Recombinant porcine rotavirus expressing different VP7 of G type simultaneously and construction method and application thereof

Abstract

The application discloses a recombinant porcine rotavirus capable of simultaneously expressing different G type VP7s and a construction method and application thereof, and relates to the technical field of biotechnology. The construction method comprises the following steps: taking a G9 type porcine rotavirus as a skeleton, inserting a coding gene of a G5 type porcine rotavirus VP7 protein into a 3' end of an NSP1 gene open reading frame of the G9 type porcine rotavirus, inserting a coding gene of a G4 type porcine rotavirus VP7 protein into a 3' end of an NSP3 gene open reading frame of the G9 type porcine rotavirus, and packaging and constructing to obtain a recombinant porcine rotavirus capable of simultaneously expressing G4, G5 and G9 type VP7 proteins. Immunization of the recombinant porcine rotavirus can simultaneously induce high-level antibodies against the G4, G5 and G9 types, the antibodies have a long duration, and can passively protect animals against infection of the three G type rotaviruses.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a recombinant porcine rotavirus expressing different G type VP7 simultaneously and a construction method and application thereof. BACKGROUND

[0002] Porcine rotavirus (PoRV) is one of the main pathogens causing severe diarrhea in piglets. Epidemiological investigation results of porcine rotavirus show that G4, G5 and G9 types are the main genotypes currently prevalent in pig farms. Since PoRV belongs to segmented double-stranded RNA virus, it is prone to genetic recombination during genetic evolution, producing recombinant viruses with new antigenic properties. This directly leads to the diversity of epidemic strains and poor cross-protection effect between different serotypes, so it is urgent to develop a multivalent vaccine against prevalent strains.

[0003] Reverse genetic manipulation system is one of the key technical means for designing new vaccines and other researches. Currently, rotaviruses of different species such as human, avian, murine, bovine and ovine have been successfully rescued, but only G5 type of PoRV reverse genetic manipulation system has been reported. Current vaccine research on PoRV mainly focuses on inactivated multivalent vaccine or subunit vaccine, and there is no research on recombinant virus vector vaccine. SUMMARY

[0004] The purpose of the present application is to provide a recombinant porcine rotavirus expressing different G type VP7 simultaneously and a construction method and application thereof, in order to solve the problems existing in the prior art. Immunization with the recombinant porcine rotavirus can induce high levels of antibodies against G4, G5 and G9 types, and the antibodies have a long duration and can passively protect animals against infection with three G type rotaviruses.

[0005] To achieve the above purpose, the present application provides the following solutions:

[0006] The present application provides a construction method of a recombinant porcine rotavirus expressing different G type VP7 simultaneously, comprising the following steps: taking G9 type porcine rotavirus as a backbone, inserting the coding gene of G5 type porcine rotavirus VP7 protein into the 3' end of the NSP1 gene open reading frame of the G9 type porcine rotavirus, inserting the coding gene of G4 type porcine rotavirus VP7 protein into the 3' end of the NSP3 gene open reading frame of the G9 type porcine rotavirus, and packaging to obtain a recombinant porcine rotavirus capable of expressing G4, G5 and G9 type VP7 proteins simultaneously.

[0007] Further, the G9 type porcine rotavirus is porcine rotavirus NJ2012.

[0008] Further, the nucleotide sequence of the coding gene of the G5 type porcine rotavirus VP7 protein is shown as SEQ ID NO. 14; and the nucleotide sequence of the coding gene of the G4 type porcine rotavirus VP7 protein is shown as SEQ ID NO. 13.

[0009] Further, the construction method specifically comprises the following steps:

[0010] The NJ2012-VP1 gene, the NJ2012-VP2 gene, the NJ2012-VP3 gene, the NJ2012-VP4 gene, the NJ2012-VP6 gene, the NJ2012-VP7 gene, the NJ2012-NSP1 gene, the NJ2012-NSP2 gene, the NJ2012-NSP3 gene, the NJ2012-NSP4 gene and the NJ2012-NSP5 gene with the nucleotide sequences shown as SEQ ID NO. 1-11, respectively, are connected to the pT7 vector, respectively, to obtain the recombinant plasmids pT7-NJ2012-VP1, pT7-NJ2012-VP2, pT7-NJ2012-VP3, pT7-NJ2012-VP4, pT7-NJ2012-VP6, pT7-NJ2012-VP7, pT7-NJ2012-NSP1, pT7-NJ2012-NSP2, pT7-NJ2012-NSP3, pT7-NJ2012-NSP4 and pT7-NJ2012-NSP5;

[0011] The coding gene of the G4 type porcine rotavirus VP7 protein is inserted into the 3' end of the NSP3 gene open reading frame in the pT7-NJ2012-NSP3 to obtain the recombinant plasmid pT7-NJ2012-NSP3-haG4-VP7;

[0012] The coding gene of the G5 type porcine rotavirus VP7 protein is inserted into the 3' end of the NSP1 gene open reading frame in the pT7-NJ2012-NSP1 to obtain the recombinant plasmid pT7-NJ2012-NSP1-fG5-VP7;

[0013] co-transfect cells stably expressing T7 RNA polymerase with the pT7-NJ2012-VP1, the pT7-NJ2012-VP2, the pT7-NJ2012-VP3, the pT7-NJ2012-VP4, the pT7-NJ2012-VP6, the pT7-NJ2012-VP7, the pT7-NJ2012-NSP1, the pT7-NJ2012-NSP2, the pT7-NJ2012-NSP3, the pT7-NJ2012-NSP4, the pT7-NJ2012-NSP5, the pT7-NJ2012-NSP3-haG4-VP7 and the pT7-NJ2012-NSP1-fG5-VP7 to construct a recombinant porcine rotavirus expressing G4, G5 and G9 type VP7 proteins simultaneously.

