Infective clones of CA16 virus carrying the luciferase gene Nluc, their construction and application

Abstract

This invention relates to an infectious clone of CA16 virus carrying the luciferase gene Nluc, its construction, and its applications. The P1 region of the CA16 genome from strain OP293089 was constructed into pcDNA, and the P2 and P3 regions were constructed into the pUC57 vector. The P1 and P2+P3 regions were recombined with the pSVA vector to construct an infectious CA16 clone. The Nluc luciferase reporter gene was then introduced to construct the pSVA-CA16-Nluc infectious clone. After rescuing the CA16-Nluc virus, its activity was experimentally verified to be similar to that of the parent virus. After continuous passage in Vero cells, the genetic stability of CA16-Nluc in in vitro cell culture was experimentally verified. Furthermore, this infectious clone can be used for high-throughput drug screening and has wide applications in basic research on viral replication and pathogenesis mechanisms, high-throughput drug screening, and neutralizing antibody screening.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology or medicine, and in particular to an infectious clone of a CA16 virus carrying the luciferase gene Nluc, its construction, and its application. Background Technology

[0002] Coxsackievirus A16 (CA16) is a single-stranded positive-sense RNA virus belonging to the genus Enterovirus and the family Picornaviridae. The CA16 viral genome is approximately 7.5 kb in length and consists of a 5' untranslated region (5'UTR), a structural protein-coding region (P1 region), non-structural protein-coding regions (P2+P3), and a 3' untranslated region (3'UTR). After infecting a cell, the virus releases its genomic RNA into the cytoplasm for replication and translation. During translation, since CA16 contains only one reading frame, it first translates into a polyprotein comprising both structural and non-structural proteins. Subsequently, the viral proteases 2A and 3C cleave the polyprotein, forming four mature structural proteins—VP1, VP2, VP3, and VP4—and seven non-structural proteins—2A, 2B, 2C, 3A, 3B, 3C, and 3D. The fully cleaved structural proteins form an icosahedral capsid structure, which encapsulates the replicated RNA genome to form a mature viral particle, which is then released extracellularly. Studies have shown that CA16, along with three other enteroviruses—EV71, CVA10, and CVA6—is the main pathogen of hand-foot-and-mouth disease (HFMD). Most HFMD cases caused by CA16 infection are mild and self-limiting. However, in recent years, CA16 and EV71 have alternated in the Western Pacific region, seriously endangering children's health. Numerous cases have been reported with severe and even fatal consequences caused by CA16 infection. Currently, inactivated vaccines against EV71 are available to effectively prevent disease caused by EV71 infection, but there is still no effective vaccine available for CA16. Therefore, strengthening research on CA16 is urgently needed.

[0003] Infectious clones have been widely used as a powerful tool in virus research. Using reverse genetics, scientists can easily construct viral genomes into plasmids that are easy to amplify and store. They can also perform various targeted modifications to the viral genome, such as gene deletion mutations, foreign tag insertions, and reporter gene insertions. Furthermore, compared to the high mutation rate of viruses, infectious clones have a lower mutation rate, thus avoiding mutations during viral passage. In conclusion, infectious clones, as a tool for virus research, have broad application value in areas such as viral pathogenesis mechanisms, drug development, and neutralizing antibody screening.

[0004] Infectious clones carrying reporter genes are constructed by inserting exogenous reporter genes into infectious viral clones. Widely used reporter genes are generally divided into two main categories: fluorescent proteins and luciferases. Fluorescent proteins include green fluorescent protein (GFP) and red fluorescent protein (mCherry), while luciferases include firefly luciferase (Fluc), kidney luciferase (Rluc), Gaussian luciferase (Gluc), and novel luciferases (NanoLuc, Nluc). Currently published infectious clones of enteroviruses carrying reporter genes include EV71 and CA16 infectious clones carrying GFP. Viruses carrying GFP can be tracked in vivo and in vitro by observing their luminescence, but quantitative analysis of the virus using fluorescent proteins is difficult, thus limiting the application of viruses carrying reporter genes in virological research and drug screening. Furthermore, studies have reported an infectious EV71 clone carrying Gluc, whose Gluc reporter gene simplifies the viral quantitative analysis process; however, the large size of the Gluc gene fragment leads to poor stability of viruses carrying Gluc.

[0005] Chinese patent CN103805634A discloses an infectious CA16 clone with a green fluorescent protein gene, its construction method, and its application. This patent uses the low-copy plasmid pACYC17 as a vector, inserts the full-length cDNA of the CA16 / GD09 / 24 viral strain, and inserts an eGFP reporter gene between the 5' UTR and VP4, thereby obtaining a full-length infectious CA16 clone with the eGFP reporter gene. However, this patent lacks research on the genetic stability of the eGFP-carrying CA16 reporter virus; a reporter virus that cannot be stably passaged is not suitable for high-throughput screening of antiviral drugs. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the existing technology by providing an infectious clone of CA16 virus carrying the luciferase gene Nluc, its construction, and its application. The rescued CA16-Nluc virus can be used in the screening of high-throughput antiviral drugs and neutralizing antibodies. Furthermore, the infectious CA16-Nluc clone constructed in this invention will provide important tools and insights for basic and applied research on CA16 virus and other similar enteroviruses.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] An infectious clone of a CA16 virus carrying the luciferase gene Nluc, the nucleic acid sequence of which is shown in SEQ ID NO.1.

