Infectious clone plasmid of human fiveleaf virus or human norovirus, reverse genetic virus rescue system and application of infectious clone plasmid and reverse genetic virus rescue system

Abstract

The invention discloses an infectious clone plasmid of a human fiveleaf virus or a human norovirus, a reverse genetic virus rescue system and application of the infectious clone plasmid and the reverse genetic virus rescue system. The infectious clone plasmid is based on a clone vector and is inserted into a cDNA genome of a human fiveleaf virus or human norovirus; the 5'end of the cDNA genome is connected in series with two T7 promoters, and an internal ribosome entry site is inserted between the two T7 promoters; an HDV ribozyme sequence and a T7 terminator sequence are sequentially connected in series behind a PolyA tail of the cDNA genome. The reverse genetic virus rescue system comprises a human fiveleaf virus or human norovirus infectious clone plasmid, a plasmid for expressing T7RNA polymerase and a BHK-T7 cell. The reverse genetic virus rescue system provided by the invention can efficiently and quickly rescue filial generation virions of human fiveleaf viruses or human noroviruses, and can quickly obtain a large number of viruses.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of virus culture, and particularly relates to an infectious clone plasmid of human sapovirus or human norovirus, a reverse genetic virus rescue system and application thereof. BACKGROUND

[0002] Human sapoviruses (HuSaVs) and human noroviruses (HuNoVs) are members of the Caliciviridea family and are important non-bacterial pathogens causing acute gastroenteritis outbreaks in human. According to genetic characteristics, sapoviruses can be divided into five genotypes (GI, GII, GIII, GIV and GV), among which G I, GII, GIV and GV mainly infect humans, GIII mainly infects pigs, and G I and GII are the main genotypes of human sapoviruses. Noroviruses can be divided into 10 genogroups (GI-GX), which are known to infect humans, mice, dogs, cats and cattle. GI and GII are the two main genogroups causing acute gastroenteritis in humans.

[0003] The currently widely accepted surrogate model of human norovirus, murine norovirus (MNV), can support virus replication under conventional culture [such as RAW26.7 (macrophages) and MSC-1 (small intestinal epithelial cells)] and obtain a large amount of progeny virus. However, human noroviruses and human sapoviruses lack cell lines for infection. Since the discovery of HuNoVs in 1972 and HuSaVs in 1976, various classic primary cells and immortalized cells have been tried for culture, such as immune cells (T cells, B cells, macrophages and DC cells), HEK293, HEK293T, Hela, THuh7, Caco-2, etc., but all failed to obtain successful results. Under these conventional culture conditions, the virus does not replicate or proliferate, and it is difficult to obtain progeny virus. At present, only human intestinal epithelial tissues (HIEs) can support several strains of human norovirus (but not all strains), but HIEs have the disadvantages of high cost, complicated operation and long cycle.

[0004] The infection symptoms of MNV are different from those of HuNoV, and cannot reflect the molecular biological characteristics of HuNoV. The maturation and assembly of virus particles in the virus rescue process of human norovirus and human sapovirus are different from those of murine norovirus: murine norovirus can better complete the maturation and assembly of virus particles in host cells, and the produced virus particles have good infectivity and stability. However, human norovirus and human sapovirus have some obstacles in the replication and assembly process in cells, resulting in insufficient maturation of virus particles, incorrect conformation of surface proteins (poor integrity), and affecting their infectivity and transmission ability.

[0005] Therefore, due to the lack of stable and effective cell culture system and reliable small animal infection model, the infection, pathogenesis, epidemic mechanism and potential anti-disease drugs of the two viruses have developed very slowly.

[0006] Reverse genetics technology can manipulate RNA virus genome at DNA level in vitro, which is an important method for studying RNA virus gene expression regulation mechanism, virus protein structure and function, pathogen and host interaction, host signaling pathway, virus pathogenesis and antiviral drug screening. The existing norovirus and sapovirus reverse genetics systems mainly include T7 RNA polymerase in vivo transcription system, T7 RNA polymerase in vitro transcription system, T7 RNA polymerase stable expression cell line system and eukaryotic cell promoter in vivo transcription system. Specifically as follows:

[0007] 1) T7 RNA polymerase in vivo transcription system: full-length or segmented virus cDNA plasmid is transfected into a pre-infected auxiliary virus (such as vaccinia virus, fowlpox virus) cell line expressing T7 polymerase. This method was first used to rescue GI.1 type HuNoV in HEK293T cells (human embryonic kidney cells expressing SV40 large T antigen mutant), but no virus particles containing viral nucleic acid were obtained by CsCl density gradient purification. Thereafter, the same method was used for GII.3 type HuNoV, but vaccinia virus caused cytopathic effect, and the virus density obtained was lower than that in the clinical sample, so vaccinia virus is not suitable for virus rescue. Later studies found that vaccinia virus also inhibited MNV replication. Therefore, fowlpox virus expressing T7 RNA polymerase (FPV-T7) was used for MNV rescue, and the full-length cDNA of MNV CW1 strain was transfected into hamster kidney cells (BHK-21) and hamster kidney cells (BSR-T7) pre-infected with FPV-T7, which first rescued MNV in vitro, and the infection of FPV-T7 had no effect on MNV replication. The in vivo virus rescue method dependent on auxiliary virus has low efficiency, and the rescued virus needs to be separated from the auxiliary virus through complex steps, and the auxiliary virus can cause cytopathic effect, thereby affecting the proliferation of the rescued virus. Therefore, this system has been rarely used in current research.

