of inactivated vaccines

[0098] 1.18.1 Mouse immunization 20 BALB / C mice weighing 16-18g were collected and blood was collected before immunization. They were randomly divided into 2 groups, 10 mice in each group. In the vaccine group, 10 mice were injected with HN-28 virus inactivated vaccine in the axilla of each limb, 200μL / mouse. After an interval of 28 days, the second and third injections were given with the same dose. The negative control group was injected with RPMI-1640 culture medium 200μL / dose in the same way. After each dose of vaccination, the mice were observed every day for their reactions, mental state, and whether they had symptoms such as hair loss, hair loss, and loss of appetite.

[0099] 1.18.2 Immune response antibody detection and virus challenge Before each immunization and virus challenge, the tail vein blood of mice was collected and incubated at 37°C for 2 h, 4°C overnight, centrifuged for 5 min, and serum was collected. At the end of the experiment, the mice were killed after blood was collected from the eyeballs. The antibody titer was detected by ELISA, and the A value was detected by an enzyme marker at a wavelength of 450 nm. A 450 The wells with a value greater than 2.1 times the negative value were considered positive. The maximum dilution multiple of the positive wells in each serum sample was the antibody titer of the sample. After the first injection, the mice were weighed every day for 28 days, and the mental state and dietary status of the mice were observed during the entire immunization period. All mice were immunized with a virus titer of 5.5lgCCID on the 28th day after the third injection. 50 / mL G9 virus solution was gavaged, and the overdose of 500μL / mouse was used for virus challenge. The feces of mice were observed for 0-96h after virus challenge. The feces of the day were collected 24h, resuspended in PBS, centrifuged and the supernatant was extracted. The RNA in the supernatant was detected by RT-PCR method for the detoxification of mice at 24h. The body temperature was measured every day, and the mental state, diet and clinical manifestations of the mice were recorded in detail. The mice were observed for 28 consecutive days.

[0100] 2 Results

[0101] 2.1 Cell morphological changes during HN-28 culture

[0102] After the clinical sample filtrate was subcultured twice on MA-104 cells, a few cells showed CPE under the microscope; after five consecutive generations, typical CPE caused by rotavirus was produced. In the early stage of the lesion, the cell morphology changed, the cells became hypertrophic and elongated, and in the later stage of culture, the intercellular spaces became loose, and some cells shrank, lysed, and fell off to form large empty plaques. Compared with the control cells, the light transmittance of the culture supernatant was weakened; the cells in the control group grew well, and no CPE was observed. Figure 1 .

[0103] 2.2 RNA-PAGE assay

[0104] The results of RNA-PAGE detection of HN-28 strain showed that the electrophoresis pattern was a unique group A rotavirus pattern (11 bands arranged in 4:2:3:2): 4 high molecular weight dsRNA segments (gene segments 1 to 4); 5 medium-sized segments (gene segments 5 to 9), of which 7 to 9 together formed a characteristic trisome; 2 small segments (gene segments 10 and 11). See Figure 2

[0105] 2.3 Plaque purification results

[0106] The results of the plaque test showed that HN-28 strains formed plaques after adsorption on MA-104 cells for 3 to 4 hours and cultured at 37°C 5% CO2 for 5 to 6 days. The plaques formed with longer adsorption time were larger and more obvious, with round or quasi-round shapes, neat edges, and clear and transparent. The number of plaques gradually decreased with the increase of virus dilution (see Figure 3 ), select a single plaque for amplification and culture, and after ELISA detection, clone the strongest positive well again, clone 5 times in a row, and select the clone strain with high virus titer for further amplification and culture for strain identification and preservation.

[0107] 2.4 RT-PCR identification

[0108] The VP7 RT-PCR amplification product of HN-28 strain was analyzed by 1% agarose gel electrophoresis, and a 1062 bp target fragment was observed, which was consistent with the expected size. Figure 4 .

[0109] 2.5 Determination of the whole genome nucleotide sequence and sequence analysis

[0110] The whole genome nucleotide sequence of HN-28 strain was determined by next-generation sequencing (the specific process was completed by Shanghai Bojie Medical Technology Co., Ltd.). The results of the 11 full-length gene determinations were compared with GenBank, and the evolutionary relationship analysis was performed on BLAST (see Figure 5 The results showed that the HN-28 strain was in a large branch with group A RV and a small branch with group A G9 RV. It was most closely related to RVA / Human / G9P[8]. This isolate was identified as G9P[8].