[0009] Furthermore, the infectious clone has similar growth characteristics to wild-type CA16.

[0010] Furthermore, the infectious clone exhibits passage stability within cells.

[0011] A method for constructing an infectious clone of CA16 virus carrying the luciferase gene Nluc, comprising the following steps:

[0012] S1 and CA16 (OP293089) viral genome RNA extraction;

[0013] Construction of S2 and CA16 subclones;

[0014] Construction of full-length infectious clones of S3 and pSVA-CA16;

[0015] Construction of infectious clones of S4 and pSVA-CA16-Nluc.

[0016] Further, in step S1, RNA from the CA16 virus genome is extracted using a kit. After the concentration of the obtained RNA is determined, it is verified by agarose gel electrophoresis. The verified RNA is aliquoted and stored at -80°C.

[0017] Furthermore, in step S2, the RNA extracted in step S1 is reverse transcribed into first-strand cDNA, and a CA16 subclone with the correct sequence is constructed using seamless cloning technology.

[0018] Furthermore, the RNA extracted in step S1 is reverse transcribed into first-strand cDNA. Using the cDNA as a template, fragments P1, P2, and P3 are amplified, with sequences of SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively. Then, seamless cloning technology is used to amplify the pcDNA vector fragment and the pUC57 vector fragment, respectively. Fragment P1 is recombined with the pcDNA fragment, and fragments P2 and P3 are recombined with pUC57 to obtain CA16 subclones with correct sequences, named pcDNA-P1 and pUC57-P2+P3, respectively. In this process, the long full-length viral genome is reverse transcribed and divided into three smaller fragments for amplification, which improves the fidelity of sequence amplification.

[0019] Further, in step S3, using the CA16 subclone prepared in step S2 as a template, the P1 region, P2+P3 region, and pSVA vector are amplified respectively. The purified P1, P2+P3, and pSVA are recombined and transformed into Trans2-Blue E. coli competent cells. After shaking, the plasmid is extracted and sequenced for verification. Then, using the verified plasmid as a template, the upstream and downstream primers SEQ ID NO.21 and SEQ ID NO.22 are used to insert a hammerhead ribozyme sequence into the plasmid. After sequencing verification, the plasmid with successfully inserted hammerhead ribozyme is named pSVA-CA16, which is the full-length infectious clone of pSVA-CA16.

[0020] Furthermore, a poly(A)25 tail is added after the 3'UTR following the P2+P3 link, and then recombined with pSVA.

[0021] Furthermore, the hammerhead ribozyme sequence is inserted before the 5'UTR, and the nucleotide sequence of the hammerhead ribozyme is shown in SEQ ID NO.27.

[0022] Furthermore, a nucleotide sequence with a T7 RNA polymerase promoter is inserted before the hammerhead ribozyme sequence, as shown in SEQ ID NO.28.

[0023] Further, in step S4, based on the full-length infectious clone of pSVA-CA16 prepared in step S3, the Nluc gene fragment is recombined with the full-length fragment of pSVA-CA16 using seamless cloning technology. The clone that is correctly sequenced is named pSVA-CA16-Nluc, which is the infectious clone of pSVA-CA16-Nluc.

[0024] Furthermore, the Nluc gene fragment is inserted before the P1 fragment.

[0025] Nluc, a novel luciferase, possesses several advantages, including catalyzing its substrate, furimazine, to emit brighter light compared to those of sea urchin and firefly luciferases, and exhibiting higher detection sensitivity. These characteristics make Nluc a uniquely advantageous luciferase, which can be used as a reporter gene to construct infectious viral clones, providing significant application value for antiviral drug screening and neutralizing antibody screening.

[0026] Application of an infectious clone of a CA16 virus carrying the luciferase gene Nluc: applying the infectious clone to the screening of antiviral drugs.

[0027] Furthermore, the antiviral drugs include ribavirin and chloroquine.

[0028] Application of an infectious clone of CA16 virus carrying the luciferase gene Nluc: applying the infectious clone CA16-Nluc to mechanisms such as viral invasion, replication and translation.

[0029] High-throughput drug screening refers to the evaluation of a large drug library in microplates to achieve rapid and highly sensitive screening. High-throughput screening of antiviral drugs relies on viruses carrying reporter genes, especially those carrying luciferase. As the second leading cause of HFMD, CA16 makes screening for effective antiviral drugs crucial for HFMD treatment. Therefore, constructing stable, Nluc-carrying infectious CA16 clones provides a powerful technical platform not only for studying viral pathogenic mechanisms but also for high-throughput antiviral drug screening.