[0008] 2) T7 RNA polymerase in vitro transcription system: the full-length cDNA sequence is controlled by T7 promoter, and the enzyme cutting site introduced at the 3' end of the cDNA is used for in vitro transcription to obtain viral RNA, which is then transfected into sensitive cells to rescue infectious virus. Using this method, the RNA transcribed in vitro from MNV was capped, and the conditions for its electroporation into murine macrophages (Raw264.7) were optimized, establishing a stable and efficient method for rescuing MNV, with a yield of 6x106 Virus yield of 10 7 TCID 50 Meanwhile, the capped viral RNA was transfected into HEK-293T and BHK-21 cells, and still able to rescue virus. The same method was used to clone GII.3 HuSaV full-length cDNA into a vector with T7 promoter, in vitro transcribed and capped, and transfected into HuTu-80 cells with added cholate, and rescued HuSaV was detected after 12 days. In addition, porcine sapovirus (PEC) can also be capped in vitro by T7 polymerase, transfected into porcine kidney cells (LLC-PK) with added cholate, and infectious PEC was rescued. Although the T7 RNA polymerase in vitro transcription system can directly obtain infectious RNA, and can rescue virus after transfection of cells, the system is time-consuming and has low rescue efficiency.

[0009] 3) T7 RNA polymerase stable expression cell line system:

[0010] The T7 RNA polymerase stable expression cell line system constructs a cell line stably expressing T7 RNA polymerase, which directly drives the assembly of infectious cDNA clones into infectious virions in cells. Using this method, a cDNA plasmid containing the full-length MNV was transfected into the cell line, and infectious MNV was successfully obtained.

[0011] 4) Eukaryotic cell promoter in vivo transcription system: Eukaryotic cell promoter in vivo transcription system, RNA polymerase I, II is widely distributed in eukaryotic cells, and when constructing RNA virus full-length cDNA clone, eukaryotic cell specific recognition promoter can be introduced into the vector to replace T7 promoter to directly drive the in vivo transcription of viral genome. The virus cDNA constructed by this strategy no longer needs in vitro transcription and can be directly introduced into host cells; the virus transcript produced in vivo can act as messenger RNA to express viral proteins using the host cell translation system. Although the function of eukaryotic cell RNA polymerase to initiate transcription of foreign genes is not as powerful as T7 RNA polymerase, it has the advantages of correction function, wide cell adaptability, and avoidance of long-term operation of RNA in vitro. Although the eukaryotic polymerase rescue system is simple to operate, its transcription ability is limited, the transcript is at risk of being cut, and the virus rescue efficiency is not high. Since human sapovirus and human norovirus lack an effective cell culture system, the virus cannot be amplified in vitro, and the amount of sample obtained is very limited. The existing reverse genetics system of human norovirus or human sapovirus has low efficiency in recovering viruses. In addition, although it has been confirmed that NoV can replicate in human-derived B cell lines and stem cell-derived non-transformed human intestinal monolayer cell culture systems, it is difficult to be widely used for continuous passage of NoV due to its support for replication of only a small number of virus strains and harsh culture conditions, and there are still certain limitations. Therefore, it is still necessary to develop a robust human sapovirus and human norovirus in vitro cell culture system.

[0012] Therefore, the present application is proposed. SUMMARY

[0013] The technical problem solved by the present application is to overcome the shortcomings of the prior art and provide an infectious clone plasmid of human sapovirus or human norovirus, a reverse genetic virus rescue system and applications thereof.

[0014] The first object of the present application is to provide an infectious clone plasmid of human sapovirus or human norovirus.

[0015] The second object of the present application is to provide a reverse genetic virus rescue system comprising the infectious clone plasmid of human sapovirus or human norovirus, which can efficiently and quickly rescue progeny virions and quickly obtain a large amount of virus, which is beneficial to the further research of the virus and the development of related drugs, vaccines and the like.

[0016] At present, there are many difficulties in rescuing human norovirus and human sapovirus viruses: 1, due to the lack of an infected cell line, it is difficult to maintain stable replication and amplification, resulting in extremely low yield of the rescued virus; 2, human norovirus and human sapovirus RNA is easy to degrade, compared with murine norovirus, its RNA is more resistant and not easy to degrade; 3, the condition optimization of the rescue process, the rescue of human norovirus and human sapovirus needs to simultaneously satisfy the accurate space-time coordination of RNA transcription, translation, replication and virus particle assembly, compared with murine norovirus, its life cycle is more easily regulated in a cell line; 4, detection and verification are complex, human norovirus and human sapovirus cannot form visible plaques, and need to be detected by RT-qPCR or immunofluorescence to detect infectivity, and sometimes due to extremely low virus yield, immunofluorescence cannot be detected. Therefore, the sensitivity of their detection and verification is much lower than that of murine norovirus.

[0017] To solve the above technical problems, the basic idea of the technical scheme of the present application is:

[0018] In a first aspect, the present application provides an infectious clone plasmid of human sapovirus or human norovirus, which is based on a cloning vector, and a cDNA genome of human sapovirus or human norovirus is inserted; two T7 promoters are connected in series at the 5' end of the cDNA genome, and an internal ribosome entry site is inserted between the two T7 promoters; and an HDV ribozyme sequence and a T7 terminator sequence are connected in series after the PolyA tail of the cDNA genome.

[0019] The infectious clone plasmid of human sapovirus (HuSaVs) or human norovirus (HuNoVs) of the present application includes a full-length cDNA genome of the virus, a double T7 promoter is constructed upstream of the genome, and an internal ribosome entry site is inserted between the two T7 promoters, which is beneficial to rapidly and efficiently initiate the translation expression of human sapovirus or human norovirus; and an HDV ribozyme sequence and a T7 terminator sequence are connected in series downstream of the genome, which can ensure transcription termination, ensure high-fidelity cleavage efficiency, and ensure the accuracy of expression. Therefore, the infectious clone plasmid of the present application can not only efficiently initiate expression, but also accurately terminate, thereby ensuring the accuracy of virus expression.

[0020] It is found in the present application that the double T7 promoter can successfully express the VP1 protein of the virus, while the CMV promoter does not observe the expression of the VP1 protein. The internal ribosome entry site (IRES) derived from EMCV, which is inserted after the first T7 promoter, can efficiently mediate the translation of the viral genome protein, which is beneficial to promoting the translation of the viral genome; and the second T7 promoter mediates the replication of the viral genome.

