A human-sheep rotavirus gene reassortment attenuated strain and its application

Abstract

The present invention discloses a human-sheep rotavirus gene reassortment attenuated strain and application. The human-sheep rotavirus gene reassortment attenuated strain comprises: i) the coding gene of the structural protein VP7 of the human G9 type rotavirus HN-28 strain; and ii) the coding genes of the structural proteins VP1, VP2, VP3, VP4, VP6 and the non-structural proteins NSP1, NSP2, NSP3, NSP4, and NSP5 of the sheep rotavirus LLR strain. The present invention successfully constructs for the first time a human-sheep rotavirus gene reassortment strain with a sheep rotavirus strain as a skeleton, containing the human G9-VP7 gene and capable of stably passaged on Vero cells. The reassortment strain has both the safety of animal strains and the immunogenicity of human G9 type RV, and can effectively prevent infection of human G9 type wild strains, laying a foundation for the research and development of a multivalent RV vaccine covering G9 type.

Description

Technical Field

[0001] The invention belongs to the technical field of classical gene reassortment, and particularly relates to a human-sheep rotavirus gene reassortment attenuated strain and application thereof. Background Art

[0002] Rotavirus (RV) belongs to the genus Rotavirus of the family Reoviridae. The 11 gene segments of the viral genome encode six structural proteins (VP ​​1-4, VP6, VP7) and six non-structural proteins (NSP1-6). [1] There are three inner core proteins (VP1 to VP3), one inner capsid protein (VP6), and two outer capsid proteins (VP4 and VP7). VP6 protein is a group antigen, which is an antigen shared by different strains in the same group. The outer capsid proteins VP7 and VP4 are the main cross-neutralizing antigens of RV and are the main targets for vaccine design. [1] According to the antigenic specificity of VP6, RV can be divided into 9 groups (A~D and F~J), and group A RV (rotavirus A, RVA) is the most common. [3] The types are divided according to the specificity and diversity of the virus coat proteins VP7 and VP4, the G serotypes (G1 to G35) are divided according to the VP7 sequence, and the P genotypes are divided according to the VP4 sequence. [2] (P[1]~P

[50] ). Most human and animal RVs are RVA. The main difference between human RVA and animal RVA is the specific surface antigen type. Humans are the only host of human RV. Epidemiology has not yet confirmed that RV is transmitted from animals to humans. [4] Human RVA is one of the leading pathogens of severe gastroenteritis and death in children under 5 years old worldwide. [5] , G1, G2, G3, G4, and G9 constitute the main epidemic strains in my country [6] , of which G9 and P[8] RV infections have now spread throughout the world and have become the dominant strains in some countries and / or regions. [7-8] .

[0003] Although RV has been studied for decades, there is still no specific treatment. Vaccination is the most economical and effective way to prevent RV infection. [9]The RV vaccines currently in use around the world mainly include: 1) LLR: isolated lamb RV strain G10P

[12] type is inoculated into newborn bovine kidney cells, cultured, the virus fluid is harvested, and stabilizers are added. It was developed by Lanzhou Institute of Biological Products Co., Ltd., China; 2) Rotarix: the strain is RIX444 (G1P[8] type), developed by GlaxoSmithKline of Belgium; 3) RotaTeq: based on bovine rotavirus WC3, the genes of VP4 and VP7 in human rotavirus are replaced by the corresponding genes of the original strain through gene recombination. , a pentavalent vaccine of G1, G2, G3, G4, and P[8] was obtained, developed by Merck in the United States; 4) Rotavac: a monovalent liquid frozen attenuated live vaccine of G9P

[11] , developed by Bharat Company in India; 5) ROTASIIL: a pentavalent live attenuated vaccine of bovine-human rotavirus, whose serotypes include G1, G2, G3, G4, and G9, developed by the Serum Institute of India; 6) Rotavin-M1: a frozen attenuated live vaccine made from the G1P[8] strain isolated from children, developed by POLYVAC Company in Vietnam.

[0004] At present, the country still lacks an ideal vaccine strain for the G9 rotavirus live attenuated vaccine that has good biological properties, immunogenicity and protective effects, and is safe and efficient. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a human-sheep rotavirus gene reassortment attenuated strain and its application. The strain uses the sheep rotavirus strain as the skeleton, contains the human G9-VP7 gene and can be stably propagated on Vero cells. It has good immunogenicity and protective effect in BALB / c mice and is an ideal vaccine candidate strain for a safe and efficient G9 type rotavirus attenuated live vaccine.

[0006] In one aspect of the present invention, a human-sheep rotavirus gene reassortment attenuated strain is provided, comprising: i) a gene encoding the structural protein VP7 of the human G9 rotavirus HN-28 strain; and ii) genes encoding the structural proteins VP1, VP2, VP3, VP4, VP6 and non-structural proteins NSP1, NSP2, NSP3, NSP4, NSP5 of the sheep rotavirus LLR strain.

[0007] According to a specific embodiment of the present invention, the human-sheep rotavirus gene reassortment attenuated strain is the LG9 strain, and the preservation number is CCTCC NO: V202429.

[0008] Another aspect of the present invention provides a method for constructing the aforementioned human-sheep rotavirus gene reassortment attenuated strain, comprising: co-infecting a monolayer of MA-104 cells with a human G9 rotavirus HN-28 strain and a sheep rotavirus LLR strain; harvesting the mixed culture when the monolayer MA-104 cell lesions are ≥75%; and screening the target gene reassortment strain from the mixed culture by limiting dilution and plaque assays.