[0030] Virus rescue: The full-length cDNA of the virus is cloned into a plasmid vector, and the virulent virus is rescued in vitro using reverse genetics.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] 1. The CA16 infectious clone with the luciferase Nluc reporter gene constructed in this invention is prepared based on the pSVA vector as the backbone. The long full-length viral genome is reverse transcribed and divided into three smaller segments for amplification, which improves the fidelity of sequence amplification.

[0033] 2. A poly(A)25 tail was added after the 3'UTR, and an Nluc gene fragment was inserted between the 5'UTR and the P1 fragment; in addition, a T7 RNA polymerase promoter sequence and a hammerhead ribozyme sequence were inserted before the 5'UTR. The T7 RNA polymerase promoter can be used to obtain mRNA in vitro; the hammerhead ribozyme enhances the replication ability of viral RNA.

[0034] 3. After transfecting cells with mRNA obtained by in vitro transcription, the mRNA replicates efficiently in the cells and can be correctly assembled into mature viral particles;

[0035] 4. This invention, combined with cytopathic effect experiments, one-step growth curves, and plaque experiments, verifies that the CA16 infectious clone constructed in this invention, carrying the luciferase Nluc reporter gene, can produce infectious CA16-Nluc virus particles. It also demonstrates that the CA16-Nluc virus rescued through reverse genetics has similar growth characteristics and other biological features to the parent virus. The infectious clone constructed in this invention is a powerful tool for the study of CA16 virus-related mechanisms and applied research.

[0036] 5. This invention, through experiments including viral genome copy number detection, luciferase detection, and specific PCR amplification, demonstrates that the CA16-Nluc virus rescued by the CA16 infectious clone with the luciferase Nluc reporter gene constructed in this invention can be continuously and stably passaged for more than 10 generations in in vitro cell culture without losing the exogenous Nluc gene. In existing reports, the virus rescued by enterovirus infectious clones with exogenous genes can only be stably passaged in cells a maximum of 5 times. The CA16-Nluc virus rescued by this invention greatly improves the stability of the virus. Stable CA16-Nluc virus is an important foundation for viral mechanism research and antiviral drug screening.

[0037] 6. Drug inhibition studies have shown that the CA16-Nluc virus can be effectively used for drug screening research. Compared with published CA16 viruses carrying GFP, the CA16-Nluc virus of this invention can be used for large-scale screening of antiviral drugs quickly and conveniently, greatly reducing the time and economic cost of drug screening. At the same time, the rescued CA16-Nluc virus can be used to screen drugs at different stages of the viral replication cycle, such as drugs that inhibit membrane fusion, acidification inhibitors, and drugs that inhibit RNA replication. This infectious clone has significant application value in drug screening. Attached Figure Description

[0038] Figure 1 A schematic diagram illustrating the construction of an infectious CA16 clone carrying the luciferase reporter gene Nluc;

[0039] Figure 2 A schematic diagram illustrating the basic growth characteristics of CA16-Nluc virus rescued from an infectious CA16 clone carrying the Nluc reporter gene.

[0040] A shows the cytopathic effects induced in Vero after infection with the uninfected group, the parental line, and recombinant CA16-Nluc virus.

[0041] B represents the plaque morphology of the parental and recombinant CA16-Nluc viruses.

[0042] C represents the one-step growth curve of the parental line and recombinant CA16-Nluc virus in Vero cells.

[0043] D represents the luciferase activity at different time points after cells were infected with parental and recombinant CA16-Nluc viruses;

[0044] Figure 3 A schematic diagram illustrating the passage stability of CA16-Nluc virus rescued from an infectious CA16 clone carrying the Nluc reporter gene.

[0045] A represents the gene copy number after different generations of CA16-Nluc virus infection of cells.

[0046] B represents the luciferase activity of cells infected with different generations of CA16-Nluc virus.

[0047] C represents the PCR amplification of the Nluc gene of different generations of CA16-Nluc virus;

[0048] Figure 4 A schematic diagram illustrating the screening of antiviral drugs for CA16-Nluc virus rescued from an infectious CA16 clone carrying the Nluc reporter gene.

[0049] A represents the inhibitory effect of ribavirin at different concentrations on CA16-Nluc virus replication.

[0050] B represents the cytotoxicity of ribavirin on Vero cells at different concentrations.