[0021] DHV ribozyme is a self-cleaving RNA enzyme, which can accurately cleave its own RNA after transcription, and generate a sequence identical to the 3' end of the natural viral RNA, and can also increase the infectivity of viral RNA; T7 terminator sequence can specifically terminate the transcription of T7 RNA polymerase, prevent specific transcription extension, and maintain RNA stability.

[0022] In a further aspect, the cloning vector comprises one of pBluescript II-SK(+) vector, pBluescript II-SK(-) vector, pT7-M-ASKA (Reverse genetics vector pT7-M-ASKA), pT7-L (Reverse genetics vector pT7-L), and a reverse genetics vector.

[0023] Preferably, the cloning vector is pBluescript II-SK(+) vector.

[0024] As a preferred aspect, the cloning vector is pBluescript II-SK(+) vector, which itself contains a T7 promoter, and when constructing the infectious clone plasmid, the IRES, the second T7 promoter, and the cDNA genome can be connected in series downstream of the T7 promoter.

[0025] In a further aspect, there is no spacer nucleotide between the first T7 promoter sequence and the internal ribosome entry site sequence.

[0026] In the present application, the redundant nucleotide sequence between the first T7 promoter sequence and the internal ribosome entry site sequence is deleted by mutation PCR, so that the fidelity of the transcribed viral RNA in the cell can be ensured, that is, the transcribed viral RNA does not have other sequences in addition to the viral RNA itself.

[0027] In a further aspect, the cDNA genome of the human sapovirus is a full genome sequence of GI.1 human sapovirus.

[0028] Preferably, the full genome sequence of the GI.1 human sapovirus is shown in SEQ ID NO: 14.

[0029] In a further aspect, the cDNA genome of the human norovirus is a full genome sequence of GII.4 human norovirus.

[0030] Preferably, the full genome sequence of the GII.4 human norovirus is shown in SEQ ID NO: 17.

[0031] Further, a reporter gene is inserted into the cDNA genome of the human sapovirus.

[0032] Preferably, the reporter gene comprises an EGFP gene.

[0033] In a second aspect, the present application provides a method for constructing the human sapovirus or human norovirus infectious clone plasmid, comprising:

[0034] (1) designing a double T7 promoter, internal ribosome entry site driven human sapovirus or human norovirus cDNA genome strategy;

[0035] (2) cloning the double T7 promoter, internal ribosome entry site, human sapovirus or human norovirus cDNA genome, HDV ribozyme sequence and T7 terminator sequence into a cloning vector to construct the human sapovirus infectious clone plasmid or human norovirus infectious clone plasmid;

[0036] Preferably, the cloning vector is pBluescript II-SK(+).

[0037] As a specific embodiment,

[0038] In step (1), according to the full-length genome sequence of the human sapovirus or human norovirus and the selection of the restriction site of the vector, the full-length genome sequence is divided into two segments for the construction of the full-length infectious cDNA clone, which are named as A segment and B segment, respectively.

[0039] In step (2), the internal ribosome entry site-second T7 promoter-human sapovirus or human norovirus cDNA genome A segment is first constructed into the cloning vector (pBluescript II-SK(+)) (downstream of the first T7 promoter), and then the redundant nucleotide sequence between the first T7 promoter and the internal ribosome entry site is removed by mutant PCR, and then the B segment-HDV ribozyme sequence and T7 terminator sequence are connected to the vector to obtain the infectious clone plasmid.

[0040] In a third aspect, the present application provides a reverse genetic virus rescue system of the human sapovirus or human norovirus, comprising:

[0041] The human sapovirus or human norovirus infectious clone plasmid, the plasmid expressing T7 RNA polymerase and the BHK-T7 cell.

[0042] The reverse genetic virus rescue system of human sapovirus or human norovirus of the present application comprises a human sapovirus or human norovirus infectious clone plasmid, a plasmid expressing T7 RNA polymerase and BHK-T7 cells. In one aspect, the transfection efficiency can be improved by co-transfecting the plasmid expressing T7 RNA polymerase together with the infectious clone plasmid. In another aspect, the present application finds that the target virus can be successfully rescued by using BHK-T7 cells, which is efficient and fast, and a large amount of virus can be rescued.

[0043] The present application finds that the amount of rescued virus is significantly higher than that of the virus infectious clone plasmid alone when the human sapovirus or human norovirus infectious clone plasmid and the plasmid expressing T7 RNA polymerase are co-transfected into cells, which indicates that the transfection efficiency and the amount of rescued virus can be significantly improved by adding the plasmid expressing T7 RNA polymerase.

[0044] The present application uses different cell lines for transfection, including BHK-T7, 293T, HuTu-80, CaCo2 and Hela cells. It is unexpectedly found that the number of viral genome copies of the plasmid transfected into BHK-T7 cells is increased by about 3.7 and 3.9 Log10 / μL, respectively; while the number of RNA is not increased in other cell lines.

[0045] In a fourth aspect, the present application provides a method for rescuing virus by using the reverse genetic virus rescue system of human sapovirus or human norovirus, comprising:

[0046] The human sapovirus or human norovirus infectious clone plasmid and the plasmid expressing T7 RNA polymerase are mixed and transfected into BHK-T7 cells, and after culture, the cells and supernatant are collected, centrifuged, and the rescued virus particles are purified and recovered;

[0047] Preferably, the mass concentration ratio of the human sapovirus or human norovirus infectious clone plasmid and the plasmid expressing T7 RNA polymerase is 1:1.

[0048] In a fifth aspect, the present application provides the use of the human sapovirus or human norovirus infectious clone plasmid or the reverse genetic virus rescue system of human sapovirus or human norovirus in the preparation of prophylactic and / or therapeutic vaccines and / or drugs.

[0049] In addition, the human sapovirus or human norovirus infectious clone plasmid or the reverse genetic virus rescue system of human sapovirus or human norovirus of the present application can also be used as an important tool and method for studying the gene expression regulation mechanism of virus, the structure and function of viral proteins, the interaction between pathogen and host, and the pathogenic mechanism of virus.