[0014] Further, the cells stably expressing T7 RNA polymerase are BHK-T7 cells.

[0015] Further, in the pT7-NJ2012-NSP3-haG4-VP7, a P2A sequence is further inserted between the NSP3 gene open reading frame and the coding gene of the G4 type porcine rotavirus VP7 protein;

[0016] In the pT7-NJ2012-NSP1-fG5-VP7, a P2A sequence is further inserted between the NSP1 gene open reading frame and the coding gene of the G5 type porcine rotavirus VP7 protein;

[0017] The nucleotide sequence of the P2A sequence is shown in SEQ ID NO. 12.

[0018] Further, in the pT7-NJ2012-NSP3-haG4-VP7, a HA sequence is further inserted between the P2A sequence and the coding gene of the G4 type porcine rotavirus VP7 protein; the nucleotide sequence of the HA sequence is shown in SEQ ID NO. 58;

[0019] In the pT7-NJ2012-NSP1-fG5-VP7, a 3×Flag sequence is further inserted between the P2A sequence and the coding gene of the G5 type porcine rotavirus VP7 protein; the nucleotide sequence of the 3×Flag sequence is shown in SEQ ID NO. 59.

[0020] The application also provides a recombinant porcine rotavirus expressing different G type VP7 proteins simultaneously, which is constructed by the above-mentioned construction method.

[0021] The application also provides application of the recombinant porcine rotavirus expressing different G type VP7 simultaneously in preparation of a porcine rotavirus vaccine.

[0022] The application also provides a porcine rotavirus vaccine, wherein the active ingredient comprises the recombinant porcine rotavirus expressing different G type VP7 simultaneously.

[0023] The application discloses the following technical effects:

[0024] The application constructs a recombinant porcine rotavirus expressing different G type VP7 simultaneously by a reverse genetics system, and the recombinant porcine rotavirus is efficient and stable. Specifically, on the basis of a successfully established G9 type porcine rotavirus reverse genetic operation system, the virus gene is reformed, G5 and G4 type VP7 genes are expressed on the NSP1 and NSP3 genes of the virus respectively, and a recombinant virus expressing G4, G5 and G9 type VP7 simultaneously is rescued. The recombinant virus has good genetic stability. Through an animal passive immunization protection experiment, it is proved that the recombinant virus can induce high-level antibodies against the three G types simultaneously, the antibodies have a long duration, and the animals can be passively protected against infection of the three G type rotaviruses. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0026] Figure 1 A design mode diagram of the recombinant plasmid constructed for example 1;

[0027] Figure 2 A mode diagram of the recombinant virus prepared for example 1;

[0028] Figure 3 A result identification diagram of the recombinant virus prepared for example 1; wherein, A is a dsRNA-PAGE identification result diagram; B is a Western blot identification result diagram;

[0029] Figure 4Figure of genetic stability identification results of recombinant viruses prepared for Example 1; wherein, A-C are respectively dsRNA-PAGE identification results of recombinant viruses rNJ2012-NSP1-fG5-VP7, rNJ2012-NSP3-haG4-VP7 and ArNJ2012-fG5-VP7 / haG4-VP7A; D-F are respectively Western blot identification results of recombinant viruses rNJ2012-NSP1-fG5-VP7, rNJ2012-NSP3-haG4-VP7 and ArNJ2012-fG5-VP7 / haG4-VP7A;

[0030] Figure 5 Figure of diarrhea rate of each group of mammalian mice in Example 2; wherein, A-C are respectively detection results of challenge with JSJR2023 (G4P

[23] ), JSNJ2024 (G5P[7]) and NJ2012 (G9P[7]);

[0031] Figure 6 Figure of weight change of each group of mammalian mice in Example 2; wherein, A-C are respectively detection results of challenge with JSJR2023 (G4P

[23] ), JSNJ2024 (G5P[7]) and NJ2012 (G9P[7]);

[0032] Figure 7 Figure of excretion detection results of fecal samples of each group of mammalian mice in Example 2; wherein, A-C are respectively detection results of challenge with JSJR2023 (G4P

[23] ), JSNJ2024 (G5P[7]) and NJ2012 (G9P[7]);

[0033] Figure 8 Figure of indirect ELISA (IgG) titer determination results of maternal antibody level of each group of mammalian mice in Example 2; wherein, A-C are respectively detection results of challenge with JSJR2023 (G4P

[23] ), JSNJ2024 (G5P[7]) and NJ2012 (G9P[7]);

[0034] Figure 9 Figure of neutralizing antibody titer determination results of maternal antibody level of each group of mammalian mice in Example 2; wherein, A-C are respectively detection results of challenge with JSJR2023 (G4P

[23] ), JSNJ2024 (G5P[7]) and NJ2012 (G9P[7]);

[0035] Figure 10Figures of intestinal virus load detection results of each group of the nursing mice in Example 2; wherein, A-C are detection results of the mice challenged with JSJR2023 (G4P

[23] ), JSNJ2024 (G5P[7]) and NJ2012 (G9P[7]) respectively;

