CN110195093B - A drug screening system based on recombinant Zika virus expressing green fluorescent protein and its application - Google Patents

Abstract

The invention discloses a drug screening system based on recombinant Zika virus expressing green fluorescent protein and application thereof. The drug screening system of the invention consists of recombinant Zika virus (reporter Zika virus) expressing Green Fluorescent Protein (GFP) and a BHK-21 cell line expressing DC-SIGNR. The reported Zika virus expresses green fluorescent protein, has strong fluorescence number, is uniformly colored in cytoplasm, is easy to observe and is easy for high-throughput screening analysis. The BHK-21 cell line stably expressing DC-SIGNR improves the sensitivity to the infection of the reported Zika virus, improves the virus replication efficiency, reduces the virus replication pressure and also improves the stability of the recombinant virus. The drug screening system can directly observe and analyze the virus replication conditions before and after the drug action in the living cell state without fixing and immunostaining the cells, so that the whole operation process is simpler, more convenient and faster, the operation steps of the living viruses are reduced, and the safety of the whole operation process is improved.

Description

A Drug Screening System Based on Recombinant Zika Virus Expressing Green Fluorescent Protein and its application

technical field

The present invention relates to an antiviral drug screening system and its application, in particular to a drug screening system based on recombinant Zika virus expressing green fluorescent protein and its application. The invention belongs to the field of biotechnology.

Background technique

Zika virus (ZIKV) is a zoonotic pathogen mainly transmitted by mosquitoes, and it is related to dengue virus (DENV), West Nile virus (WNV), Japan B Japanese encephalitis virus (JEV) and yellow fever virus (YFV) belong to the Flavivirus genus of the family Flaviviridae. In 1947, researchers such as Dick isolated it from rhesus monkeys in the Zika Forest of Uganda. ZIKV can cause Zika fever after infecting the host. Most infected people have no obvious symptoms or mild symptoms, including conjunctivitis, rash, fever, joint and muscle pain and other symptoms. The course of the disease is about 2-7 days. However, if ZIKV infection occurs during pregnancy, the newborn may suffer from microcephaly and other congenital malformations, and it may also cause serious symptoms such as premature delivery and miscarriage in the pregnant woman. Studies have shown that ZIKV exists in about 86 countries or regions in the world, which shows that ZIKV is spreading globally. There are currently no vaccines and antiviral drugs approved for use. In order to prevent and control the disease more effectively, it is necessary to adopt a variety of advanced technical means for vaccine research and drug screening. The reverse genetic operating system is used to construct a reporter virus to speed up the vaccine research of the virus disease. The reporter virus also provides an important technical platform for drug screening and is an effective and advanced means of virology research. However, due to the instability of the genome and the instability of recombinant viruses carrying foreign genes, it has always been a difficult problem and challenge to construct a stable reporter virus among flaviviruses.

The ZIKV virion is spherical in shape, with a diameter of about 40-70nm. The virion is surrounded by a layer of lipid capsule derived from the host. The genome is a single-stranded positive-sense RNA with a length of about 11kb, and its coding gene sequence is 5'-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3'. RNA is translated into a polyprotein, and then cleaved by protease into structural proteins capsid protein (capsid, C), precursor membrane protein (precursor membrane, prM), envelope protein (envelope, E) and NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5 seven non-structural proteins. Among them, C protein combines with ZIKV genomic RNA to form the nucleocapsid core, prM protein and E protein form the virus surface protein, and in the host cell, non-structural proteins participate in the process of virus replication and polyprotein shear processing. At present, according to molecular biology and bioinformatics, it can be divided into two subtypes, Asian type and African type. The difficulty in constructing a stable viral genome clone is due to the existence of coding sequences that are toxic to the host bacteria in the E protein gene and NS1 protein gene of the viral genome. Moreover, studies have shown that the reporter Zika virus expressing luciferase gene or fluorescent protein gene constructed by reverse genetic manipulation technology has decreased replication ability, unstable expression of foreign genes, and rapid loss of foreign genes after passage.

The present invention constructs the recombinant Zika virus ZIKV-GFP expressing green fluorescent protein (GFP) through the intracellular recombination method. Moreover, a cell line expressing DC-SIGNR was constructed. The cell line promoted the replication of the reporter Zika virus ZIKV-GFP, reduced the genetic stability pressure of the recombinant virus, and improved the stability of the recombinant reporter virus. The recombinant virus and the stable expression cell line constitute a convenient Zika virus drug screening system, which can be used for anti-Zika virus drug screening, and can also be used for the detection of Zika virus neutralizing antibodies, which can be used for Zika virus Vaccine efficacy evaluation.

Contents of the invention

The purpose of the present invention is to provide a system for screening anti-Zika virus drugs to overcome the defects of low replication ability and instability of the reporter Zika virus.