[0050] Compared with the prior art, the present application has the following beneficial effects:

[0051] 1、The present application constructs a human sapovirus infectious clone plasmid or a human norovirus infectious clone plasmid, inserts a full-length cDNA genome of the human sapovirus or the human norovirus, and constructs a double T7 promoter upstream of the full-length cDNA genome, and inserts an internal ribosome entry site between the two T7 promoters, which is beneficial to rapidly and efficiently starting expression of the human sapovirus or the human norovirus; a HDV ribozyme sequence and a T7 terminator sequence are further connected in sequence after a PolyA tail of the cDNA genome, which can guarantee transcription termination, ensure high-fidelity cleavage efficiency, and ensure expression accuracy. Therefore, the infectious clone plasmid of the present application can not only efficiently start expression, but also accurately terminate, thereby guaranteeing expression accuracy of the virus.

[0052] 2、The reverse genetic virus rescue system of the human sapovirus or the human norovirus of the present application comprises a human sapovirus or human norovirus infectious clone plasmid, a plasmid expressing T7 RNA polymerase, and BHK-T7 cells. On the one hand, the plasmid expressing T7 RNA polymerase is added to co-transfect cells together with the infectious clone plasmid, which can improve transfection efficiency. On the other hand, it is found that the BHK-T7 cells can successfully rescue the target virus. The present application can efficiently and rapidly rescue progeny virus particles and rapidly obtain a large amount of virus.

[0053] 3、The reverse genetic virus rescue system of the human sapovirus or the human norovirus of the present application can rapidly obtain a large amount of virus, and can be used as a robust human sapovirus and human norovirus in-vitro cell culture system, which provides a basis for further research on the structure and function of the virus genome, the interaction between the virus and the host, the pathogenic mechanism of the virus, and the like, and can also be used for screening of antiviral drugs, research and development of vaccines, and the like, and has important significance.

[0054] The specific embodiments of the present application will be further described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0055] The accompanying drawings, which are part of the present application, serve to provide a further understanding of the present application, and the schematic embodiments of the present application and the descriptions thereof serve to explain the present application, but do not constitute an improper limitation on the present application. Obviously, the accompanying drawings in the following description are only some embodiments, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings. In the drawings:

[0056] Figure 1 is a schematic diagram of a GI.1 type HuSaV full-length infectious cDNA clone construction strategy;

[0057] Figure 2Figure 1 is a schematic diagram of the construction strategy of GI.1 type HuSaV full-length infectious cDNA clone carrying EGFP gene;

[0058] Figure 3 Figure 2 is a schematic diagram of the construction strategy of GII.4 type HuNoV full-length infectious cDNA clone;

[0059] Figure 4 Figure 3 is a Western blotting identification of pBlue-GI.1-HuSaV or pBlue-GI.1-HuSaV-EGFP and RNA polymerase plasmid co-transfected cells; A: HuSaV expressed protein and GI.1 type HuSaV-VP1 antibody reactivity; B: recombinant HuSaV-EGFP expressed protein and GI.1 type HuSaV-VP1 antibody reactivity; C: recombinant HuSaV-EGFP expressed protein and EGFP antibody reactivity;

[0060] Figure 5 Figure 4 is a Western blotting identification of pBlue-GII.4-HuNoV and RNA polymerase plasmid co-transfected cells;

[0061] Figure 6 Figure 5 is a Western blotting identification of rescued GI.1-HuSaV virus and recombinant GI.1-HuSaV-EGFP virus after purification; A: rescued GI.1-HuSaV virus and GI.1 type HuSaV-VP1 antibody reactivity; B: recombinant GI.1-HuSaV-EGFP virus and GI.1-0013-VP1 and VP2 antibody reactivity;

[0062] Figure 7 Figure 6 is a Western blotting identification of rescued GII.4-HuNoV virus after purification; A: Western blotting identification of rescued GII.4-HuNoV virus after concentrated by 20% sucrose cushion; B: Western blotting identification of rescued GII.4-HuNoV virus after purified by CsCl;

[0063] Figure 8 Figure 7 is an electron microscope observation of rescued GI.1-HuSaV, recombinant GI.1-HuSaV-EGFP and GII.4-HuNoV virus particles; A: rescued GI.1-HuSaV virus particles; B: rescued recombinant GI.1-HuSaV-EGFP virus particles; C: rescued GII.4-HuNoV virus particles;

[0064] Figure 9Figure 1 is a result chart of RT-qPCR experiment of BHK-T7 cells transfected with pBlue-GI.1-HuSaV and pBlue-GI.1-HuSaV-EGFP plasmids respectively or co-transfected with T7 RNA polymerase plasmid in Comparative Example 1, wherein the cell samples were harvested at 24 h and subjected to RT-qPCR experiment;

[0065] Figure 10 Figure 2 is a schematic diagram of cloning strategy of pcDNA3.1-GI.1-HuSaV and pcDNA3.1-GII.4-HuNoV in Comparative Example 2;

[0066] Figure 11 Figure 3 is a Western blotting identification of BHK-T7 cells transfected with pcDNA3.1-GI.1-HuSaV or pcDNA3.1-GII.4-HuNoV plasmid in Comparative Example 2. A: HuSaV expressed protein reacted with GI.1 type HuSaV-VP1 antibody; B: HuNoV expressed protein reacted with GII.4 type HuNoV-VP1 antibody.

[0067] Figure 12 Figure 4 is a result chart of transfected different cell lines in Comparative Example 3, wherein ****, P<0.0001; ***, P<0.001; **, P<0.01; *, P<0.05; ns, not significant.

[0068] It should be noted that these drawings and written descriptions are not intended to limit the scope of the concept of the present application in any way, but to illustrate the concept of the present application to those skilled in the art by referring to specific embodiments. DETAILED DESCRIPTION

[0069] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments will be described clearly and completely below in conjunction with the drawings of the embodiments of the present application, and the following embodiments are used to illustrate the present application but not to limit the scope of the present application.