[0009] Another aspect of the present invention provides the use of the aforementioned human-sheep rotavirus gene reassortment attenuated strain in the preparation of rotavirus vaccine.

[0010] Another aspect of the present invention provides the use of the aforementioned human-sheep rotavirus gene reassortment attenuated strain in the preparation of rotavirus diagnostic reagents.

[0011] Another aspect of the present invention provides a vaccine comprising an immunogenic effective amount of the aforementioned human-sheep rotavirus gene reassortment attenuated strain.

[0012] According to a specific embodiment of the present invention, the vaccine further comprises at least one acceptable adjuvant.

[0013] According to a specific embodiment of the present invention, the vaccine further comprises at least one pharmaceutically acceptable carrier or diluent.

[0014] According to a specific embodiment of the present invention, the dosage form of the vaccine is an oral dosage form or an injection dosage form.

[0015] Another aspect of the present invention provides a diagnostic reagent comprising the aforementioned human-sheep rotavirus gene reassortment attenuated strain.

[0016] The beneficial effects of the present invention are:

[0017] The present invention successfully constructed for the first time a human-sheep rotavirus gene reassortment attenuated strain with a sheep rotavirus strain as the skeleton, containing the human G9-VP7 gene and capable of stably propagating on Vero cells. The reassortment attenuated strain carries the VP7 gene of human RV G9 (stimulating human G9-specific neutralizing antibodies), and the other 10 gene fragments are all from the sheep rotavirus strain LLR. It has both the safety of animal strains and the immunogenicity of human G9 RV, and can effectively prevent infection by human G9 wild strains, laying a foundation for the development of a multivalent RV vaccine covering G9. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The RNA-PAGE genomic nucleic acid banding pattern of the gene reassortment mixture; wherein, lanes 1 to 2 are the gene reassortment mixture; lane 3 is the HN-28 control; lane 4 is the LLR control;

[0019] Figure 2 The gene reassortment mixture was adsorbed for 4 hours and cultured for 4 days to form plaques;

[0020] Figure 3 The RNA-PAGE genomic nucleic acid bands of different clones; 1 is the HN-28 control; 2 is the LLR control; lanes 3 to 8 are different clones;

[0021] Figure 4 It is the RNA-PAGE genomic nucleic acid banding pattern of the LG9 strain; among them, lanes 1 to 2 are the virus harvest fluids cultured in MA-104; lanes 3 to 4 are the virus harvest fluids cultured in Vero; G9 is the HN-28 control; L is the LLR control;

[0022] Figure 5 The results of mycoplasma test, where 1 is LG9 virus harvest fluid; 2 is negative control; M is DNA Marker; 3 is oral mycoplasma; 4 is mycoplasma pneumoniae;

[0023] Figure 6 This is the electron microscopy observation of the LG9 strain;

[0024] Fig. 7A To purify the viral protein of LG9 strain - SDS-PAGE analysis;

[0025] Figure 7B To purify the viral protein of LG9 strain - Western-blot analysis;

[0026] Fig. 8A This is a photo of Vero cells 6 days after infection with LG9 strain (×100);

[0027] Figure 8B The photo is of the control Vero cells after 6 days of culture (×100);

[0028] Fig. 9 This is the virus proliferation curve of LG9 strain;

[0029] Fig.10 RNA-PAGE profiles of different generations of LG9 strains; lanes 1 to 20 are the LG9 virus harvest fluids of 1 to 20 generations of continuous passages; G9 is the HN-28 strain control; L is the LLR control;

[0030] Fig.11A It is the homology analysis map of VP1 gene of LG9 strains P5, P10, P15, and P20;

[0031] Fig. 11B It is the homology analysis map of VP2 gene of LG9 strains P5, P10, P15 and P20;

[0032] Fig. 11CIt is the homology analysis map of VP3 gene of LG9 strains P5, P10, P15 and P20;

[0033] Fig.11D It is the homology analysis map of VP4 gene of LG9 strains P5, P10, P15 and P20;

[0034] Fig.11E It is the homology analysis map of NSP1 gene of LG9 strains P5, P10, P15 and P20;

[0035] Fig.11F It is the homology analysis map of VP6 gene of LG9 strains P5, P10, P15 and P20;

[0036] Fig.11G It is the homology analysis map of NSP2 gene of LG9 strains P5, P10, P15 and P20;

[0037] Fig.11H It is the homology analysis map of NSP3 gene of LG9 strains P5, P10, P15 and P20;

[0038] Fig.11I It is the homology analysis map of VP7 gene of LG9 strains P5, P10, P15 and P20;

[0039] Fig.11J It is the homology analysis map of NSP4 gene of LG9 strains P5, P10, P15 and P20;

[0040] Figure 11K It is the homology analysis map of NSP5 gene of LG9 strains P5, P10, P15 and P20;

[0041] Fig.12 The virus titers of LG9 strain at different passages in Vero cells;

[0042] Fig.13 The ELISA test results of fecal toxin excretion in each group of mice after immunization;

[0043] Fig.14 The average body temperature changes of mice in each group within 28 days after immunization;

[0044] Fig.15 The average body weight changes of mice in each group within 28 days after immunization;

[0045] Fig.16A The ELISA test results of IgG antibody response in the serum of each group of mice;

[0046] Fig. 16B ELISA test results of IgA antibody response in the serum of mice in each group;

[0047] Fig.17 The ELISA test results of fecal toxin excretion in each group of mice after infection;

[0048] Fig.18 The average body temperature changes of mice in each group within 7 days after infection;

[0049] Fig.19 The average body temperature changes of mice in each group within 7 days after infection. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] In the following examples, the materials and methods involved are as follows:

[0052] 1. Cells: Rhesus monkey kidney cells (MA-104, P30); African green monkey kidney cells (Vero, P135) were preserved and provided by the Second Research Laboratory of Lanzhou Institute of Biological Products Co., Ltd.