[0051] C represents the inhibitory effect of chloroquine on CA16-Nluc virus replication at different concentrations, and D represents the cytotoxicity of chloroquine on Vero cells at different concentrations. Detailed Implementation

[0052] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0053] In the following examples, the nucleotide sequences are shown below:

[0054] The nucleotide sequence of CA16-Nluc is shown in SEQ ID NO.1;

[0055] The nucleotide sequence of fragment P1 is shown in SEQ ID NO.2;

[0056] The nucleotide sequence of fragment P2 is shown in SEQ ID NO.3;

[0057] The nucleotide sequence of fragment P3 is shown in SEQ ID NO.4;

[0058] The nucleotide sequence of the hammerhead ribozyme is shown in SEQ ID NO.27;

[0059] The nucleotide sequence of the T7 RNA polymerase promoter is shown in SEQ ID NO.28;

[0060] The nucleotide sequence of the CA16 virus genome is shown in SEQ ID NO.33;

[0061] The nucleotide sequence of the pcDNA vector is shown in SEQ ID NO.34;

[0062] The nucleotide sequence of the pUC57 vector is shown in SEQ ID NO.35;

[0063] The nucleotide sequence of the Nluc gene fragment is shown in SEQ ID NO.36.

[0064] Unless otherwise specified, the experimental methods used in this embodiment are conventional methods in the art. The present invention will be described in detail below through several specific embodiments, but the present invention is not limited to these embodiments.

[0065] Example 1: Preparation of an infectious CA16 clone carrying the luciferase reporter gene Nluc. The steps are as follows:

[0066] 1. Extraction of CA16 (OP293089) viral RNA:

[0067] RNA was extracted from 200 μl of CA16 virus sample using the RNeasy Mini Kit. Following the manufacturer's instructions, RNA was extracted and eluted with 25 μl of nuclease-free water. The RNA concentration was determined using a Nanodrop 2000. 500 ng of RNA was then subjected to agarose gel electrophoresis to determine if it was a single, correctly sized band. The quality-controlled RNA was aliquoted and stored at -80°C.

[0068] 2. Construction of CA16 subclones:

[0069] The extracted RNA was reverse transcribed into first-strand cDNA using a reverse transcription kit. Using the cDNA as a template, fragments P1, P2, and P3 were amplified, with sequences SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively. The amplified fragments were subjected to 1% (w / v) agarose gel electrophoresis. Based on the DNA marker bands, the correctly sized P1, P2, and P3 amplification products were cut and the DNA fragments were recovered and purified using an enhanced gel recovery kit.

[0070] Using seamless cloning technology, pcDNA and pUC57 vector fragments were amplified separately. The amplified fragments were verified by agarose gel electrophoresis at a concentration of 0.7% (w / v). Based on the DNA marker bands, the pcDNA and pUC57 amplification products of the correct size were cut and the DNA fragments were recovered and purified using the Tiangen Enhanced Gel Recovery Kit.

[0071] The purified P1 fragment was recombined with the pcDNA fragment, and the P2 and P3 fragments were recombined with pUC57. The recombinant products were transformed into Trans5α competent E. coli cells. After picking single clones, the clones that were preliminarily identified by PCR were expanded and cultured. The plasmids were extracted and sequenced for verification. The plasmids that were correctly sequenced were CA16 subgenome clones, named pcDNA-P1 and pUC57-P2+P3, respectively.

[0072] The upstream amplification primers for P1 have the sequence shown in SEQ ID NO.5:

[0073] 5'-CCGCCTTAAAACAGCCTGTGGGTTGTTCCC-3';

[0074] The downstream amplification primers for P1 have the sequence shown in SEQ ID NO.6:

[0075] 5'-GACCGGTTTACAGCGTTTGTTATCTTGTCTCTACTAGTGC-3';

[0076] The upstream amplification primers for P2 have the sequence shown in SEQ ID NO.7:

[0077] 5'-CTCACTATAGGGGAGAGTTTGGACAGCAATCGG-3';

[0078] The downstream amplification primers for P2 have the sequence shown in SEQ ID NO.8:

[0079] 5'-GACTGTGGCTGTCCTTAGCACAGGCTTTTTGAG-3';

[0080] The upstream amplification primers for P3 have the sequence shown in SEQ ID NO.9:

[0081] 5'-TAAGGACAGCCACAGTCCAAGGACCG-3';

[0082] The downstream amplification primers for P3 have the sequence shown in SEQ ID NO.10:

[0083] 5'-ACATGAGAATTGTCGACTTTTTTTTTTTTTTTTTTTTTTCTGCTATTCTGGTTATAACAAATTTACCCCC-3';

[0084] The upstream amplification primers for the pcDNA vector have the sequences shown in SEQ ID NO.11:

[0085] 5'-CAACGCTGTAAACCGGTCATCATCACCATCACC-3';

[0086] The downstream amplification primers for the pcDNA vector have the sequences shown in SEQ ID NO.12:

[0087] 5'-GGCTGTTTTAAGGCGGTGCTAGCAGC-3';

[0088] The upstream amplification primers for the pUC57 vector have the sequence shown in SEQ ID NO.13:

[0089] 5'-GTCGACAATTCTCATGTTTGACAGCT-3';

[0090] The downstream amplification primers for the pUC57 vector are shown in SEQ ID NO.14:

[0091] 5-CTCTCCCCTATAGTGAGTCGTATTACGCGGC-3';

[0092] The reaction system for TR-PCR was as follows: Oligo(dT)23VN (10μM): 1μl, RNA: 3μl, RNase-free ddH2O: 4μl, 65℃, 5min;

[0093] The above mixture: 8 μl, 2×RT buffer: 10 μl, Enzyme: 2 μl; 50℃, 45 min; 85℃, 5 min.