[0070] Embodiment 1

[0071] 1. Main experimental methods

[0072] 1.1 Construction of GI.1 type HuSaV full-length infectious cDNA clone

[0073] According to the full-length genome sequence of GI.1 type HuSaV 0013 strain and the selection of vector enzyme cutting sites, the full-length genome sequence of HuSaV was divided into two segments for the construction of full-length infectious cDNA clone, which were named as A segment and B segment. The A segment was 2294 bp, and the B segment was 5127 bp.

[0074] 1.1.1A fragment gene cloning

[0075] Prepare pBluescript II-SK(+) vector digested by Sal I / Sac II and purify; pBluescript II-SK(+) vector itself has a T7 promoter, so redesign the internal ribosome entry site (IRES) + T7 promoter + HuSaV-A fragment sequence, and send to Genescript Biotech Co., Ltd. for gene synthesis and ligation, to obtain pBlue-GI.1-HuSaV-A (construction strategy as shown in Figure 1 Transform Top10 competent cells, coat Amp + Incubate the plate overnight, pick colonies for expansion culture, extract plasmids, and perform sequencing identification.

[0076] Use primer T7-Tu-F and T7-Tu-R to perform mutation PCR to remove the redundant nucleotide sequence between IRES and the first T7 promoter, using the identified correct plasmid as the template (see Table 1 for primers), to finally obtain plasmid pBlue-GI.1-HuSaV-Aa, transform competent cells, and perform sequencing.

[0077] 1.1.2B segment gene cloning and full-length infectious cDNA cloning

[0078] In order to terminate the mRNA transcribed after the viral genome is transfected into cells, and terminate the T7 promoter, HDV and T7 polymerase termination sequences will be added after the PolyA tail of the viral genome.

[0079] Amplify B fragment using homologous arm primers B-F (containing Sac II restriction site) and B-R; amplify HDV and T7 RNA polymerase termination fragment using non-homologous arm primer HDV-F and homologous arm primer T7-Ter-R (Sal I restriction site) and purify (see Table 1 for primers), and prepare pBlue-GI.1-HuSaV-Aa vector digested by Sac II / Sal I and purify; use homologous recombination method to ligate the two amplified target fragments with pBlue-GI.1-HuSaV-Aa vector, to obtain full-length pBlue-GI.1-HuSaV-A-B plasmid (construction strategy as shown in Figure 1 Transform TOP10 competent cells, coat Amp + Incubate the plate overnight, pick colonies for expansion culture, extract plasmids, and perform sequencing again.

[0080] Table 1 Primer sequences

[0081]

[0082] The nucleic acid sequence of the T7 promoter is shown as SEQ ID NO: 12, the nucleic acid sequence of the internal ribosome entry site (IRES) is shown as SEQ ID NO: 13, the nucleic acid sequence of the full-length genome sequence of the GI.1 type HuSaV is shown as SEQ ID NO: 14; the nucleic acid sequence of the HDV ribozyme is shown as SEQ ID NO: 15, and the nucleic acid sequence of the T7 terminator sequence is shown as SEQ ID NO: 16.

[0083] 1.2 Construction of a full-length infectious cDNA clone of GI.1 type HuSaV-EGFP

[0084] 1.2.1 Cloning of the A segment gene

[0085] The pBluescript II-SK(+) vector digested by Xho I / Sac II was prepared and purified; the internal ribosome entry site (IRES) + T7 promoter + HuSaV-A-EGFP (NSP1 / 2-EGFP-NSP3) segment sequence was designed and sent to Genescript Biotech Co., Ltd. for gene synthesis and ligation to obtain the pBlue-GI.1-HuSaV-A-EGFP plasmid (pBlue-GI.1-HuSaV-A-EGFP) Figure 2 ); the Top10 competent cells were transformed, the colonies were expanded and cultured, the plasmid was extracted, and sequencing identification was performed. The identified correct plasmid was used as a template for mutation PCR to remove the redundant nucleotide sequence between IRES and the first T7 promoter (see Table 1 for primers), and finally the recombinant plasmid pBlue-GI.1-HuSaV-Aa-EGFP was obtained, which was transformed into competent cells and then sequenced again.

[0086] 1.2.2 Cloning of the B segment gene and construction of a full-length infectious cDNA clone carrying EGFP

[0087] The two target fragments were amplified by the method of 1.1.2 above and ligated with the pBlue-GI.1-HuSaV-Aa-EGFP vector to obtain the full-length pBlue-GI.1-HuSaV-A-B-EGFP Figure 2 ), i.e., pBlue-GI.1-HuSaV-EGFP; the TOP10 competent cells were transformed, the plasmid was extracted, and sequencing was performed.

[0088] 1.3 Construction of a full-length infectious cDNA clone of GII.4 type HuNoV

[0089] The full-length genomic nucleic acid sequence of the GII.4 type HuNoV is shown as SEQ ID NO: 17. According to the full-length genomic sequence of the GII.4 type HuNoV and the selection of the restriction sites of the vector, the full-length genomic sequence of the HuNoV is divided into two segments for the construction of the full-length infectious cDNA clone, which are named as segment A and segment B. The segment A is 3403 bp, and the segment B is 4156 bp.

[0090] The same construction strategy as the GI.1 type HuSaV is adopted. The pBlue-SK(+) vector digested by Not I / BamH I is prepared and purified; the internal ribosome entry site (IRES) + T7 promoter + HuNoV-A segment sequence is designed and sent to GenScript Biotech Co., Ltd. for gene synthesis and ligation to obtain the pBlue-GII.4-HuNoV-A Figure 3 ) vector. The segment B is amplified by using NV-4156-F (containing a BamH I restriction site) and NV-HDV-R; the HDV and T7 RNA polymerase termination segment is amplified by using HDV-F and NV-Ter-R (containing an EcoR I restriction site) and purified (see Table 1 for primers). Then the two segments are ligated with the pBlue-GII.4-HuNoV-A vector to obtain the plasmid pBlue-GII.4-HuNoV Figure 3 ), which is sequenced.