[0053] 2. Virus strains and strains: Virus strains: sheep strain LLR, commercially available monovalent human rotavirus vaccine strain, Lanzhou Institute of Biological Products Co., Ltd., is group A G10P

[12] type, isolated from sheep; HN-28 strain: deposited in the China Center for Type Culture Collection on January 13, 2022, with the deposit number: CCTCC NO: V202203, is group A G9P[8] type, isolated from the feces of children with diarrhea. Strains: Mycoplasma pneumoniae (strain number ATCC15531) and Mycoplasma oralis (strain number: ATCC23714) were purchased from the China Food and Drug Administration.

[0054] 3. Main reagents and instruments: RPMI-1640, MEM and trypsin were purchased from Gibco, USA; newborn calf serum was purchased from Lanzhou Rongye Technology Bioengineering Co., Ltd.; low melting point agarose, HRP-labeled goat anti-rabbit IgG and IgA were purchased from Sigma, USA; anti-RV LLR rabbit serum was prepared by the Second Research Laboratory of Lanzhou Institute of Biological Products Co., Ltd.; Lumi-Light Western blotting substrate was purchased from Thermo, USA; total RNA extraction kit RNeasyMini Kit and Agarose Gel DNA recovery kit were purchased from Qiagent, Germany; One-Step PrimeScript RT-PCR Kit (Perfect Real Time) was purchased from TaKaRa, Japan; rotavirus ELISA kit was provided by the Diagnostic Supplies Room of Lanzhou Institute of Biological Products Co., Ltd. (National Medicine Approval Number: S10870016). Fluorescence cell analyzer was purchased from Shanghai Ruiyu Biotechnology Co., Ltd.; CO2 INC-108 incubator was purchased from Memmert, Germany; XD-30 inverted microscope was purchased from Ningbo Sunny Optical Instrument Co., Ltd.; SpectraMax M3 microplate reader was purchased from Molecular, USA; S1000 Thermal Cycler PCR amplifier was purchased from BIO-RAD, USA; NANODROP 2000 nucleic acid quantification instrument was purchased from Thermo Fisher, USA; ImageQuant LAS 4000 gel imager was purchased from General Electric (GE), USA.

[0055] 4. Data processing method: Prism 8.0.1 software was used to perform statistical analysis on the experimental data of different test groups and control groups. The difference was statistically significant when P < 0.05.

[0056] Example 1 Construction of LG9 strain

[0057] 1. Classical gene reassortment

[0058] 1.1 MA-104 cell culture: The MA-104 cell cryopreservation tube was taken out from liquid nitrogen, revived in a 37.0°C water bath, cultured with RPMI-1640 (containing 10% newborn calf serum) for 24 h, and then replaced with the corresponding culture medium (containing 5% newborn calf serum) and cultured for 72 h. After the cells grew to a monolayer, the growth medium was discarded and the cells were digested with 0.25% Trypsin-0.02% EDTA. 5×10 4 The cells were inoculated into a 24-well plate at a concentration of cell / mL, 1 mL / well, and cultured at 37.0°C for 96 h. After the cells were observed to be confluent as a monolayer under a microscope, they were washed once with RPMI-1640 medium and set aside.

[0059] 1.2 HN-28 and LLR virus gene reassortment: Add trypsin with a final concentration of 15 μg / mL to the parent strain LLR and parent strain HN-28, incubate in a 37.0℃ water bath for 60 min, and then perform 10-fold serial dilutions from a dilution of 10 4 The two strains were mixed to infect MA-104 cells at a virus quantity of HN-28 / LLR = 1:1 to 1:50000, and the cells were adsorbed at 37.0°C 5.0% CO2 for 2 hours, gently shaken once for 20 minutes, and the adsorption liquid was discarded after 2 hours. The cells were washed twice with RPMI-1640 liquid, and serum-free RPMI-1640 culture medium containing 0.5 μg / mL was added to 1 mL / well. The cells were cultured in a 37.0°C 5.0% CO2 cell culture incubator, and the mixed culture was harvested when CPE ≥ 75% was observed under a microscope.