[0094] 3. Construction of CA16 infectious clones:

[0095] Using seamless cloning technology, the CA16 subclone prepared in step 2 was used as a template to amplify the P1 region, P2+P3 region, and pSVA vector, respectively. The amplified fragments were subjected to 0.7% (w / v) agarose gel electrophoresis. Based on the DNA marker bands, the P1, P2+P3, and pSVA amplification products of the correct size were cut and the DNA fragments were recovered and purified using the Tiangen Enhanced Gel Extraction Kit.

[0096] Purified P1, P2+P3, and pSVA were recombined, and the recombinant products were transformed into Trans2-Blue E. coli competent cells. Single clones were picked, and clones that were preliminarily identified by PCR were expanded and cultured. Plasmids were extracted and sequenced for verification. Using the correctly sequenced plasmid as a template, a hammerhead ribozyme sequence was inserted into the plasmid using upstream and downstream primers SEQ ID NO.21 and SEQ ID NO.22. Sequencing verification confirmed that the plasmid with successfully inserted hammerhead ribozyme was named the pSVA-CA16 infectious clone.

[0097] The upstream amplification primers for the P1 region are shown in SEQ ID NO.15:

[0098] 5'-CTATAGGTTAAAACAGCCTGTGGGTTGTTCC-3';

[0099] The downstream amplification primers for the P1 region are shown in SEQ ID NO.16:

[0100] 5'-GCTGTCCAAACTCTCCCAGCGTTGTTATCTTGTCTCTACTAGTGC-3';

[0101] The upstream amplification primers for the P2+P3 region are shown in SEQ ID NO.17:

[0102] 5'-TGGGAGAGTTTGGACAGCAATCGGG-3';

[0103] The downstream amplification primers for the P2+P3 region are shown in SEQ ID NO.18:

[0104] 5'-CATGAGAATTGTCGACTTTTTTTTTTTTTTTTTTTTTTTTCTGCTATTCTGGTTATAACAAATTTACCCCC-3';

[0105] The upstream amplification primers for the pSVA vector have the sequences shown in SEQ ID NO.19:

[0106] 5'-GTCGACAATTCTCATGTTTGACAGCT-3';

[0107] The downstream amplification primers for the pSVA vector are shown in SEQ ID NO.20:

[0108] 5'-CAGGCTGTTTTAACCTATAGTGAGTCGTATTACGCGGC-3';

[0109] The upstream primer introduced by the hammerhead ribozyme has the sequence shown in SEQ ID NO.21:

[0110] 5'-GCCGAAAGGCCGAAAACCCGGTATCCCGGGTTCTTAAAACAGCCTGTGGGTTGTTCCC-3';

[0111] The downstream primer introduced by the hammerhead ribozyme has the sequence shown in SEQ ID NO.22:

[0112] 5'-TTTCGGCCTTTCGGCCTCATCAGTTAAAACACCCCCTATAGTGAGTCGTATTACGCGGC-3';

[0113] The recombinant reaction system was as follows: P1: 1 μl, P2+P3: 1 μl, support: 2 μl, water: 6 μl; 2×Hieff MultiSEnzyme Premix: 10μl, 50℃, 30min.

[0114] 4. Construction of a full-length infectious clone of CA16-Nluc:

[0115] Using seamless cloning technology, based on the infectious clone prepared in step 3, the full-length pSVA-CA16 plasmid was amplified. Using the pcDNA-Nluc plasmid as a template, the Nluc gene fragment was amplified. The two PCR amplified fragments were subjected to DNA gel electrophoresis. The size of the amplified fragments was determined according to the DNA marker. The Nluc fragment and the full-length pSVA-CA16 fragment of the correct size were recovered from the gel. The recovered fragments were purified into DNA. The two fragments were recombined and transformed into Trans2-Blue E. coli competent cells. Colony PCR was used to preliminarily identify the correct clone for expansion culture. The endotoxin-free plasmid was extracted and sent for sequencing. The clone that was verified by sequencing was named pSVA-CA16-Nluc, which is the CA16-Nluc infectious cloning plasmid.