[0036] Figure 11 Figures of HE staining of pathological histological lesions of intestinal tissues of each group of the nursing mice in Example 2;

[0037] Figure 12 Figures of detection results of cellular immune responses of each group of the nursing mice in Example 2; wherein, A is a figure of detection results of T lymphocyte subgroups CD3 CD4; B is a figure of detection results of T lymphocyte subgroups CD3 CD8; and C is a figure of detection results of B lymphocyte subgroups;

[0038] Figure 13 Figures of indirect ELISA (IgG) titer determination results of antibody levels of each group of the nursing mice in Example 2; wherein, A is a time sequence dynamic change of IgG antibody titer of anti-VP4*P[7] serum; B is a time sequence dynamic change of IgG antibody titer of anti-VP7*G4 serum; C is a time sequence dynamic change of IgG antibody titer of anti-VP7*G5 serum; and D is a time sequence dynamic change of IgG antibody titer of anti-VP7*G9 serum;

[0039] Figure 14 Figures of neutralizing antibody titer determination results of antibody levels of each group of the nursing mice in Example 2; wherein, A-C are detection results of the mice challenged with JSJR2023 (G4P

[23] ), JSNJ2024 (G5P[7]) and NJ2012 (G9P[7]) respectively. DETAILED DESCRIPTION

[0040] The foregoing description of various example embodiments of the application has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the application to the precise form disclosed. Many modifications and variations are possible in light of this disclosure. It was chosen to discuss some aspects of one or more embodiments of the application solely for the purpose of illustration of the conceptual subtleties. Based on the teachings provided herein, one skilled in the art should appreciate that there are many ways those teachings can be utilized without departing from the scope of the application.

[0041] It is to be understood that the terms used herein are merely descriptive, but that they are not intended to restrict the application. In addition, for numerical ranges in the present application, it is to be understood that every intermediate value between the upper and lower limits of that range and any other stated or intervening value in that stated range, is encompassed within the application. Each smaller range between any stated value or intervening value in the stated ranges and any other stated or intervening value in that stated range is also encompassed within the application. The upper and lower limits of these smaller ranges can independently be included or excluded in the range.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this application pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. In case of conflict between the content of the specification and that of any incorporated literature, the content of the specification controls.

[0043] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0044] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed materials and methods.

[0045] The present application establishes a high-efficiency and stable method for developing a multivalent recombinant rotavirus strain, specifically, on the basis of a successfully established G9 type porcine rotavirus reverse genetic manipulation system, the virus gene is modified, such as Figure 2 As shown in the figure, the G5 and G4 type VP7 genes are respectively fused and expressed on the NSP1 and NSP3 genes of the virus, and the recombinant virus capable of simultaneously expressing G4, G5 and G9 type VP7 is rescued, which has good genetic stability. It is proved by animal passive immunization protection experiment that the immunization of the recombinant virus can simultaneously induce high level of antibodies against the above three types of G, and the antibody duration is long, which can passively protect animals against three types of rotavirus infection.

[0046] The primer information related to the present application is shown in Table 1.

[0047] Table 1 Primer information related to the present application

[0048]

[0049] Example 1

[0050] 1. Main experimental materials

[0051] PoRV virus NJ2012 (G9P[7]), JSNJ2024 (G5P[7]), JSJR2023 (G4P

[23] ), MA104 cells were provided by Jiangsu Academy of Agricultural Sciences, Institute of Veterinary. BHK-T7 cell line was purchased from Yaaji Biological. pT7-SA11-VP7 plasmid and 3 helper plasmids (pCAG-D1R, pCAG-D12L and pCAG-FAST-p10) were purchased from Addgene platform. Helper plasmid C3P3-G1 was a gift from Dr. Siyuan Ding, Washington University in St. Louis (which has been disclosed in the literature "Sánchez-Tacuba L, Feng N, Meade N J, et al. An optimized reverse genetics system suitable for efficient recovery of simian, human, and murine-like rotaviruses [J]. Journal of virology, 2020, 94(18): 10.1128 / jvi. 01294-20."). pT7-NSP3-P2A-3xFUnaG, pT7-NSP3-P2A-NLuc, helper plasmid pCAGGS-HA-NSP2 and pCAGGS-HA-NSP5 were provided by Jiangsu Academy of Agricultural Sciences, Institute of Veterinary, in which pT7-NSP3-P2A-NLuc was synthesized by General Bio Corporation, pT7-NSP3-P2A-3xFUna has been disclosed in the literature "Philip A A, Patton J T. Expression of separate heterologous proteins from the rotavirus NSP3 genome segment using a translational 2A stop-restart element [J]. Journal of virology, 2020, 94(18): 10.1128 / jvi. 00959-20."; helper plasmid pCAGGS-HA-NSP2 and pCAGGS-HA-NSP5 have been disclosed in the literature "Bian X Y, Li S F, Wang J X, et al. Prokaryotic expression, antibody preparation and application of major non-structural proteins of porcine rotavirus [J]. Chinese Journal of Agricultural Sciences, 2024, 57(17): 3494-3506."