[0060] 2. Isolation and screening methods of gene reassortment attenuated strains

[0061] 2.1 Isolation of gene reassortment attenuated strains: The target reassortant strains were isolated by limiting dilution and plaque assays, i.e., the mixture was serially diluted 10-fold to 10 8 times, the dilution is 10 3 ~10 8 The virus solution was inoculated into a 6-well plate with MA-104 cells. Two duplicate wells were inoculated for each dilution, and 0.9 mL was inoculated in each well. The cells were adsorbed in a cell culture incubator at 37.0°C and 5% CO2 for 4 hours. The supernatant was discarded, and 1% low-melting point agarose prepared with RPMI-1640 was added at 2 mL / well. The cells were cultured after solidification at room temperature. After 4 to 5 days, 1% low-melting point agarose prepared with RPMI-1640 containing neutral red was added at 2 mL / well according to CPE. The cells were cultured after solidification at room temperature, and the plaques were observed for 4 to 24 hours. A single independent plaque was picked and transplanted onto MA-104 cells in a 24-well plate. One plaque was inoculated into one well. The cells were cultured in a cell culture incubator at 37.0°C and 5% CO2 for 4 to 6 days. The cells were harvested when CPE ≥ 75%, and then sampled for detection.

[0062] 2.2 RNA polyacrylamide gel electrophoresis (RNA-PAGE) detection: The plaques were transferred to the harvested fluid of the 24-well plate culture, and 400 μL was extracted from each well. The viral genomic RNA was extracted by phenol-chloroform extraction method and the standard polyacrylamide gel electrophoresis (PAGE) procedure was followed.

[10] Electrophoresis was performed with a gel concentration of 10% and a sample volume of 10 μL / lane. HN-28 and LLR lanes were added as controls. The target gene reassortant progeny strains with VP7 bands consistent with the HN-28 strain control and other bands consistent with the LLR strain were initially selected. The target gene reassortant progeny strain was cloned again, and the cloning was repeated 6 times. The positive well with the strongest OD value detected by ELISA in the 6th empty plaque clone was selected, amplified and cultured, and the virus strain was preserved after passing the test, which was named LG9 strain. Deposit date: 2024.03.15, deposit unit: China Center for Type Culture Collection, deposit address: Wuhan University, Wuhan, China, deposit number: CCTCCNO: V202429.

[0063] 3. Results

[0064] 1) RNA-PAGE electrophoresis of gene reassortment mixture: The results showed that the mixture contained all gene segments of the two virus strains, indicating that the two strains can coexist at a suitable ratio. Figure 1 .

[0065] 2) Plaque purification: Plaque assay results showed that the virus mixture formed typical plaques on MA-104 cells, which were round or quasi-round in shape, with neat edges, clear and transparent; and as the virus dilution increased, the number of plaques gradually decreased. Figure 2 A single plaque was selected and cultured in a 24-well plate. After ELISA detection, the culture fluid of the positive well was sampled and the strain was preliminarily identified by RNA-PAGE detection.

[0066] 3) Preliminary screening of target monoclonal gene reassortment attenuated strains: According to the position of the bands in the PAGE profile, the clone in lane 6 was selected as the target gene reassortment strain. Its VP7 gene band was consistent with HN-28 (G9 type), and the other bands were consistent with LLR. Figure 3 After 6 rounds of plaque purification, a pure gene reassortment attenuated strain was obtained.

[0067] Example 2 Detection of LG9 strain

[0068] 1. Virus titer determination: After freezing and thawing, the samples were collected and tested according to the potency test section of the 2000 edition and 2002 supplement of the "Regulations on Biological Products in China". [11-12] The cell culture infective dose 50% (CCID 50 ) and ELISA to determine the virus titer, and the Karber method to calculate the corresponding lgCCID 50 / mL.

[0069] Calculated using the Karber formula: lgCCID 50=Ld(s-0.5), where L = the logarithm of the highest dilution, d = the difference between the logarithms of the dilutions, s = the sum of the positive tube ratios, and the test result LG9 has a virus titer of 7.50lgCCID 50 / mL.

[0070] 2. RNA-PAGE assay: Take 400 μL of LG9 virus solution, extract the viral genomic RNA by phenol-chloroform extraction method, detect the viral nucleic acid banding type by PAGE and silver staining, and preserve it with dry gel.

[0071] PAGE showed that the electrophoretic pattern of the LG9 strain was long, and the 11 bands showed a unique 4:2:3:2 arrangement of group A rotavirus. Only the VP7 gene segment was parallel to the HN-28VP7, and the other segments were parallel to the LLR segment. The electrophoretic patterns of the viruses cultured in Vero cells and MA-104 cells were consistent, but the bands in the MA-104 cell harvest fluid were clearer. Figure 4 .

[0072] 3. Whole genome sequencing and identification: Whole genome sequencing and identification were performed using the second-generation gene sequencing method (the specific process was completed by Shanghai Berger Medical Technology Co., Ltd.). The sequence of VP1 is shown in SEQ ID NO: 1, the sequence of VP2 is shown in SEQ ID NO: 2, the sequence of VP3 is shown in SEQ ID NO: 3, the sequence of VP4 is shown in SEQ ID NO: 4, the sequence of NSP1 is shown in SEQ ID NO: 5, the sequence of VP6 is shown in SEQ ID NO: 6, the sequence of NSP3 is shown in SEQ ID NO: 7, the sequence of NSP2 is shown in SEQ ID NO: 8, the sequence of VP7 is shown in SEQ ID NO: 9, the sequence of NSP4 is shown in SEQ ID NO: 10, and the sequence of NSP5 is shown in SEQ ID NO: 11.

[0073] The results of the second-generation sequencing showed that among the 11 full-length gene fragments of the LG9 strain, the homology between VP7 and G9P[8] was 99.43%, and the homology between the remaining fragments and sheep RV was ≥99.81%, indicating that the VP7 gene of the LLR strain and the G9-type VP7 gene had been exchanged, and a human-sheep rotavirus gene reassortment attenuated strain containing the G9-type VP7 gene was successfully obtained (see Table 1).