[0116] The full-length upstream amplification primers for pSVA-CA16 are shown in SEQ ID NO.23:

[0117] 5-GCCATTACTACCCTTGGGTCACAAGTCTCCACCCA-3';

[0118] The full-length downstream amplification primers for pSVA-CA16 are shown in SEQ ID NO.24:

[0119] 5'-CACCTGGGATCCCATTTCTTACAGTTGAGGAGCAA-3';

[0120] The upstream amplification primers for the Nluc gene fragment are shown in SEQ ID NO.25:

[0121] 5'-GAAATGGGATCCCAGGTGTTCACACTCGAAG-3';

[0122] The downstream amplification primers for the Nluc gene fragment are shown in SEQ ID NO.26:

[0123] 5'-CCCAAGGGTAGTAATGGCCGCCAGAATGCGTTCGCA-3'.

[0124] Example 2: An infectious clone of CA16 carrying the luciferase reporter gene Nluc has the ability to produce CA16-Nluc virus. The steps are as follows:

[0125] 1. In vitro transcription to rescue viruses:

[0126] Using pSVA-CA16 and pSVA-CA16-Nluc as templates, the full-length CA16-Nluc DNA template was amplified by PCR. After gel extraction, proteinase K treatment, and DNA product purification, the PCR product was subjected to T7 in vitro transcription to obtain mRNA. The mRNA was purified by LiCl precipitation, and the quality was assessed by agarose gel electrophoresis. After confirming correct banding, the mRNA was aliquoted and stored at -80℃. Well-grown Vero cells were digested and seeded at 3*102 cells per well in 6-well plates. 5 After culturing Vero cells overnight at 37°C, CA16 and CA16-Nluc mRNA were transfected into Vero cells using Lipo3000 transfection reagent. The transfected Vero cells were then cultured further, and the cytopathic effect was observed. The observation continued until more than 80% of the cells showed cytopathic effects. Figure 2 -A), repeat freeze-thaw cycles at -80℃ twice, centrifuge at 4000g to collect the viral supernatant as P0 generation. The P0 generation viral solution is used to infect Vero cells. When the cytopathic effect exceeds 80%, the viral solution is collected as P1 generation virus. The same steps are continued to passage to P10 generation. The harvested viral solution is stored at -80℃.

[0127] The full-length upstream amplification primers for pSVA-CA16 and pSVA-CA16-Nluc are shown in SEQ ID NO.29:

[0128] 5'-CACGAGGCCCTTTCGTCTTC-3';

[0129] The full-length downstream amplification primers for pSVA-CA16 and pSVA-CA16-Nluc are shown in SEQ ID NO.30:

[0130] 5'-TTTTTTTTTTTTTTTTTTTTTTTCTGCTATTCTGGTTATAACAAATTTACCCCC-3';

[0131] T7 in vitro transcription reaction system: template: 8 μl, A: 1.5 μl, G: 1.5 μl, C: 1.5 μl, U: 1.5 μl, buffer: 2 μl, T7 Enzyme: 1 μl; RNase-free ddH2O: 3 μl, 37℃, 2 h.

[0132] 2. Parental CA16 and recombinant CA16-Nluc plaques:

[0133] The growth characteristics of parental CA16 and recombinant CA16-Nluc viruses were compared using monolayer plaques: Vero cells were seeded in 12-well plates overnight, and parental CA16 and CA16-Nluc were serially diluted in 2% DMEM medium (10⁻¹⁰ and 10⁻¹⁰ respectively). -1 -10 -7 Cells were infected with 300 μl of diluted virus solution at 37°C for 2 hours. The viral supernatant was discarded, and the cells were washed once with PBS. A mixture of Avicel and 2×4% DMEM (1:1 ratio) was added to cover the cells, and the cells were incubated at 37°C for 3-4 days. After plaque formation, the covering material was discarded, and the cells were fixed with 4% paraformaldehyde for 2 hours. The paraformaldehyde was then discarded, and crystal violet staining was added for 2 hours. After staining, the crystal violet was rinsed off with running water, and the culture plate was allowed to dry before photographing and observation. Figure 2 -B).

[0134] 3. One-step growth curves of parental CA16 and recombinant CA16-Nluc:

[0135] In a 12-well plate, each well is inoculated with 2.5 x 10⁻⁶ seeds. 5 Vero cells were cultured overnight at 37°C. Rescued P5 generation CA16-Nluc cells were infected with CA16 at an MOI of 1. Simultaneously, laboratory-preserved parental CA16 cells were used as a control, also infected with the same MOI. Cells were collected at 3h, 6h, 12h, 24h, and 36h after infection. RNA was extracted from the samples, and viral gene copy number was quantitatively detected using a one-step RT-PCR method. The one-step viral growth curve was analyzed. Figure 2 -C).

[0136] 4. Luciferase activity of parental CA16 and recombinant CA16-Nluc viruses:

[0137] Digest well-growing Vero cells and seed 2 x 10⁶ cells per well in a 96-well plate. 4 Cells were cultured overnight at 37°C. The rescued P5 generation CA16-Nluc and parental CA16 virus were then used to infect Vero cells with an MOI of 1. Cells were cultured at 37°C, and at 6, 12, 18, 24, 30, and 36 hours post-infection, cells were treated with luciferase lysis buffer. Lysis products were collected, and substrate was added. Fluorescence values ​​were then measured. Figure 2 -D).