[0091] 1.4 Transfection of BHK-T7 monolayer cells in vitro

[0092] The full-length infectious cDNA clone plasmids of 1.1.2, 1.2.2 and 1.3 which were sequenced correctly and T7 RNA polymerase plasmid (the specific plasmid name used was pCI-T7, reference Rescue and characterization of a recombinant HY12 bovine enterovirus carrying a foreign HA epitope in the 3A nonstructural protein) were amplified, and pBlue-GI.1-HuSaV, pBlue-GI.1-HuSaV-A-B-EGFP, pBlue-GII.4-HuNoV were mixed with T7 RNA polymerase plasmid respectively according to the Lipofectamine 3000 transfection reagent instructions, and co-transfected into 6-well culture plates of BHK-T7 cells with a density of 80%-90%. Then at different time points after transfection, the cells were lysed with lysis buffer, and Western Blotting experiments were performed; untransfected plasmid cells were set up as blank wells as negative controls, and the rescued viruses were named GI.1-HuSaV, GI.1-HuSaV-EGFP and GII.4-HuNoV respectively.

[0093] 1.5 Western Blotting experiment

[0094] After the cell lysates harvested at different time points in 1.4 were subjected to SDS-PAGE electrophoresis, the proteins were transferred to PVDF membranes by semi-dry transfer. Then the PVDF membranes were blocked in PBS containing 50 g / L skimmed milk powder overnight, washed with PBS-T three times for 5 min each time; rabbit-derived GI.1 HuSaV-VP1 antibody or mouse-derived GII.4 HuNoV-VP1 antibody or EGFP antibody (1:1000) was added as the primary antibody, incubated at room temperature for 1 h, washed with PBS-T three times for 5 min each time; horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:5000) or goat anti-mouse IgG (1:1000) was added as the secondary antibody, incubated at room temperature for 45 min. After thorough washing with PBS-T, ECL exposure solution was added and incubated for 1 min, and analyzed by a Tianen imaging system.

[0095] 1.6 Virus particle purification and electron microscopy observation

[0096] The 1.1.2, 1.2.2 and 1.3 full-length infectious cDNA clone plasmids were respectively co-transfected with T7 RNA polymerase plasmid in large quantities, and 24-32 h, the cells and supernatant were collected about 280 mL. The cell precipitate was discarded by centrifugation at 8000 rpm for 20 min at 4°C, and the supernatant was passed through a 20% sucrose pad and centrifuged at 10 0000 g for 3 h at 4°C. The supernatant was discarded, and the precipitate was dissolved in PBS overnight. The precipitate (turbid liquid) was further purified by centrifugation at 11 2000 g for 22 h at 4°C using a CsCl density gradient (1.36 g / mL). After gradient layering, the sample was sequentially aspirated from top to bottom (500 μL each time for rescued HuSaV and HuSaV-EGFP virus; 1 mL each time for rescued HuNoV), and each layer was diluted 10 times with PBS. The virus particles were recovered by centrifugation at 10 0000 g / min for 3 h at 4°C. After Western blotting detection, electron microscopy observation was performed.

[0097] 2Results

[0098] 2.1Western blotting identification after pBlue-GI.1-HuSaV or pBlue-GI.1-HuSaV-EGFP plasmid transfection of cells

[0099] After co-transfecting pBlue-GI.1-HuSaV and pBlue-GI.1-HuSaV-EGFP plasmids with T7 RNA polymerase plasmid into BHK-T7 cells, cell lysates were harvested at different time points, and Western blotting experiments were performed.

[0100] The results show that, whether it is pBlue-GI.1-HuSaV or pBlue-GI.1-HuSaV-EGFP plasmid, the expression of VP1 protein can be observed at 8 h after transfection of cells, the protein expression reaches the highest at 24 h, the protein expression decreases at 48 h, and only a small amount of VP1 protein expression can be detected at 72 h. The size of VP1 is about 60 kDa Figure 4 A, B); and specific bands were not detected in the negative control (Mock) BHK-T7 cells. Compared with the control group (Mock), the expression of NSP1 / 2 and EGFP fusion protein can be observed by EGFP antibody, with a size of about 65 kDa; the expression of NSP1 / 2 and GFP and NSP3 fusion precursor protein, with a size of about 102 kDa; but the EGFP protein after the virus itself encoding protease cleavage of ORF1, with a size of about 27 kDa Figure 4 C), indicating that the pBlue-GI.1-HuSaV or pBlue-GI.1-HuSaV-EGFP plasmid transfected into BHK-T7 cells may package progeny viruses.

[0101] 2.2 Western blotting identification of pBlue-GII.4-HuNoV transfected cells

[0102] After co-transfecting BHK-T7 cells with pBlue-GII.4-HuNoV plasmid and T7 RNA polymerase plasmid, we collected 12h, 24h and 36h cell lysate samples for protein detection by Western Blotting. The results showed that a small amount of VP1 protein expression was detected in the transfected cells at 12h, and the target band size was about 60kDa. The VP1 expression was the highest at 24h and 36h, and the BHK-T7 cell negative control (Mock) did not detect specific bands. Figure 5 The results showed that the VP1 protein expression of GII.4 type HuNoV virus could be detected in BHK-T7 cells, and the transfected plasmid packaged progeny virus.

[0103] 2.3 Western blotting identification of rescued GI.1-HuSaV and its recombinant GI.1-HuSaV-EGFP virus after purification

[0104] To further determine whether the pBlue-GI.1-HuSaV or pBlue-GI.1-HuSaV-EGFP plasmid transfected into BHK-T7 cells packaged progeny virus particles, we performed ultracentrifugation on a large amount of supernatant after transfection. The results showed that the rescued GI.1-HuSaV virus ( Figure 6 A) and the recombinant GI.1-HuSaV-EGFP virus ( Figure 6 B) both produced a clear band with VP1 antibody after purification, and the target band was most obvious in the 8th-10th layer, with the 8th layer being the most.