[0052] The endotoxin-removing plasmid extraction kit and the gel recovery kit are products of Omega Bio-tek, Inc; 2×GreenTaqMix, 2×Phanta Max Master Mix high-fidelity enzyme, homologous recombinase, RNA extraction kit and reverse transcription kit are products of Vazyme Biotech Co., Ltd; pancreatin and trypsin are products of Sigma Company; TransIT-LT1 transfection reagent is a product of Mirus Bio Company; RIPA protein lysis buffer is a product of Shanghai Biyun Tian Biological Technology Co., Ltd; 7.5% PAGE gel rapid preparation kit is a product of Shanghai Yezhen Biological Medicine Technology Co., Ltd; GAPDH, Flag-tagged antibody and HA-tagged antibody are products of Proteintech Group, Inc; goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP are products of Beijing Solaybao Technology Co., Ltd; nucleic acid dye is a product of Shanghai Yisen Biological Technology Co., Ltd. Rotavirus VP6 protein mouse monoclonal antibody, VP4*P[7] and VP7*G9 coating proteins are provided by Jiangsu Academy of Agricultural Sciences, Institute of Animal Health, wherein the VP6 protein mouse monoclonal antibody has been disclosed in the literature “Li K M, Zhou J Z, Zhou J M, et al. Preparation and preliminary identification of porcine group A rotavirus VP6 monoclonal antibody [J]. Animal Husbandry and Veterinary Medicine, 2023, 55(04): 79-85.”; VP4*P[7] and VP7*G9 coating proteins have been disclosed in the literature “Tang X, Li S, Zhou J, et al. Recombinant bivalent subunit vaccine combining truncated VP4 from P [7] and P

[23] induces protective immunity against prevalent porcine rotaviruses [J]. Journal of Virology, 2024, 98(5): e00212-24.” and “Li S, Bian X, Wang J, et al. VP4-Specific IgA level as a correlate of neutralizing antibody and fecal shedding of porcine rotavirus infection [J]. Veterinary Microbiology, 2025, 304: 110501.”.

[0053] SPF level 4-week-old BALB / c mouse strain mice were purchased from Jiangsu Jizhuangkang Biotechnology Co., Ltd. and were raised in the animal experiment base of the Jiangsu Academy of Agricultural Sciences Veterinary Institute.

[0054] 2. Plasmid construction

[0055] Based on the whole genome sequences of PoRV strains at home and abroad collected by GenBank database, 11 pairs of convergent amplification primers were designed by using SnapGene4.1.8 software, and the primer sequences are shown in Table 1. The cDNA product of virus NJ2012 was used as a template to obtain all 11 fragments, i.e. NJ2012-VP1, NJ2012-VP2, NJ2012-VP3, NJ2012-VP4, NJ2012-VP6, NJ2012-VP7, NJ2012-NSP1, NJ2012-NSP2, NJ2012-NSP3, NJ2012-NSP4 and NJ2012-NSP5 by PCR amplification, and the nucleotide sequences thereof are shown in SEQ ID NO. 1-11, respectively.

[0056] PCR amplification method is described by taking the amplified NJ2012-VP1 fragment as an example:

[0057] The PCR system is as follows: NJ2012-VP1-F / R 2.5 μL, cDNA 3 μL, 2×Phanta Max Master Mix high-fidelity enzyme 25 μL, and sterile water 17 μL.

[0058] The PCR program is as follows: 95℃ pre-denaturation for 3 min; 95℃ for 15 s, 51℃ for 15 s, 72℃ for 2 min, 35 cycles; 72℃ for 5 min, and 4℃ storage.

[0059] The pT7-SA11-VP7 plasmid was used as a template for pT7 vector PCR amplification, and the PCR system was as follows: p3E5-F / R 2.5 μL, cDNA 3 μL, 2×Phanta Max Master Mix high-fidelity enzyme 25 μL, and sterile water 17 μL; the PCR program was as follows: 95℃ pre-denaturation for 3 min; 95℃ for 15 s, 51℃ for 15 s, 72℃ for 2 min, 35 cycles; 72℃ for 5 min, and 4℃ storage.

[0060] After gel electrophoresis verification, the target band was cut and purified for recovery. The amplified fragments and the amplification vector were connected by homologous recombination to obtain the recombinant plasmid pT7-NJ2012-VP1. After the constructed plasmids were sequenced and checked for no errors, they were extracted by using an endotoxin-free plasmid extraction kit.

[0061] Similarly, the recombinant plasmids pT7-NJ2012-VP2, pT7-NJ2012-VP3, pT7-NJ2012-VP4, pT7-NJ2012-VP6, pT7-NJ2012-VP7, pT7-NJ2012-NSP1, pT7-NJ2012-NSP2, pT7-NJ2012-NSP3, pT7-NJ2012-NSP4 and pT7-NJ2012-NSP5 encoding rotavirus VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4 and NSP5 were constructed.

[0062] 2A peptide is a cis-acting hydrolytic enzyme element, commonly known as a "self-cleavage" peptide. It functions through a "ribosome skipping" mechanism, mediating the cleavage of the peptide bond between Gly and Pro residues. This enables a single transcript to produce multiple different proteins. Positioning a GSG fragment upstream of P2A can improve the self-cleavage efficiency of P2A, so it is necessary to insert GSG before the P2A sequence. The nucleotide sequence of the P2A sequence is shown in SEQ ID NO. 12.