In order to achieve the above object, the present invention adopts the following technical means:

A drug screening system based on recombinant Zika virus (Zika virus, ZIKV) expressing green fluorescent protein (GFP) of the present invention, the drug screening system is composed of recombinant Zika virus expressing green fluorescent protein and expressing DC-SIGNR Composition of the BHK-21 cell line.

Wherein, preferably, the recombinant Zika virus expressing green fluorescent protein is prepared by the following method:

1) According to the Zika virus gene sequence, other genomic sequences except the structural protein gene and the GFP reporter gene sequence gene of the structural protein gene position of the insertion and deletion were synthesized by segmented gene synthesis technology, and connected into the mammalian through the multiple cloning restriction site Animal expression vector, obtained replicon recombinant plasmid, the open reading frame of the replicon recombinant plasmid is "CMV promoter-restriction site-5'UTR-nucleotide sequence encoding 20 amino acids at the N-terminal of C protein-GFP- FMDV2A-Nucleotide sequence encoding 22 amino acids at the C-terminal of E protein-NS1~5 (nonstructural protein 1~5 gene sequence)-3'UTR-HDVr (hepatitis delta virus ribozyme gene sequence)-enzyme cutting site" ;

2) According to the Zika virus gene sequence, the structural protein gene sequence and the GFP sequence were synthesized by gene synthesis technology, and the connection sequence of each element was 5'UTR-the nucleotide sequence encoding the 38 amino acids at the C-terminal of the ZIKV C protein-GFP- FMDV2A-C-prM-E-NS1 gene 1-134 nucleotide sequence, the fragment is cloned into a mammalian expression vector through multiple cloning restriction sites, and a plasmid containing ZIKV structural gene and green fluorescent protein is obtained;

3) Rescue of reporter ZIKV

Using the plasmid obtained in step 2) as a template, perform PCR amplification, and amplify to obtain the nucleotide sequence containing CMV promoter and 5'UTR-coding 38 amino acids at the C-terminal of ZIKV C protein-GFP-FMDV2A-C-prM - The PCR product of the 1-134 nucleotide sequence of the E-NS1 gene, which is used for the rescue of the reporter virus;

Rescue of reporter ZIKV: HEK-293T cells were cultured in 24-well plates with 10% v/v FBS DMEM medium to a density of 70%-90%, and the obtained PCR products and linear The transformed replicon recombinant plasmids were transfected respectively. After 72 hours of transfection, the cells and supernatant were harvested and stored at -70°C to obtain recombinant Zika virus expressing green fluorescent protein.

Wherein, preferably, the Zika virus gene sequence is the gene sequence of the ZIKV strain popular in Brazil in 2015, and the Genbank accession number is: KX280026.

Wherein, preferably, step 1) is to connect the mammalian expression vector pCI-neo through the multiple cloning restriction sites Sac I and Not I to obtain the recombinant plasmid of the replicon, and the open reading frame of the recombinant plasmid of the replicon is successively initiated by CMV Son-SacI-5'UTR-the nucleotide sequence encoding the 20 amino acids at the N-terminal of the C protein-GFP-FMDV2A-the nucleotide sequence encoding the 22 amino acids at the C-terminal of the E protein-NS1～5 (nonstructural protein 1～5 gene sequence)-3'UTR-HDVr (hepatitis delta virus ribozyme gene sequence)-Not I.

Among them, preferably, in step 2), the nucleotide sequence of 5'UTR-encoding the C-terminal 38 amino acids of ZIKV C protein-GFP-FMDV2A-C-prM-E- The 1-134 nucleotide sequence of NS1 gene was cloned into pCI-neo vector.

Among them, preferably, in step 3), the plasmid obtained in step 2) is used as a template, and primers P1: 5'-GGCCTTTTGCTCACATGGCTCGACAG-3' and P2: 5'GACTGCTGCTGCCAATCTACGGGGG-3' are used for PCR amplification, and the PCR product is used for Rescue of the reporter virus.

Wherein, preferably, the BHK-21 cell line expressing DC-SIGNR is constructed by the following method:

1) constructing an expression plasmid containing DC-SIGNR;

2) Linearize the prepared DC-SIGNR expression plasmid, recover from gel, and transfect BHK-21 cells;

3) Screening to obtain a BHK-21 cell line stably expressing DC-SIGNR.

Wherein, preferably, the coding nucleotide sequence of DC-SIGNR is shown in SEQ ID NO.1, and the synthesized DC-SIGNR sequence is cloned into pCAGneo vector.

Furthermore, the present invention also proposes the application of the drug screening system in screening anti-Zika virus drugs.