[0105] 2.4 Western blotting identification of rescued GII.4-HuNoV virus after purification

[0106] To determine whether the pBlue-GII.4-HuNoV plasmid transfected into BHK-T7 cells also produced progeny virus particles, we performed ultracentrifugation on the supernatant collected after large-scale transfection of the plasmid. Then we concentrated the culture through a 20% sucrose pad and performed Western Blotting identification together with the cell lysate sample collected at 24h after transfection. The results showed that compared with the BHK-T7 cell negative control group (Mock), both the sucrose pad concentrate and the 24h cell lysate sample produced the correct size target band with VP1 antibody, and the band of the concentrate was more obvious ( Figure 7 A).

[0107] The sample purified by sucrose cushion was further purified by CsCl density gradient centrifugation. The results are shown in Figure 7 As shown in Figure B, the target band began to appear in the 8th layer, and the target band was most obvious in the 10th layer, and the subsequent target band began to weaken. It is shown that the purified virus after density gradient centrifugation is mainly concentrated in the 8th-12th layer, and the most in the 10th layer.

[0108] 2.5 Electron microscope observation of rescued virus particles.

[0109] The purified GI.1-HuSaV virus, recombinant GI.1-HuSaV-EGFP virus and GII.4-HuNoV virus were negatively stained and observed under an electron microscope, as shown in Figure 8 , we can observe that the circular virus particles with a diameter of about 30-40 nm contain nucleic acid solid virus particles and hollow virus particles without nucleic acid, and there is no contamination of other viruses.

[0110] Comparative Example 1 Single plasmid transfection of cell line

[0111] The pBlue-GI.1-HuSaV and pBlue-GI.1-HuSaV-EGFP plasmids prepared in 1.1.2 and 1.2.2 were respectively single-transfected into BHK-T7 cells, or the pBlue-GI.1-HuSaV and pBlue-GI.1-HuSaV-EGFP plasmids were respectively co-transfected with T7 RNA polymerase plasmid into BHK-T7 cells, and the cell samples were harvested 24 h after transfection for RT-qPCR experiment.

[0112] Results: As shown in Figure 9 , in the figure, BHKT7+T7+0013 represents co-transfection of pBlue-GI.1-HuSaV and T7 RNA polymerase plasmid into BHK-T7 cells, BHKT7+0013 represents single transfection of pBlue-GI.1-HuSaV into BHK-T7 cells, BHKT7+T7+0013EGFP represents co-transfection of pBlue-GI.1-HuSaV-EGFP and T7 RNA polymerase plasmid into BHK-T7 cells, and BHKT7+0013EGFP represents single transfection of pBlue-GI.1-HuSaV-EGFP into BHK-T7 cells. As can be clearly seen from the figure, whether it is pBlue-GI.1-HuSaV or pBlue-GI.1-HuSaV-EGFP plasmid co-transfected with T7 RNA polymerase plasmid into BHK-T7 cells, the copy number of virus is significantly higher than that of BHK-T7 cells transfected with single plasmid pBlue-GI.1-HuSaV or pBlue-GI.1-HuSaV-EGFP. It is shown that adding a plasmid expressing T7 RNA polymerase for co-transfection with the infectious clone plasmid into cells can significantly improve the virus yield.

[0113] Comparative Example 2 uses CMV promoter

[0114] According to the construction strategy in Reference Example 1, pcDNA3.1 Zeo(+) vector was prepared and purified by double digestion of Afl II / Sac II and Not I / BamH I; HuSaV-A and HuNoV-A fragment sequences were designed respectively and sent to GenScript for gene synthesis and ligation to obtain pcDNA3.1-GI.1-HuSaV-A plasmid and pcDNA3.1-GII.4-HuNoV-A Figure 10 ) respectively; Top10 competent cells were transformed, and the colonies were expanded and cultured to extract plasmids and perform sequencing identification. The B fragments of the two genes were amplified by the methods of 1.1.2 and 1.3 in Example 1, and the HDV fragment was amplified by HDV-F and SapT3.1-HDV-R (containing Kpn I digestion site) or NVT3.1-HDV-R (containing EcoR I digestion site) and purified (see Table 1 for primers), and then connected with pcDNA3.1-GI.1-HuSaV-A plasmid and pcDNA3.1-GII.4-HuNoV-A vector respectively to obtain full-length pcDNA3.1-GI.1-HuSaV-A-B and pcDNA3.1-GII.4-HuNoV-A-B, i.e. pcDNA3.1-GI.1-HuSaV and pcDNA3.1-GII.4-HuNoV; TOP10 competent cells were transformed, and plasmids were extracted and sequenced.

[0115] According to the method of 1.4 in Reference Example 1, BHK-T7 cells were transfected, and at different time points after transfection, the cells were lysed with lysis buffer and Western Blotting was performed.

[0116] As shown in Figure 11 , no VP1 protein expression was observed at each time point after transfection of pcDNA3.1-GI.1-HuSaV and pcDNA3.1-GII.4-HuNoV plasmids in the transfected cells.

[0117] Comparative Example 3 transfects different cell lines

[0118] According to the method of 1.4 in Reference Example 1, 2.5 μg of pBlue-GI.1-HuSaV and 2.5 μg of pBlue-GI.1-HuSaV-EGFP infectious clone plasmid were separately transfected into BHK-T7, BHK-21, 293T, HuTu-80, CaCo2 and Hela cells with a density of about 80% in a 6-well culture plate.

[0119] Or 2.5 pg pBlue-GI.1-HuSaV and 2.5 pg pBlue-GI.1-HuSaV-EGFP infectious clone plasmid respectively with 2.5 pg T7 polymerase plasmid, using Lipofectamine 3000 to co-transfect into 6-well culture plates of BHK-T7, 293T, HuTu-80, CaCo2 and Hela cells with a density of about 80%.