[0063] To construct the NSP3 plasmid carrying VP7*G4, the P2A sequence, HA sequence (SEQ ID NO. 58) and G4-VP7 fragment of PoRV strain JSJR2023 (G4P

[23] ) were co-inserted into the 3' end of the NSP3 gene open reading frame (ORF) in pT7-NJ2012-NSP3 by homologous recombination, and the recombinant plasmid pT7-NJ2012-NSP3-haG4-VP7 was constructed. Figure 1 Using the pT7-NSP3-P2A-NLuc infectious clone plasmid as a template, the fragment 1 was amplified using primers pT7-NSP3-F and haG4-R; the cDNA product of JSJR2023 was used as a template, and the fragment 2 was amplified using primers haG4-F and G4-NSP3-R; the pT7-NJ2012-NSP3 infectious clone plasmid was used as a template, and the fragment 3 was amplified using primers G4-NSP3-F and pT7-NSP3-R, and finally the final target fragment was amplified using pT7-NSP3-F / R as a template with fragment 1, fragment 2 and fragment 3.

[0064] In view of the fact that the RV NSP1 fragment can also accommodate exogenous genes, the present application co-inserts a P2A sequence, a 3xFlag sequence (SEQ ID NO. 59) and a G5-VP7 fragment of PoRV strain JSNJ2024 (G5P[7]) into the 3' end of the NSP1 gene ORF in pT7-NJ2012-NSP1 to construct a recombinant plasmid pT7-NJ2012-NSP1-fG5-VP7 Figure 1 The fragment 1 is amplified from the pT7-NJ2012-NSP1 infectious clone plasmid as a template using primers pT7-NSP1-F and NSP1-G5-R; the fragment 2 is amplified from the pT7-NSP3-P2A-3xFunaG as a template using primers NSP1-G5-F and flagG5-R; the fragment 3 is amplified from the cDNA product of the JSNJ2024 strain viral genome as a template using primers flagG5-F and G5-NSP1-R; and the fragment 4 is amplified from the pT7-NJ2012-NSP1 infectious clone plasmid as a template using primers G5-NSP1-F and pT7-NSP1-R. Finally, the final target fragment is amplified from the fragment 1, the fragment 2, the fragment 3 and the fragment 4 using pT7-NSP1-F / R.

[0065] All plasmid sequences are verified by DNA sequencing. Finally, identification primers are designed for verifying whether the strain is successfully mutated. The primer sequences are shown in Table 1.

[0066] 3. Preparation of recombinant virus

[0067] Construction method of recombinant virus rNJ2012-fG5-VP7 / haG4-VP7:

[0068] BHK-T7 cell monolayer was inoculated in cell plates, and when the cell confluence in the plates reached 65%-75%, transfection was prepared. TransIT-LT1 transfection reagent was mixed with recombinant plasmids pT7-NJ2012-VP1, pT7-NJ2012-VP2, pT7-NJ2012-VP3, pT7-NJ2012-VP4, pT7-NJ2012-VP6, pT7-NJ2012-VP7, pT7-NJ2012-NSP3-haG4-VP7, pT7-NJ2012-NSP1-fG5-VP7, pT7-NJ2012-NSP2, pT7-NJ2012-NSP4, and pT7-NJ2012-NSP5, and various auxiliary plasmids C3P3-G1, pCAG-FAST-p10, pCAG-D1R, pCAG-D12L, pCAGGS-HA-NSP2, and pCAGGS-HA-NSP5 were added to serum-reduced medium (Opti-MEM) together, and after being mixed and allowed to stand at room temperature for 30 min, they were uniformly added dropwise to each cell well. After 24 h of transfection, the supernatant was discarded, and the cells were washed twice with PBS, and the culture medium was replaced with 1 mL of DMEM basic medium containing trypsin at a final concentration of 0.1 μg / mL. After 24 h of culture, MA104 cells were added to the transfected BHK-T7 cells, and 4 h later, the trypsin concentration in each well was adjusted to 0.5 µg / mL, and trypsin was added at a ratio of 1:5000. After 3 d of co-culture of BHK-T7 and MA104 cells, the cells were repeatedly frozen and thawed twice, centrifuged at 12000 rpm for 5 min to discard the cell debris, and the supernatant was recovered to obtain recombinant virus (named rNJ2012-fG5-VP7 / haG4-VP7), which was stored at -80℃.

[0069] The construction method of recombinant virus rNJ2012-WT was the same as that of recombinant virus rNJ2012-fG5-VP7 / haG4-VP7, except that TransIT-LT1 transfection reagent was mixed with recombinant plasmids pT7-NJ2012-VP1, pT7-NJ2012-VP2, pT7-NJ2012-VP3, pT7-NJ2012-VP4, pT7-NJ2012-VP6, pT7-NJ2012-VP7, pT7-NJ2012-NSP1, pT7-NJ2012-NSP2, pT7-NJ2012-NSP3, pT7-NJ2012-NSP4, and pT7-NJ2012-NSP5, and various auxiliary plasmids C3P3-G1, pCAG-FAST-p10, pCAG-D1R, pCAG-D12L, pCAGGS-HA-NSP2, and pCAGGS-HA-NSP5 were added to serum-reduced medium together.