[0111] 2.6 Virus purity test results

[0112] 2.6.1 Sterility test results After 14 days, the positive control tube was turbid, while the test sample (HN-28 virus suspension) tube and the negative control tube were clear, and the HN-28 strain was judged to have passed the sterility test.

[0113] 2.6.2 Mycoplasma test results

[0114] The results of Nested-PCR showed that the negative control and HN-28 strain harvest fluid were both negative, and the positive control result was positive, which indicated that the mycoplasma test of HN-28 strain was negative. Figure 6 .

[0115] 2.6.3 Results of exogenous factor detection

[0116] 2.6.3.1 Cell culture results

[0117] The results of the non-blood adsorption virus test: negative and positive controls were established, and no cytopathic effect was observed in each cell culture inoculated with the HN-28 virus suspension. The results were negative and met the requirements. The results of the blood adsorption virus test: negative and positive controls were established, and each cell culture inoculated with the HN-28 virus suspension was negative. Figure 7 .

[0118] 2.6.3.2 Animal testing results

[0119] During the observation period, the 10 suckling mice initially vaccinated and the 10 mice vaccinated additionally all survived, and neither the suckling mice nor the mice showed any transmissible factors or other viral infections unrelated to the HN-28 strain, which met the requirements.

[0120] 2.6.3.3 Results of the chicken embryo test

[0121] During the observation period, each group of inoculated chicken embryos survived and the allantoic fluid hemagglutination test was negative, which met the requirements.

[0122] 2.6.4 Mycobacterium test results

[0123] The positive control tube grew well, and the negative control tube and the HN-28 strain harvest fluid inoculation tube were both clear, judging that the HN-28 strain harvest fluid met the requirements.

[0124] 2.7 Virulence test of viruses: Karber method

[0125] Calculation formula: lgCCID50=Ld(s-0.5), where L=logarithm of the highest dilution, d=difference between dilution logarithms, s=total ratio of positive tubes. The virulence of the isolated strains was 5.50-6.50 lgCCID 50 / mL.

[0126] 2.8 Proliferation curve of HN-28 strain

[0127] The HN-28 strain was cultured on MA-104 cells for 9 consecutive days, and CCID samples were collected every day. 50 The virus titer was detected by the method. As the number of culture days increased, the virus titer gradually increased and then decreased. The highest virus infection titer was 6.50lgCCID on the 5th and 6th days. 50 / mL, draw the virus proliferation curve, see Figure 8.

[0128] 2.9 Indirect immunofluorescence assay

[0129] Indirect immunofluorescence test with G9 rabbit serum showed obvious green fluorescence at the cell membrane of the HN-28 infection group at 18h. The fluorescence was more obvious at the place where the cells were aggregated due to CPE, and the higher the degree of aggregation, the stronger the fluorescence. No green fluorescence was observed in the negative control cells, indicating that the cells were infected and replicated by rotavirus. Fig. 9

[0130] 2.10 SDS-PAGE and Western-blot identification

[0131] The results showed that the rabbit anti-G9 polyclonal antibody reacted with the structural proteins of the HN-28 strain, and protein blotting bands with relative molecular weights of 45KD (VP6), 88KD (VP4) and 30KD-40KD (VP7) were observed, which were consistent with the expected molecular weights of the three coat proteins. Fig.10 .

[0132] 2.11 Electron microscopy results

[0133] Typical intact rotavirus particles and damaged virus particles can be clearly seen under the electron microscope. The diameter of intact virus particles is about 75nm, which is consistent with the size of RV. Fig.11 .

[0134] 2.12 Stability Passaging Results

[0135] The HN-28 strain was cultured for 10 generations, and the RNA-PAGE patterns of the 10 generations were consistent (see Fig.12 ); The results of second-generation sequencing showed that the homology of the 11 fragments of the virus seed, 5th generation, and 10th generation viruses was greater than 99.30% (see Fig.13 ); The titer of the virus remained stable at 5.5-6.00 lgCCID50 / mL for 10 consecutive generations (see Fig.14 ).

[0136] 2.13 Detection of inactivation effect of HN-28 strain

[0137] The inactivated stock solution of HN-28 strain was blindly propagated on MA-104 cells for three generations, and no cytopathic changes were observed under the microscope. Fig.15 , the third generation of blind virus liquid was passed by CCID 50 The results of the test were all negative, indicating that the rotavirus antigen in the original solution was completely inactivated.

[0138] 2.14 Results of vaccine immunization in mice

[0139] 2.14.1 Observation results of mice’s reactions after vaccination After vaccination (3 doses), the mice in the experimental group did not show abnormal symptoms such as loss of appetite, slow activity, hair loss, etc. There was no significant difference compared with the mice in the control group.