Furthermore, the present invention also proposes a method for screening anti-Zika virus drugs using the drug screening system, comprising the following steps:

1) Drug treatment and virus infection: Inoculate the BHK-21 cell line expressing DC-SIGNR in a black 96-well plate with 2% v/v FBS100ul medium, and when the cell density reaches 90%, use 50ul containing 2% v/v The drug to be tested diluted in FBS DMEM medium was acted for 1 hour, and after 1 hour, the recombinant Zika virus expressing green fluorescent protein was added for infection, and cultured in a 5% CO ₂ incubator at 37°C for 48 hours;

2) Statistical analysis of the high-content system: After the cells were treated with drugs and infected with the virus for 48 hours, the nuclei were stained with Hoechst, and the cell plate was placed in a high-content cell screening system (PerkinElmer) for observation, and the fluorescent channel of green fluorescent protein and the fluorescent channel of cell nucleus staining were used to scan For analysis, the number of infected positive cells and the total number of cells in each well were counted, the virus infection rate was calculated, and the inhibition rate of the drug to be tested on virus replication was calculated.

Compared with the prior art, the beneficial effects of the present invention are:

Reporter flaviviruses constructed with the reverse genetics system, such as recombinant viruses expressing luciferase and fluorescent proteins, are unstable and their replication ability is greatly weakened. Under such replication pressure, foreign genes are easily lost. Restore the mutants to wild-type virus. The system of the present invention constructs a cell line stably expressing DC-SIGNR molecules, which improves the sensitivity to reporter Zika virus infection, improves virus replication efficiency, reduces virus replication pressure, and improves the stability of recombinant virus . The reporter virus of the present invention expresses green fluorescent protein, has a strong fluorescence number, is uniformly colored in the cytoplasm, and is easy to observe and high-throughput screening and analysis. The reporter virus system of the present invention can visually observe and analyze the virus replication in the state of living cells without fixing and immunostaining the cells, which makes the whole operation process easier and faster, reduces the operation steps of live viruses, and improves the The security of the entire operation process.

Description of drawings

Fig. 1 is the identification of pCAG-DC-SIGNR plasmid;

M1: DL 10000 DNA standard; 1: Sac I and Xho I digestion identification; 2: pCAG-neo plasmid;

Figure 2 is a schematic diagram of the construction of the ZIKV replicon pZIKVrepdCME and pC38GFP-CME-NS1 ₁₃₄ plasmids;

Fig. 3 is the enzyme digestion identification result of ZIKV replicon pZIKVrepdCME (A), pC38GFP-CME-NS1 ₁₃₄ (B) plasmid;

In Figure 3A, M ₁ : DNA maker, 1: undigested plasmid control; 2: Xhol I and Not I double enzyme digestion identification results;

In Figure 3B, M ₂ : DNA maker, 1: Sac I and Not I double digestion identification results; 2: undigested plasmid control;

Fig. 4 is the Western blot identification of recombinant virus ZIKV-GFP;

M: protein molecular weight standard; 1: virus-infected cells; 2: virus-uninfected cells;

Fig. 5 is the observation of recombinant virus ZIKV-GFP expressing green fluorescent protein;

Fig. 6 is the plaque formation of recombinant virus ZIKV-GFP infection BHK-21, Vero and BHK-DR cells;

Fig. 7 is the determination of the growth kinetics curve of recombinant virus ZIKV-GFP;

Fig. 8 is Ribavirin anti-ZIKV drug experiment;

Wherein Fig. 8A is the scanning analysis result using the green fluorescent protein fluorescent channel and the nuclear staining fluorescent channel; Fig. 8B is the statistical result of the virus infection rate; Fig. 8C is the inhibition rate of the drug to the virus replication;

Fig. 9 is 6-Azauridine anti-ZIKV drug experiment;

Wherein Fig. 9A is the scanning analysis result using the green fluorescent protein fluorescent channel and the nuclear staining fluorescent channel; Fig. 9B is the statistical result of the virus infection rate; Fig. 9C is the inhibition rate of the virus replication by the drug;

Fig. 10 is Chloroquine anti-ZIKV drug experiment.

Figure 10A shows the scanning analysis results using green fluorescent protein fluorescent channel and nuclear staining fluorescent channel; Figure 10B shows the statistical results of virus infection rate; Figure 10C shows the inhibition rate of the drug on virus replication.

Detailed ways

In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

Example 1 Construction of a cell line stably expressing DC-SIGNR

1.1 Experimental materials

1.1.1 Main materials and reagents

BHK-21 cells; DMEM, fetal bovine serum, and Opti-MEM were purchased from Gibco; the mixture of trypsin and penicillin was purchased from Gibco; polylysine was purchased from Sigma; TritoX-100 was purchased from Sigma Infrared fluorescence-labeled anti-rabbit IgG antibody was purchased from KPL Company; FITC-labeled goat anti-mouse IgG was purchased from Zhongshan Jinqiao Company; QIAGEN plasmid extraction kit was purchased from QIAGEN Company; TransIT-293Transfection Reagent was purchased from Mirus Company.