[0074] Table 1 Comparison results of 11 full-length gene sequences with GenBank accession sequences

[0075]

[0076] 4. Sterility test and mycoplasma test: Sterility test shall be carried out in accordance with the 2020 edition (Part III) of the Chinese Pharmacopoeia

[13] ; Nested-PCR method

[14] For mycoplasma detection, the primers were designed by Lanzhou Institute of Biological Products Co., Ltd. and verified by mycoplasma positive standard strains. The primer sequences are shown in Table 2.

[0077] Table 2 Mycoplasma primer names and sequences

[0078]

[0079] After 14 days of culture, the positive control tube was turbid, while the test sample (LG9 virus suspension) tube and the negative control tube were clear, indicating that the LG9 strain passed the sterility test. The Nested-PCR results showed that there were no PCR amplification bands in the LG9 harvest fluid and the negative control, and the positive control amplified clear bands of the corresponding size, indicating that the LG9 strain was not contaminated with mycoplasma. Figure 5 .

[0080] 5. Electron microscope (EM) observation: The LG9 virus harvest solution was frozen once at -20°C, thawed at room temperature, and centrifuged at 8000r / min and a centrifugal radius of 7cm for 30min at 4°C. The supernatant was collected and subjected to 50% sucrose-pad ultracentrifugation. The solution was centrifuged at 35000r / min and a centrifugal radius of 4.5cm for 4h at 4°C. The supernatant was discarded and the precipitate was resuspended in RPMI-1640 to obtain purified virus. The purified virus particles were diluted to 100μg / mL with PBS, and a 200-mesh carbon-free Formvar copper mesh was placed in 50μL of the virus solution for 5min. The residual virus solution on the copper mesh was absorbed with filter paper, and then the copper mesh was placed in 2% phosphotungstic acid for negative staining for 5min. The residual phosphotungstic acid on the copper mesh was absorbed with filter paper. After drying at room temperature overnight, transmission photography was performed (Electron Microscopy Center of Lanzhou University).

[0081] Under electron microscopy, the LG9 virus was clearly visible as a typical complete rotavirus particle. The diameter of the complete virus particle was about 75nm, which was consistent with the size of RV. Figure 6 .

[0082] 6. Virus protein detection: Take an appropriate amount of ultracentrifugation purified LG9 virus sample and dilute it to 1 mg / mL for SDS-PAGE and Western-blot detection. SDS-PAGE electrophoresis: Take 40 μL of the diluent, add SDS loading buffer, boil at 100°C for 10 minutes, 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: Take the protein sample for SDS-PAGE electrophoresis, transfer it to PVDF membrane, wash 3 times with PBST, block with PBST buffer containing 1% BSA at room temperature for 1 hour; add LLR rabbit serum (1:1000 dilution) and incubate for 1 hour; wash 3 times with PBST, add HRP-labeled goat anti-rabbit IgG (1:1000 dilution) and incubate for 1 hour; wash 3 times with PBST, develop with DAB colorimetric solution, and perform Western-blot analysis.

[0083] SDS-PAGE results showed that the antigenic components of the LG9 virus purified by ultracentrifugation were complex. Western-blot analysis showed that the ultracentrifugation purified LG9 virus and anti-RV LLR rabbit serum showed specific reaction bands at the expected relative molecular mass positions of VP2, VP3, VP4, NSP1, VP6, NSP3, NSP2 and NSP4 proteins, and no VP7 protein band, which was consistent with the expected results, see Figure 7A to Figure 7B .

[0084] Example 3 Study on the stability of LG9 strain

[0085] 1. Vero cell culture: The Vero cells (passage 135) were taken out of the cryopreserved tubes of liquid nitrogen and revived in a 37.0°C water bath. Vero cells were cultured with MEM (containing 10% newborn calf serum) for 24 h and then replaced with the corresponding culture medium (containing 5% newborn calf serum). The culture was continued for 72 h. After the cells grew to a monolayer, the growth medium was discarded and the cells were digested with 0.25% Trypsin-0.02% EDTA. The cells were cultured at 1×10 5 T25-FLASK was inoculated with 10 mL / bottle at a concentration of cell / mL, and cultured at 37.0°C for 96 h. After the cells were fully covered in a monolayer under a microscope, they were washed once with MEM and set aside.

[0086] 2. Adaptive culture of LG9 strain on Vero cells: Take 1 mL of LG9 strain P5 harvest fluid cultured with MA-104 cells, add 50 μL of 300 μg / mL trypsin to a final concentration of 15 μg / mL, incubate in a 37.0°C water bath for 60 min, then inoculate into a bottle of Vero cells that have grown into a monolayer, place at 37.0°C for adsorption for 1 h, discard the adsorption fluid, add MEM maintenance fluid to 10 mL (the final concentration of trypsin is 0.5 μg / mL), and place in a 36.5°C, 5% CO2 incubator for culture. Observe the cell status and CPE every day, harvest according to CPE after 5 to 7 days of culture, take the previous generation harvest fluid as the virus seed, and pass it for more than 5 times in a row.