[0120] After transfection for 24-32 h, the cells and supernatants were harvested respectively for RT-qPCR experiments; the cells with only 2.5 pg pBlue-GI.1-HuSaV or 2.5 pg pBlue-GI.1-HuSaV-EGFP (without T7 polymerase) or 2.5 pg empty vector plasmid (Empty, EV) were used as negative controls.

[0121] Results: As shown in the figure, the names of each group in the figure, +T7 means adding T7 RNA polymerase plasmid when transfecting the infectious clone plasmid, +0013 means pBlue-GI.1-HuSaV plasmid, 0013EGFP means pBlue-GI.1-HuSaV-EGFP plasmid, +EV means empty vector plasmid. Figure 12

[0122] Among them, BHK-21 cells are standard cell lines that do not express T7 RNA polymerase in their cells; BHK-T7 (labeled as BHKT7 in the figure) is a genetically engineered BHK-21 cell that only integrates the T7 RNA polymerase expression gene. Therefore, BHK-21+0013 or BHK-21+0013EGFP as a control can eliminate the background value brought by transfecting pBlue-GI.1-HuSaV or pBlue-GI.1-HuSaV-EGFP plasmid cells (through RT-qPCR not only can detect viral RNA but also can detect residual plasmid).

[0123] When pBlue-GI.1-HuSaV or pBlue-GI.1-HuSaV-EGFP plasmid is co-transfected with T7 RNA polymerase plasmid into BHK-T7 cells, compared with the control group (only transfecting pBlue-GI.1-HuSaV or pBlue-GI.1-HuSaV-EGFP plasmid), the number of viral genome copies proliferated by about 5.0 and 5.5 Log 10 / μL respectively; secondly, transfecting pBlue-GI.1-HuSaV or pBlue-GI.1-HuSaV-EGFP plasmid into BHK-T7 cells, the number of viral genome copies proliferated by about 3.7 and 3.9 Log 10 ​ / μL; while the other transfected cell lines did not obtain higher RNA quantity proliferation.

[0124] The above description is only the preferred embodiment of the present application, not any form of the present application, although the present application has been disclosed as above, however, not to limit the present application, any skilled in the art within the scope of the present application, when the above-mentioned technical content of the prompt can be made some more changes or modifications for equivalent embodiments, but as long as it does not deviate from the technical solution of the present application, according to the technical essence of the present application, any simple modification, equivalent change and modification of the above embodiment, still belongs to the scope of the present application.

Claims

1. An infectious clonal plasmid of human norovirus or human zazovirus, characterized in that, Based on a cloning vector, a cDNA genome of human zarrovirus or human norovirus is inserted; the 5' end of the cDNA genome is tandemly linked to two T7 promoters, and an internal ribosome entry site is inserted between the two T7 promoters; the cDNA genome is followed by a PolyA tail and then tandemly linked to an HDV ribozyme sequence and a T7 terminator sequence.

2. The infectious clonal plasmid of human norovirus or human zoonavirus according to claim 1, characterized in that, The cloning vectors mentioned include one of the following: pBluescript II-SK(+) vector, pBluescript II-SK(-) vector, pT7-M-ASKA, pT7-L, and reverse genetics vector; Preferably, the cloning vector is the pBluescript II-SK(+) vector.

3. The infectious clonal plasmid of human norovirus or human zoonavirus according to claim 1 or 2, characterized in that, There are no spacer nucleotides between the first T7 promoter sequence and the sequence at the internal ribosome entry site.

4. The infectious clonal plasmid of human norovirus or human zonavirus according to any one of claims 1-3, characterized in that, The cDNA genome of the human zarrovirus is the complete genome sequence of human zarrovirus type GI.

1. Preferably, the complete genome nucleic acid sequence of the GI.1 type human zovirus is shown in SEQ ID NO:

14.

5. The infectious clonal plasmid of human norovirus or human zonavirus according to any one of claims 1-3, characterized in that, The cDNA genome of the human norovirus is the complete genome sequence of human norovirus type GII.4; Preferably, the complete genome nucleic acid sequence of the GII.4 human norovirus is shown in SEQ ID NO:

17.

6. The infectious clonal plasmid of human zazovirus according to any one of claims 1-4, characterized in that, A reporter gene was also inserted into the cDNA genome of the human zarif virus. Preferably, the reporter gene includes the EGFP gene.

7. A method for constructing an infectious clonal plasmid of human norovirus or human zonavirus as described in any one of claims 1-6, characterized in that, include: (1) Design a strategy to drive human zazovirus or human norovirus cDNA genome using a dual T7 promoter and internal ribosome entry site. (2) The double T7 promoter, internal ribosome entry site, human zarrovirus or human norovirus cDNA genome, HDV ribozyme sequence and T7 terminator sequence are cloned into a cloning vector to construct human zarrovirus infectious cloning plasmid or human norovirus infectious cloning plasmid.

8. A reverse genetic virus rescue system for human norovirus or human rorovirus, characterized in that, include: The human zazovirus or human norovirus infectious clone plasmid as described in any one of claims 1-6, the plasmid expressing T7 RNA polymerase, and BHK-T7 cells.

9. A method for rescuing a virus using the reverse genetic virus rescue system for human norovirus or human norovirus as described in claim 8, characterized in that, include: The infectious clone plasmid of human zarrovirus or human norovirus as described in any one of claims 1-6 is mixed with a plasmid expressing T7 RNA polymerase and transfected into BHK-T7 cells. After culturing, the cells and supernatant are collected, centrifuged, and the rescued virus particles are purified and recovered. Preferably, the mass concentration ratio of the human norovirus or human clonal virus plasmid to the T7 RNA polymerase expression plasmid is 1:

1.

10. The use of an infectious clonal plasmid of human zarrovirus or human norovirus as described in any one of claims 1-6, or a reverse genetic virus rescue system of human zarrovirus or human norovirus as described in claim 8, in the preparation of preventive and / or therapeutic vaccines and / or medicaments.

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