[0070] Construction method of recombinant virus rNJ2012-NSP1-fG5-VP7: same as the construction method of recombinant virus rNJ2012-fG5-VP7 / haG4-VP7, the only difference is that the TransIT-LT1 transfection reagent is added into the reduced serum medium together with the recombinant plasmids pT7-NJ2012-VP1, pT7-NJ2012-VP2, pT7-NJ2012-VP3, pT7-NJ2012-VP4, pT7-NJ2012-VP6, pT7-NJ2012-VP7, pT7-NJ2012-NSP1-fG5-VP7, pT7-NJ2012-NSP2, pT7-NJ2012-NSP3, pT7-NJ2012-NSP4 and pT7-NJ2012-NSP5, and various auxiliary plasmids C3P3-G1, pCAG-FAST-p10, pCAG-D1R, pCAG-D12L, pCAGGS-HA-NSP2 and pCAGGS-HA-NSP5.

[0071] Construction method of recombinant virus rNJ2012-NSP3-haG4-VP7: same as the construction method of recombinant virus rNJ2012-fG5-VP7 / haG4-VP7, the only difference is that the TransIT-LT1 transfection reagent is added into the reduced serum medium together with the recombinant plasmids pT7-NJ2012-VP1, pT7-NJ2012-VP2, pT7-NJ2012-VP3, pT7-NJ2012-VP4, pT7-NJ2012-VP6, pT7-NJ2012-VP7, pT7-NJ2012-NSP3-haG4-VP7, pT7-NJ2012-NSP1, pT7-NJ2012-NSP2, pT7-NJ2012-NSP4 and pT7-NJ2012-NSP5, and various auxiliary plasmids C3P3-G1, pCAG-FAST-p10, pCAG-D1R, pCAG-D12L, pCAGGS-HA-NSP2 and pCAGGS-HA-NSP5.

[0072] 4. Recombinant virus identification

[0073] 4.1 dsRNA-PAGE: The dsRNA of the parent strain and the recombinant virus was extracted respectively by using an RNA extraction kit, and a 7.5% polyacrylamide gel (PAGE) was prepared. 12 μL of the virus RNA was mixed with 4 μL of 5x loading buffer and loaded, 1x TBE electrophoresis buffer was added, and electrophoresis was performed at 180 V for 4 h. After electrophoresis, the product was stained with physiological saline containing 0.03% nucleic acid dye for 30 min, and was displayed by using a gel imaging system, and the results are shown in FIG. 2A. Figure 3

[0074] 4.2 Western blotting identification: The parent strain and the recombinant virus were inoculated into MA104 cells at a dose of 0.05 MOI, and 12 h after infection, RIPA protein lysis solution containing phenylmethylsulfonyl fluoride (PMSF) was added, and after complete lysis at 4°C, the supernatant was taken, protein loading buffer containing a thiol reducing agent was added, and after complete denaturation at 100°C for 10 min, SDS-PAGE was performed, and after electrophoresis, the product was transferred to a 0.45 μm PVDF membrane. After transfer, VP6 protein mouse monoclonal antibody (1:4000 dilution), HA protein rabbit polyclonal antibody (1:4000 dilution), and Flag protein mouse monoclonal antibody (1:4000 dilution) were used as the primary antibody, goat anti-mouse IgG-HRP (1:10000 dilution) was used as the secondary antibody, and a chemiluminescence kit was used for detection in a chemiluminescence detector, and the results are shown in FIG. 2B. Figure 3

[0075] The results of dsRNA-PAGE and Western blotting identification show that the recombinant virus prepared in the present application is successfully constructed.

[0076] 5. Detection of genetic stability of recombinant virus

[0077] The identified recombinant virus was continuously passaged for five times. When the cytopathic effect reached complete (usually 3-5 days), the cells were freeze-thawed three times, and the supernatant was recovered. The recombinant virus of each generation was identified by dsRNA-PAGE and Western blotting. The results are shown in FIG. 3. Figure 4 The results show that the recombinant virus constructed in the present application has good genetic stability.

[0078] Example 2

[0079] 1. Construction of passive protection model of suckling mice and experimental grouping

[0080] ​​4-week-old female BALB / c mice were purchased from Jiangsu Jizhuangkang Biotechnology Co., Ltd. and randomly divided into 4 groups, namely, a blank group, a challenge group (PBS), a G9 immunization group (rNJ2012-WT), and a G4 / G5 / G9 immunization group (rNJ2012-fG5-VP7 / haG4-VP7), with 8 mice in each group. The virus stock solution was inoculated at 10 50 / mL by standardizing the determination method. 6 TCID 50 / mL. The G4 / G5 / G9 immunization group was injected with 300 μL of recombinant virus rNJ2012-fG5-VP7 / haG4-VP7 (constructed in Example 1) subcutaneously, the G9 immunization group was given an equal amount of wild-type virus rNJ2012-WT (constructed in Example 1), and the challenge group and the blank group were given an equal volume of PBS. The mice were immunized twice at an interval of 14 days. After the second immunization, female mice were mated with male mice, and no further immunization was performed during this period. After the offspring mice were born for 5 days, all nursing mice were orally inoculated with 100 μL of rotavirus strain NJ2012 (G9P[7]), JSNJ2024 (G5P[7]), or JSJR2023 (G4P

[23] ) with a titer according to their group assignment.

[0081] 2. Index detection

[0082] 2.1 Diarrhea rate and body weight change monitoring

[0083] All nursing mice were monitored every day for 5 consecutive days after inoculation, general activity was recorded, and fecal characteristics were evaluated using a diarrhea scoring system. The severity of diarrhea in nursing mice was evaluated using the fecal scoring system described by Boshuizen.