[0087] 3. Study on the stability of LG9 strain in Vero cells: The LG9 strain was inoculated into Vero cells at an MOI of 0.01 and passed for 20 consecutive generations, and RNA-PAGE detection was performed; samples from P5, P7, and P9 were randomly selected every day to detect the virus titer and draw the virus proliferation curve; the P5, P10, P15, and P20 virus harvest fluids were subjected to second-generation sequencing and homology analysis; and the virus titer of single harvest fluids was determined for 20 consecutive generations.

[0088] 4. Results:

[0089] 1) Morphological changes of Vero cells infected by LG9 strain: Microscopic observation showed that a large number of Vero cells detached and elongated when LG9 infected Vero cells were cultured for 6 days, and some cells lysed, resulting in poor light transmittance of the liquid; cells in the control group grew well and no CPE was observed, indicating that the LG9 strain can adapt well to Vero cells. Figure 8A to Figure 8B .

[0090] 2) Proliferation curve of LG9 strain in Vero cells: LG9 virus was cultured on Vero cells for 9 days, CCID 50 The virus titer of each sample was detected by the method, and the virus proliferation curve was drawn. Fig. 9 The virus titer gradually increased with the increase of culture days, and the highest infection titer of the virus was 7.50lgCCID at 5-8 days. 50 / mL, and then began to show a downward trend after maintaining a plateau for 2-3 days.

[0091] 3) Stability passage results: RNA-PAGE results showed that the electrophoresis patterns of the viral genomes of each generation were consistent, see Fig.10 The results of the second generation sequencing showed that the homology of the 11 fragments of P5, P10, P15, and P20 viruses was 100%, indicating that the LG9 strain had good genetic stability on Vero cells and no gene mutation occurred during the passage process. Figures 11A to 11K CCID cells cultured for 20 generations on Vero cells50 The results showed that the virus titer was stable at 7.50lgCCID 50 / mL, indicating that LG9 maintained good proliferation stability on Vero cells. Fig.12 .

[0092] Example 4 Immunogenicity test of LG9 strain

[0093] 1. Mouse immunization: 30 BALB / c mice weighing 18-20g were randomly divided into 3 groups, 10 mice in each group, and blood was collected before immunization. Experimental group 1 was immunized by gavage with 200μL / mouse to simulate oral vaccination; experimental group 2 was immunized by intraperitoneal injection at 200μL / mouse; group 3 served as the negative control group and was gavaged with MEM culture medium at 200μL / mouse. The body temperature and weight of each mouse were tested 0-28 days after immunization. After 28 days, each group was immunized with the second dose at the same dose. Tail blood was collected before each dose of immunization (diluted 4 times when collected); after immunization, the mouse's reaction, mental state, and symptoms such as hair loss, hair loss, and loss of appetite were observed every day; because rotavirus infection is manifested in the early stages as watery diarrhea, fever, and vomiting, and diarrhea symptoms will last for 4-5 days. [15-19] Therefore, in this experiment, the feces characteristics of mice were observed from 0 to 7 days after immunization. Fecal samples were collected from 4 hours to 7 days, and fresh feces were collected every 24 hours. After resuspending in PBS, the suspensions of the same mouse were combined and centrifuged to obtain the supernatant. ELISA detection was performed and the readings were read on the microplate reader (the blank well was adjusted to zero and each well was read at a wavelength of 450nm). If the OD value of the fecal supernatant well (S) / the OD value of the negative control well (N) ≥ 2.1, it was considered positive, and the mouse corresponding to the fecal sample was judged to have excreted toxins during the observation period.

[0094] 2. Serum antibody detection: using indirect ELISA [9] Detect the IgG and IgA antibody titers in the serum. Plate: Dilute the LG9 virus antigen to a virus titer of 5.5lgCCID 50 / mL coated ELISA plate, 100μL / well, placed at 37℃ for 2h, overnight at 4℃, washed with PBST 5 times, blocked with 20% newborn calf serum after patting dry, placed at 37℃ for 2h, overnight at 4℃, patted dry and stored at -20℃ for use. Mouse serum was diluted 2-fold serially and added to the ELISA plate, 100μL / well, 2 wells for each dilution, and negative control wells were set at the same time. Placed at 37℃ for 1h, washed with PBST 5 times, HRP-labeled anti-mouse IgG (1:2000) or IgA antibody (1:2000) was added, 100μL / well, placed at 37℃ for 1h, washed with PBST 5 times, added color substrate H2O2 / TMB, 50μL / well, placed at 37℃ for 10min to terminate the reaction, and the OD value was detected using an ELISA reader. Antibody titer determination standard: The highest serum dilution with serum OD value (S) / negative serum OD value (N) ≥ 2.5 times is the antibody titer.

[0095] 3. Virus challenge test in immunized mice: All mice were immunized with a virus titer of 5.5lgCCID on the 28th day after the second dose. 50 / mL HN-28 wild strain virus liquid was gavaged (simulating fecal-oral infection), 500μL / mouse. The body temperature, weight, mental state, diet, feces characteristics, etc. of the mice were recorded every day within 7 days after the infection. Feces were collected from 4h to 7d, and collected every 24h. The detoxification of each group of mice after infection was tested as in "1".

[0096] 4. Results

[0097] 1) Observation results of clinical symptoms of mice after immunization: After two doses of antigen were administered to mice in test group 1 and test group 2, no abnormal symptoms such as vomiting, loss of appetite, slow movement, ruffled hair, or hair loss occurred. Some mice developed mild diarrhea, but the amount of diarrhea was not significantly different from that in the control group.