[0138] In summary, both CA16 and recombinant CA16-Nluc viruses exhibited cytopathic effects after infecting Vero, and CA16-Nluc virus showed similar growth characteristics to wild-type CA16.

[0139] Example 3: CA16-Nluc virus produced by an infectious clone of CA16 carrying the luciferase reporter gene Nluc exhibits passage stability in cells.

[0140] 1. Stability of CA16-Nluc virus genome copy number across different generations:

[0141] 2.5*10 5 Vero cells were seeded into 12-well plates and cultured overnight at 37°C. Cells were then infected with different passages of CA16-Nluc virus for 24 hours. Cells were digested with trypsin and collected. RNA was extracted using an RNA extraction kit, and eluted with 20 μl of nuclease-free water. The obtained RNA was quantified using one-step qPCR to calculate the CA16-Nluc virus genome copy number. Figure 3 -A).

[0142] 2. Stability of luciferase activity expressed by different generations of CA16-Nluc virus:

[0143] 2.5*10 4 Vero cells were seeded into 12-well plates and cultured overnight at 37°C. The cells were then infected with different passages of CA16-Nluc virus for 24 hours. 20 μl of lysis buffer was added to each well, and the cells were incubated at room temperature for 5 minutes. Then, 20 μl of substrate was added, and fluorescence values ​​were immediately detected. The stability of the virus was reflected by comparing the fluorescence values ​​produced after infection with different passages of CA16-Nluc virus. Figure 3 -B).

[0144] 3. Stability of the Nluc gene in different generations of CA16-Nluc virus:

[0145] 2.5*10 5 Vero cells were seeded into 12-well plates and cultured overnight at 37°C. Cells were then infected with different generations of CA16-Nluc virus for 24 hours. Cells were digested with trypsin and collected. RNA was extracted using an RNA extraction kit, and eluted with 20 μl of nuclease-free water. 2 μl of RNA was reverse transcribed into cDNA. Using the cDNA as a template, the Nluc gene was amplified at both ends. An infectious pSVA-CA16-Nluc clone was used as a positive control. The amplified products were verified by 1% (w / v) agarose gel electrophoresis. Figure 3 -C).

[0146] The upstream amplification primers for Nluc are shown in SEQ ID NO.31:

[0147] 5'-CGTCCCTCTTAATCTCAAGGCC-3';

[0148] The downstream amplification primers for Nluc are shown in SEQ ID NO.32:

[0149] 5'-GATCGCTGGGTGGAGACTTG-3'.

[0150] In summary, after 24 hours of infection of Vero cells with different generations of CA16-Nluc virus, there was no significant difference in viral gene copy number and fluorescence value, demonstrating that CA16-Nluc virus produced by CA16 infectious clones carrying the luciferase reporter gene Nluc has passage stability in cells.

[0151] Example 4: Application of CA16 infectious clones carrying the luciferase reporter gene Nluc in antiviral drug screening

[0152] 1. Toxicity analysis of ribavirin and chloroquine on Vero cells:

[0153] In a 96-well plate, seed 2*10 saturates per well. 4 Vero cells were cultured overnight at 37°C. Different concentration gradients of ribavirin (0, 0.01, 0.05, 0.1, 0.2, 0.4, 0.6 mM) and chloroquine (0, 2.5, 5, 10, 20, 40, 80 μM) were prepared in DMEM medium containing 2% FBS. The medium in the Vero cells was discarded and replaced with different concentration gradients of ribavirin or chloroquine. Cell wells without drug treatment and blank wells without cells were also prepared. Cells were cultured at 37°C for another 48 hours. CCK-8 reagent was diluted 100-fold with DMEM medium, and 100 μl of the diluent was added to each well. Cells were incubated at 37°C for 2 hours. OD values ​​were measured using a microplate reader to calculate cell viability.

[0154] 2. Inhibitory effect of ribavirin on CA16 virus carrying the luciferase reporter gene Nluc:

[0155] In a 96-well plate, seed 2*10 saturates per well. 4 Vero cells were cultured overnight at 37°C, and then infected with the rescued CA16-Nluc virus. Two hours after infection, the culture supernatant was discarded and replaced with ribavirin containing different concentration gradients (0, 0.01, 0.05, 0.1, 0.2, 0.4, 0.6 mM) for another 24 hours. The substrate was then added and the fluorescence value was detected.

[0156] 3. Inhibitory effect of chloroquine on CA16 virus carrying the luciferase reporter gene Nluc:

[0157] Inoculate 2*10 cells into 96-well plates 4 Vero cells were cultured overnight at 37°C, then co-incubated for 2 hours with chloroquine at different concentration gradients (0, 2.5, 5, 10, 20, 40, 80 μM). CA16-Nluc virus was added, and after 2 hours of viral infection, the culture supernatant was discarded and replaced with chloroquine at different concentration gradients (0, 2.5, 5, 10, 20, 40, 80 μM) for another 24 hours. Luciferase substrate was then added, and fluorescence values ​​were detected.