[0084] The results of the diarrhea rate test of the nursing mice in each group are shown in Figure 5 , and the body weight change is shown in Figure 6 . The results show that the diarrhea rate of the immunized mice was significantly lower than that of the challenge group, and the G4 / G5 / G9 immunization group had the most significant effect; the body weight gain rate of the immunized mice was significantly higher than that of the challenge group.

[0085] 2.2 Indirect ELISA (IgG) titer determination

[0086] The purified VP7*G4, VP7*G5, VP7*G9 and VP4*P[7] recombinant proteins were coated on the enzyme-labeled plate at 4°C overnight at a working concentration of 1 μg / mL, blocked with 5% skim milk for 2 hours, washed thoroughly, and then 1:100 dilution of the serum to be tested (negative serum and post-immune serum) was added to the enzyme-labeled plate, with pre-immune mouse serum as a negative control. A 1:20,000 dilution of goat anti-mouse IgG-HRP was used as a secondary antibody, and the OD 450 of each well was detected by an enzyme-labeled instrument and recorded.

[0087] The detection results of the maternal antibody levels (indirect ELISA (IgG) titer determination) of each group are shown in Table 1 Figure 8 , and the results of the detection of the antibody levels of the maternal mice at different inoculation times (indirect ELISA (IgG) titer determination) are shown in Table 2 Figure 13 . The results show that the maternal antibody and IgG antibody levels of the suckling mice in all the immunized groups were significantly higher than those in the challenge group (P<0.01), and the antibody levels induced by the G4 / G5 / G9 immunized group were the highest.

[0088] 2.3 Neutralization titer determination

[0089] After the mouse serum was inactivated at 56°C for 30 min, it was diluted with DMEM basic medium, mixed with an equal volume of activated 200 TCID 50 / 0.1 mL virus solution, and then incubated in a cell culture incubator for 1 h. The mixture was then inoculated onto a monolayer of MA104 cells, with a serum toxicity control, negative and positive serum controls, a virus control, and a normal cell control. The cell well dilution at which cytopathic effect appeared was observed daily until the 3rd day, and the serum neutralization titer was calculated according to the Kaeber method.

[0090] The detection results of the maternal antibody levels (neutralizing antibody titer determination) of each group are shown in Table 3 Figure 9 , and the results of the detection of the antibody levels of the maternal mice at different times after virus inoculation (neutralizing antibody titer determination) are shown in Table 4 Figure 14 . The results show that the serum isolated from the G4 / G5 / G9 immunized group of maternal mice and suckling mice can effectively neutralize G4, G5 and G9 rotaviruses, while the G9 immunized group has better neutralization activity against G9 strains and poorer neutralization activity against G4 and G5 strains.

[0091] 2.4 Cell immune response detection

[0092] Spleen cells were isolated from the spleen of mice using a lymphocyte isolation kit (TBD) under sterile conditions. The cells were resuspended in RPMI-1640 medium and divided into two equal parts. One set of cells was resuspended in 300 μL of PBS containing PE anti-mouse CD4 (BioLegend, Inc., USA), APC anti-mouse CD8a (BioLegend, Inc., USA), and FITC anti-mouse CD3e (Becton, Dickinson and Company Biosciences, USA) fluorescent antibodies. The other set of cells was resuspended in 300 μL of PBS containing Percp-CyTM5.5 anti-mouse B220 (Becton, Dickinson and Company Biosciences, USA) and FITC anti-mouse CD19 (BioLegend, Inc., USA) fluorescent antibodies. Both sets of cells were incubated for 30 minutes at 4°C in the dark. After washing twice with PBS, the stained cells were analyzed using a BD Accuri C6 Plus flow cytometer (BD, USA). TM C6Plus flow cytometer (BD, USA) was used for analysis.

[0093] The results of the cellular immune response detection of the mother mice in each group are shown in Figure 12 The results show that the T cell and B cell subpopulation induced by the immunized mice in the G4 / G5 / G9 immunization group was significantly higher than that in the other immunization groups.

[0094] 2.5 Analysis of fecal virus shedding amount and intestinal virus load

[0095] Fecal samples and intestinal tissues of the suckling mice were collected every 24 hours after infection for RT-qPCR analysis to evaluate the virus shedding amount and intestinal virus load. RNA extraction kit and reverse transcriptase were used to extract cDNA. Then, the expression amount of PoRV-NSP5 was detected by enzyme digestion probe method, and RT-qPCR detection was performed using the cDNA as a template.

[0096] The fecal shedding of the suckling mice in each group is shown in Figure 7 The results of the detection of the intestinal tissue virus load are shown in Figure 10 The results show that compared with the infection group, the fecal shedding amount and intestinal virus load of the G4 / G5 / G9 immunization group of suckling mice were significantly reduced under the infection of three different G-type viruses; and the fecal shedding amount and intestinal virus load of the G9 immunization group were reduced to a certain extent only under the infection of G9-type virus, and there was no significant difference in the fecal shedding amount and intestinal virus load under the infection of other types.

[0097] 2.6 Detection of pathological histological lesions of intestinal tissues

[0098] The duodenum, jejunum and ileum tissues of the mice in each group were fixed by 10% neutral formalin, and then prepared into pathological sections by using hematoxylin-eosin (H.E.) staining method to observe the pathological changes of the intestinal tracts. Figure 11 The results showed that the intestinal tract tissues of the mice in the challenge group appeared typical pathological changes such as obviously shortened and shed intestinal villi, while the intestinal tracts of the mice in the G4 / G5 / G9 immunization groups and the blank group did not appear obvious pathological changes; the intestinal tract tissues of the mice in the G9 immunization group did not appear obvious changes under the infection of the same type G9 virus, but still appeared typical damages under the infection of G4 or G5 type virus.