[0140] 2.14.2 Detection of serum antibody levels in mice after immunization

[0141] The results of ELISA test on serum antibodies showed that compared with the control group, the antibody level of mice immunized with HN-28 inactivated vaccine increased significantly with the increase of immunization doses and the extension of immunization time. The average antibody titer was 2 at 28 days after one dose of immunization. 8.44 , the average antibody titer was 2 on day 28 after the second dose of immunization 11.44 The difference between the two was statistically significant (t=5.669, P<0.05). The average antibody titer was 2 on day 28 after three doses of immunization. 15.6 The difference between the second dose and the second dose was statistically significant (t=6.454, P<0.05). Fig.16 .

[0142] 2.14.3 Changes in body weight of mice after inoculation

[0143] After inoculation of the control and inactivated vaccines, the weight of the control and vaccine groups was measured every day for 28 days. The results showed that the weight of the mice in both groups continued to increase, and there was no statistically significant difference in the change of weight (t=1.234, P=0.2225). Fig.17 .

[0144] 2.14.4 Changes in body temperature of mice after challenge

[0145] The results showed that from the first day after infection, the body temperature of most mice in the control group increased, and the average body temperature was higher than that in the vaccine group. The temperature difference reached the maximum on the second day (t=4.461, P=0.0002) and continued until the fourth day.

[0146] The average body temperature of mice in the control group was higher than that in the experimental group. After one week (8d to 28d), the body temperature of mice in both groups fluctuated within the normal range. Fig.18 .

[0147] 2.14.5 Clinical symptoms and detoxification of mice after challenge

[0148] After the HN-28 virus challenge, the mice in the vaccine group were in good spirits, with only one case of mild diarrhea; while almost all mice in the control group had mild diarrhea, one of which had watery feces, and the diarrhea lasted for 4 days. The results of RT-PCR test of resuspended fecal samples showed that after the virus challenge, only two mice in the vaccine group had rotavirus gene fragments amplified in their feces, while 80% (8 / 10) of the mice in the control group had rotavirus gene fragments amplified in their feces. Fig.19 .

[0149] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A human G9 rotavirus HN-28 strain, characterized in that: The strain is Human rotavirus (G9P8) HN-28Strain, which was deposited in the China Center for Type Culture Collection on January 13, 2022, with the deposit number: CCTCC NO: V202203.

2. Use of the HN-28 strain as claimed in claim 1 in the preparation of rotavirus vaccines.

3. The use according to claim 2, characterized in that: The rotavirus vaccine is prepared by the following steps: S1. Freeze and thaw the cell culture inoculated with HN-28 virus once at -20°C, centrifuge at 8000rpm, 4°C for 30min, collect the supernatant, and perform ultracentrifugation with sucrose as the bottom to collect virus particles. The specific steps are as follows: add 30mL of supernatant to a 50mL special centrifuge tube, gently push 20mL of 50% sucrose solution from the bottom with an 8cm long needle to keep the interface clear, place in an ultracentrifuge, ultracentrifuge at 35000rpm, 4°C for 4h, discard the supernatant, obtain the purified rotavirus particle precipitate, resuspend the precipitate with RPMI-1640, and obtain the purified virus liquid; S2. The purified virus solution was sterilized by filtering with a 0.22 μm syringe filter, and then dispensed into 100 mL sterile Erlenmeyer flasks. A 40% formaldehyde solution was added while shaking to a final concentration of 0.1%. After mixing evenly, the solution was placed in a 60°C water bath for 30 min, and then placed in a 37°C water bath for 72 h for inactivation. After inactivation, sodium bisulfite was added to the virus solution to neutralize the residual formaldehyde. The protein concentration was determined by the BCA method, and the solution was diluted with RPMI-1640 culture medium to a protein concentration of 200 μg / mL to prepare a G9 type HN-28 rotavirus vaccine stock solution, and samples were taken for virus inactivation verification. The inactivated rotavirus was blindly passaged on MA-104 cells for 3 consecutive generations to observe the cytopathic effect. The virus was harvested after 6 days of culture each time, and finally tested by CCID 50 The virus inactivation effect was detected by the method; when animals were immunized, the first dose was added with inactivated virus stock solution and Freund's complete adjuvant at a ratio of 1:1, and the second and third doses were added with inactivated virus stock solution and Freund's incomplete adjuvant at a ratio of 1:1, so that the final protein concentration of the vaccine was 100 μg / mL. The mixture was mixed on an oscillator to prepare a G9 type HN-28 rotavirus vaccine sample containing an adjuvant.

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