[0098] 2) Detoxification of mice after immunization: The results of ELISA test of resuspended feces showed that after one dose of immunization, RV was detected in the feces of mice in test group 1, test group 2 and control group, but the positive rate was low, 10% (1 / 10) in the test group and 20% (2 / 10) in the control group. The positive rate of feces samples of mice in the test group was lower than that in the control group. After two doses of immunization, the positive rate of feces in test group 1, test group 2 and control group was 10% (1 / 10). Fig.13 .

[0099] 3) Changes in body temperature and weight of mice after immunization: After LG9 immunization, the body temperature and weight of mice in the control group and the two experimental groups were tested every day for 28 days. The results showed that the body temperature of the three groups of mice fluctuated within the normal range within 28 days after vaccination. The results of variance analysis using Prism8.0.1 software showed that there was no statistically significant difference (F=2.420, P=0.0951). Fig.14 After immunization, the body weight of mice in the three groups continued to increase, and the changes in body weight were not statistically significant (F = 0.0.0004692, P = 0.9995). Fig.15 .

[0100] 4) Analysis of immune response of serum antibodies in mice after immunization: The purified LG9 virus was coated on plates, and the IgG and IgA antibody titers in the sera of immunized mice were determined by indirect ELISA. The positive conversion rates of IgG and IgA antibodies in test groups 1 and 2 were both 100%, and the results of the control group and pre-immunization serum were both negative (assigned as 0) within the set detection range. 28 days after the first dose of immunization, the average titer of serum IgG antibody in test group 1 was 1.8128lg, which was significantly different from that in the control group (t=23.13, P<0.0001); the average titer of IgG antibody in test group 2 was 2.2041lg, which was significantly different from that in the control group (t=14.18, P<0.0001); 28 days after the second dose of immunization, the average titer of serum IgG antibody in test group 1 and test group 2 was 4.5286lg, which was significantly different from that in the first dose (t=19.90 and t=10.99, respectively, P<0.0001); 28 days after the first dose of immunization, the average titer of serum IgG antibody in test group 1 and test group 2 was 4.5286lg, which was significantly different from that in the first dose (t=19.90 and t=10.99, respectively, P<0.0001); The serum IgA was 1.1694 lg, which was significantly different from the control group (t=26.34, P<0.0001); the average titer of IgA antibody in the experimental group 2 was 1.3612, which was significantly different from the control group (t=33.92, P<0.0001); 28 days after the second dose of immunization, the serum IgA in the experimental group 1 was 2.1138 lg, which was significantly different from the first dose (t=4.159, P<0.05); the average titer of IgA antibody in the experimental group 2 was 1.9031 lg, which was significantly different from the first dose (t=7.216, P<0.0001), see Figure 16A to Figure 16B .

[0101] 5) Challenge test results:

[0102] a. Clinical symptoms and detoxification of mice after infection: According to naked eye observation, only 1 mouse in test group 1 had mild diarrhea, and only 2 mice in test group 2 had mild diarrhea. The diarrhea lasted for a short time and the mice were in good spirits. In the control group, 6 mice had diarrhea, of which 4 mice had ruffled hair and loss of appetite, and 1 mouse had severe diarrhea with watery feces and depression. The diarrhea lasted until the 4th day.

[0103] After resuspending the virus, the fecal samples of each mouse in each group were tested by ELISA. The results showed that RV was positive in the feces of 1 mouse in the test group 1, RV was positive in the feces of 3 mice in the test group 2, and RV was positive in the feces of 7 mice in the control group, as shown in Table 3. Fig.17This indicates that LG9 strains can protect mice from infection by wild strains after immunization with different routes. The results of this experiment also show that oral immunization has a better protective effect than injection immunization, suggesting that oral immunization may be more protective against RV infection.

[0104] Table 3 Diarrhea and antigen detection in feces of mice in each group after infection

[0105]

[0106] b. Changes in body temperature and weight of mice in each group within 1 week after challenge: The results showed that within 5 days after challenge with the wild-type HN-28 virus, the body temperature of most mice in the control group began to rise, and the average body temperature was significantly higher than that of the vaccine group. The differences in body temperature between the experimental groups 1 and 2 and the control group were statistically significant (t=2.164 and 2.234, respectively, P<0.0001). After 6 days, the body temperatures of the mice in the three groups returned to normal and fluctuated within the normal range. Fig.18 .

[0107] c. Within 5 days after the infection, the average weight of the mice in the two groups increased slowly, with no significant weight loss; while the average weight of the mice in the control group decreased significantly 1 day after the infection, and their mental state recovered 4 days later, and their weight also increased. The statistical analysis results showed that there was no statistically significant difference in the weight change of mice in the two experimental groups (t = 0.1869, P = 0.8545), and there was a statistically significant difference in the weight change between the experimental group 1 and the experimental group 2 and the control group (t = 3.281 and 3.160, respectively, P < 0.05). Fig.19 .

[0108] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0109] References

[0110] [1]CRAWFORD SE,RAMANI S,TATE JE,et al.Rotavirus infection[J].NatRev Dis Primers,2017,3:17083.