[0099] In summary, the experimental results of Example 2 showed that the trivalent recombinant virus rNJ2012-fG5-VP7 / haG4-VP7 constructed by the application could induce the mother mice to produce relatively strong and persistent specific cellular and humoral immune responses, and the baby mice could obtain passive immunity, and could effectively resist the infection of three different G type rotaviruses.

[0100] The above-described examples only describe the preferred modes of the application, and do not limit the scope of the application. Without departing from the design spirit of the application, various modifications and improvements of the technical solutions of the application made by those skilled in the art should fall within the protection scope of the claims of the application.

Claims

1. A method for constructing recombinant porcine rotavirus simultaneously expressing different G-type VP7, characterized in that, The method includes the following steps: using G9 porcine rotavirus as a backbone, inserting the coding gene for the VP7 protein of G5 porcine rotavirus into the 3' end of the open reading frame of the NSP1 gene of G9 porcine rotavirus, inserting the coding gene for the VP7 protein of G4 porcine rotavirus into the 3' end of the open reading frame of the NSP3 gene of G9 porcine rotavirus, and packaging to construct a recombinant porcine rotavirus capable of simultaneously expressing G4, G5 and G9 VP7 proteins; The G9 type porcine rotavirus is porcine rotavirus NJ2012; The nucleotide sequence of the gene encoding the VP7 protein of the G5 porcine rotavirus is shown in SEQ ID NO.14; the nucleotide sequence of the gene encoding the VP7 protein of the G4 porcine rotavirus is shown in SEQ ID NO.

13. A P2A sequence was also inserted between the open reading frame of the NSP3 gene and the coding gene of the G4 porcine rotavirus VP7 protein; a P2A sequence was also inserted between the open reading frame of the NSP1 gene and the coding gene of the G5 porcine rotavirus VP7 protein; the nucleotide sequence of the P2A sequence is shown in SEQ ID NO.12; An HA sequence was also inserted between the P2A sequence and the coding gene of the G4 porcine rotavirus VP7 protein; the nucleotide sequence of the HA sequence is shown in SEQ ID NO.

58. A 3×Flag sequence was also inserted between the P2A sequence and the coding gene of the G5 porcine rotavirus VP7 protein; the nucleotide sequence of the 3×Flag sequence is shown in SEQ ID NO.

59.

2. The construction method according to claim 1, characterized in that, The construction method specifically includes the following steps: The NJ2012-VP1, NJ2012-VP2, NJ2012-VP3, NJ2012-VP4, NJ2012-VP6, NJ2012-VP7, NJ2012-NSP1, NJ2012-NSP2, NJ2012-NSP3, NJ2012-NSP4, and NJ2012-NSP5 genes, with nucleotide sequences as shown in SEQ ID NO. 1-11, were ligated into the pT7 vector to obtain recombinant NJ2012-VP1, NJ2012-NSP2, NJ2012-NSP3, NJ2012-NSP4, and NJ2012-NSP5 genes, respectively. Plasmids pT7-NJ2012-VP1, pT7-NJ2012-VP2, pT7-NJ2012-VP3, pT7-NJ2012-VP4, pT7-NJ2012-VP6, pT7-NJ2012-VP7, pT7-NJ2012-NSP1, pT7-NJ2012-NSP2, pT7-NJ2012-NSP3, pT7-NJ2012-NSP4 and pT7-NJ2012-NSP5; The gene encoding the VP7 protein of the G4 porcine rotavirus was inserted into the 3' end of the open reading frame of the NSP3 gene in pT7-NJ2012-NSP3 to obtain the recombinant plasmid pT7-NJ2012-NSP3-haG4-VP7. The gene encoding the VP7 protein of the G5 porcine rotavirus was inserted into the 3' end of the open reading frame of the NSP1 gene in pT7-NJ2012-NSP1 to obtain the recombinant plasmid pT7-NJ2012-NSP1-fG5-VP7. T7 was stably expressed by co-transfection with the following pT7-NJ2012-VP1, pT7-NJ2012-VP2, pT7-NJ2012-VP3, pT7-NJ2012-VP4, pT7-NJ2012-VP6, pT7-NJ2012-VP7, pT7-NJ2012-NSP1, pT7-NJ2012-NSP2, pT7-NJ2012-NSP3, pT7-NJ2012-NSP4, pT7-NJ2012-NSP5, pT7-NJ2012-NSP3-haG4-VP7 and pT7-NJ2012-NSP1-fG5-VP7. Recombinant porcine rotavirus expressing G4, G5, and G9 VP7 proteins was constructed using RNA polymerase-enhanced cells.

3. The construction method according to claim 2, characterized in that, The cells that stably express T7 RNA polymerase are BHK-T7 cells.

4. A recombinant porcine rotavirus expressing different G-type VP7 cells simultaneously, constructed using the construction method according to any one of claims 1-3.

5. The use of a recombinant porcine rotavirus expressing different G-type VP7 as described in claim 4 in the preparation of a porcine rotavirus vaccine.

6. A porcine rotavirus vaccine, characterized in that, The active ingredient includes the recombinant porcine rotavirus that simultaneously expresses different G-type VP7 as described in claim 4.

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