[0158] In summary, the study of the inhibitory effects of ribavirin and chloroquine on CA16-Nluc virus suggests that the CA16-Nluc reporter virus prepared in this invention can be effectively used for antiviral drug screening.

[0159] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An infectious clone of a CA16 virus carrying the luciferase gene Nluc, characterized in that, The nucleic acid sequence of the CA16 virus carrying the luciferase gene Nluc is shown in SEQ ID NO.1; The infectious clone was prepared using the pSVA vector as a backbone. The infectious clone had a poly(A)25 tail added after the 3'UTR and an Nluc gene fragment inserted between the 5'UTR and the P1 fragment. The infectious clone had a T7 RNA polymerase promoter sequence and a hammerhead ribozyme sequence inserted before the 5'UTR. The infectious clone rescued CA16-Nluc virus has similar growth characteristics to the parent virus. The CA16-Nluc virus can be continuously and stably passaged for more than 10 generations in in vitro cell culture without losing the Nluc exogenous gene.

2. A method for constructing an infectious clone of CA16 virus carrying the luciferase gene Nluc as described in claim 1, characterized in that, The steps are as follows: S1 and CA16 viral genomic RNA extraction; Construction of S2 and CA16 subclones; Construction of full-length infectious clones of S3 and pSVA-CA16; Construction of infectious clones of S4 and pSVA-CA16-Nluc.

3. The method for constructing an infectious clone of CA16 virus carrying the luciferase gene Nluc according to claim 2, characterized in that, In step S1, RNA from the CA16 virus genome was extracted using a kit. After the concentration of the obtained RNA was determined, it was verified by agarose gel electrophoresis. The verified RNA was aliquoted and stored at -80°C.

4. The method for constructing an infectious clone of CA16 virus carrying the luciferase gene Nluc according to claim 2, characterized in that, In step S2, the RNA extracted in step S1 is reverse transcribed into first-strand cDNA, and a CA16 subclone with the correct sequence is constructed using seamless cloning technology.

5. The method for constructing an infectious clone of CA16 virus carrying the luciferase gene Nluc according to claim 4, characterized in that, The RNA extracted in step S1 was reverse transcribed into first-strand cDNA. Using the cDNA as a template, fragments P1, P2, and P3 were amplified, with sequences of SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively. Then, using seamless cloning technology, the pcDNA vector fragment and the pUC57 vector fragment were amplified, respectively. Fragment P1 was recombined with the pcDNA fragment, and fragments P2 and P3 were recombined with pUC57 to obtain CA16 subclones with correct sequences, named pcDNA-P1 and pUC57-P2+P3, respectively.

6. The method for constructing an infectious clone of CA16 virus carrying the luciferase gene Nluc according to claim 2, characterized in that, In step S3, using the CA16 subclone prepared in step S2 as a template, the P1 region, P2+P3 region, and pSVA vector were amplified respectively. The purified P1, P2+P3, and pSVA were recombined and transformed into Trans2-Blue E. coli competent cells. After shaking, the plasmid was extracted and sequenced for verification. Then, using the verified plasmid as a template, the upstream and downstream primers SEQ ID NO.21 and SEQ ID NO.22 were used to insert a hammerhead ribozyme sequence into the plasmid. After sequencing verification, the plasmid with the successfully inserted hammerhead ribozyme was named pSVA-CA16, which is the full-length infectious clone of pSVA-CA16.

7. The method for constructing an infectious clone of CA16 virus carrying the luciferase gene Nluc according to claim 6, characterized in that, Add a poly(A)25 tail after the 3'UTR following the P2+P3 link, and then recombine it with pSVA; The hammerhead ribozyme sequence is inserted before the 5'UTR, and the nucleotide sequence of the hammerhead ribozyme is shown in SEQ ID NO. 27; A nucleotide sequence with a T7 RNA polymerase promoter was inserted before the hammerhead ribozyme sequence, as shown in SEQ ID NO.

28.

8. The method for constructing an infectious clone of CA16 virus carrying the luciferase gene Nluc according to claim 2, characterized in that, In step S4, based on the full-length infectious clone of pSVA-CA16 prepared in step S3, the Nluc gene fragment is recombined with the full-length infectious clone of pSVA-CA16 using seamless cloning technology. The clone that is correctly sequenced is named pSVA-CA16-Nluc, which is the infectious clone of pSVA-CA16-Nluc.

9. The method for constructing an infectious clone of CA16 virus carrying the luciferase gene Nluc according to claim 8, characterized in that, Insert the Nluc gene fragment before the P1 fragment.

10. Application of an infectious clone of a CA16 virus carrying the luciferase gene Nluc, wherein the infectious clone as described in claim 1 is applied to the screening of antiviral drugs.

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