[0111] [2]RCWC Current Members.Rotavirus classification working group:RC WC[EB / OL].[2020-11-25].https: / / rega.kuleuven.be / cev / viral-metagenomics / vir us-classification / rcwg.

[0112] [3] Cheng Li, Wang Song. Research progress of rotavirus vaccines for human use[J]. International Journal of Biologicals, 2019, 42(1): 24-28.

[0113] [4] Liang Xiaofeng, Luo Fengji, Feng Duojia. Vaccinology[M]. 1st edition. Beijing: People's Medical Publishing House, 2011: 779-802.

[0114] [5]GBD 2015Mortality and Causes of Death Collaborators. Global, regional, and national life expectancy, all-cause mortality, and cause-specific mortality for 249 causes of death, 1980-2015: a systematic analysis for the Global Burden of Disease Study 2015[J].Lancet,2016,388(10053):1459-1544.

[0115] [6]WU BS,HUANG ZM,WENG YW,et al.Prevalence and genotyp es ofrotavirus A and human adenovirus among hospitalized children with acu tegastroenteritis in Fujian,China,2009—2017[J].Biomed Environ Sci, 2019,32(3):210-214.

[0116] [7] TROEGER C, Khalil IA, Rao PC, et al. Rotavirus vaccination and the global burden of rotavirus diarrhea among children younger than 5years[J]. JAMA Pediatr, 2018, 172(10):958-965.

[0117] [8] Zhang Jiaying, Tian Yi, Gao Zhiyong, et al. Epidemiological characteristics of rotavirus diarrhea in Asia[J]. International Journal of Virology, 2021, 28(2): 166-169.

[0118] [9] Shen Si, Zhao Genming. Disease burden and vaccine application of rotavirus infection in children[J]. Chinese Journal of Pediatrics, 2021, 59(5): 426-430.

[0119]

[10] Jin Qi. Medical Molecular Virology[M]. Beijing: Science Press, 2001: 170, 542-563.

[0120]

[11] China Biological Products Standardization Committee. China Biological Products Regulations [S]. 2000 edition, 2002 supplement. Beijing: Chemical Industry Press, 2002: 8-10.

[0121]

[12] Kou Guiying, Yu Li, Zhu Chuanfeng, et al. Establishment and application of the limiting dilution method for screening and purification of rotavirus LD9 strain[J]. Chinese Journal of New Drugs, 2012, 21(10): 1135-1139.

[0122]

[13] Chinese Pharmacopoeia Committee. Pharmacopoeia of the People's Republic of China (Volume III)[S]. Beijing: Chinese Pharmacopoeia Science and Technology Press, 2020: 520-524.

[0123]

[14] Zhao Kai, Zhang Yihao, Li Hemin. Medical Biologicals[M]. 2nd edition. Beijing: People's Medical Publishing House, 2007: 370-392.

[0124]

[15] Crawford SE, Ramani S, Tate JE, et al. Rotavirus infection [J]. Nat RevDis Primers, 2017, 3: 17083.

[0125]

[16] TALLETT S,MACKENZIE C,MIDDLETON P,et al.Clinical,laboratory and epidemiologic features of a viral gastroenteritis In infants and children[J].Pediatrics, 1977,60(2):217-222.

[0126]

[17] CENG QB, LAI SJ, YU JX, et al. Epidemiological characteristics ofrotavirus caused diarrhea in children aged <5 years in 26 provinces in China, 2011-2014[J]. Dis Surveillance, 2016, 31(6): 463-470.

[0127]

[18] CARR ME,MCKENDRICK GD,SPYRIDAKIS T.The clinical features of infantile gastroenteritis duo to rotavirus[J].Scand J Infect Dis.1976,8(4):241-243.

[0128]

[19] Han Jing. A brief analysis of the clinical characteristics and experience of rotavirus enteritis in infants and young children[J]. Primary Medicine Forum, 2024, 28(1): 46-48,55.

Claims

1. A human-sheep rotavirus gene reassortment attenuated strain, characterized in that: The human-sheep rotavirus gene reassortment attenuated strain comprises: i) the coding gene of the structural protein VP7 of the human G9 type rotavirus HN-28 strain; and ii) the coding genes of the structural proteins VP1, VP2, VP3, VP4, VP6 and the non-structural proteins NSP1, NSP2, NSP3, NSP4, NSP5 of the sheep rotavirus LLR strain: the human-sheep rotavirus gene reassortment attenuated strain is the LG9 strain, and the deposit number is CCTCCNO: V202429.

2. Use of the human-sheep rotavirus gene reassortment attenuated strain according to claim 1 in the preparation of rotavirus vaccine.

3. Use of the human-sheep rotavirus gene reassortment attenuated strain according to claim 1 in the preparation of rotavirus diagnostic reagents.

4. A vaccine, characterized in that: The vaccine comprises an immunogenic effective amount of the human-sheep rotavirus gene reassortment attenuated strain according to claim 1.

5. The vaccine according to claim 4, characterized in that: The vaccine also comprises at least one acceptable adjuvant.

6. The vaccine according to claim 4, characterized in that: The vaccine also comprises at least one pharmaceutically acceptable carrier or diluent.

7. The vaccine according to claim 4, characterized in that: The vaccine is in the form of an oral dosage form or an injection dosage form.

8. A diagnostic reagent, characterized in that: The diagnostic reagent contains the human-sheep rotavirus gene reassortment attenuated strain according to claim 1.

Images