1.1.2 Experimental Instruments

PCR instrument (Takara); nucleic acid electrophoresis instrument (Beijing Liuyi Instrument); nucleic acid gel imaging system (Bio-Rad); pure water instrument (Millipore); optical microscope (Olympus); inverted fluorescence microscope (Life); electronic balance ( Beijing Sartorius); CO2 constant temperature incubator (Heal Force); standard pipette (Eppendorf); horizontal centrifuge (Eppendorf);

1.2 Experimental method

1.2.1 Construction of expressing DC-SIGNR plasmid

According to the DC-SIGNR gene coding sequence published by GenBank (Genbank accession number: AY042234), it was artificially synthesized, and the synthesized sequence is shown in SEQ ID NO.1. The synthesized sequence was cloned into the pCAGneo vector (this vector has been recorded in the article "Establishment of a cell line stably expressing the NS1 protein of Japanese encephalitis virus, Chen Zhenshi, master's degree dissertation", sponsored by the State Key Laboratory of Veterinary Biotechnology of Harbin Veterinary Research Institute Provided by the laboratory), the pCAG-DC-SIGNR expression plasmid was constructed.

1.2.2 Extraction of pCAG-DC-SIGNR plasmid

Resuscitate the bacteria containing the pCAG-DC-SIGNR plasmid by streaking, pick a single clone colony and place it in 4ml LB for shaking culture, then inoculate 1ml of the bacterial liquid into a 100ml Erlenmeyer flask for 4h shaking culture according to the ratio of 1:100 . After collecting the bacterial liquid, use the QIAGEN plasmid extraction kit for plasmid extraction. The extraction steps are as follows: Harvest the bacterial liquid cultured overnight in LB medium for 12-16 hours in a 50ml centrifuge tube, centrifuge at 10,000rpm, 4°C for 5min; discard After centrifugation, add 10ml Buffer P1 to the supernatant of the bacterial liquid to resuspend the bacteria; after the bacteria are completely resuspended, add 10ml Buffer P2, shake the centrifuge tube 4-6 times vigorously to mix thoroughly, and let it work at room temperature for 5 minutes. LyseBlue reagent, the solution should turn uniform blue at this time; during the incubation period, open the sealing cap of the needle port and put the filter on a suitable shelf; add 10ml of ice-bathed Buffer P3 to the lysate, shake vigorously and centrifuge Mix the tube 4-6 times. If LyseBlue reagent is added to P1, mix the reagent until there is no color; centrifuge the lysate at 6000g, 4°C for 10min; during the centrifugation of the lysate, put the QIAGEN-tip column Stand on the support frame, add 10ml Buffer QBT, and let the solution in the tube flow through by gravity; after the QBT solution equilibrates the column, add the clear supernatant of the lysate to the fully equilibrated QIAGEN-tip column and let it pass through the resin membrane Drop slowly by gravity; after the previous step is completed, wash the QIAGEN-tip column twice with 10ml Buffer QC; after the previous step is completed, use 5ml Buffer QF (incubated in water at 65°C) to elute the DNA in a 10ml centrifuge tube Medium; add 2.5ml of isopropanol to the eluent and mix well to precipitate the DNA, centrifuge at 12000rpm, 4°C for 30min; after the centrifugation is completed, gently discard the supernatant and resuspend the DNA with 2ml of 70% ethanol Precipitate, centrifuge at 12000rpm, 4°C for 10min; pour out the supernatant carefully after the centrifugation is completed, do not touch the plasmid to keep the plasmid intact, air-dry the plasmid at the bottom of the centrifuge tube in an ultra-clean bench for about 15min; use an appropriate volume of endotoxin-free ddH2O to redissolve the DNA; check the concentration of the DNA plasmid and store at -20°C.

1.2.2 Transfection of plasmids

The prepared pCAG-DC-SIGNR expression plasmid was linearized (Table 1), and after gel recovery, TransIT-293Transfection Reagent was used to transfect according to the instructions.

Table 1 Linearization system of eukaryotic expression vector pCAG-DC-SIGNR

After gel recovery, the well-grown BHK-21 cells were digested, seeded in 24-well plates, and transfected according to the following steps when the cells grew to 70%-90%: put opti-MEM and TransIT-293Transfection Reagent at 4°C Take it out and place it at room temperature for about 20 minutes, and take out the completely linearized pCAG-DC-SIGNR plasmid at the same time; take 100 μl opti-MEM in a 1.5ml EP tube, add 2 μg pCAG-DC-SIGN linearized plasmid, and gently vortex to mix Mix well; add 3 μl TransIT-293Transfection Reagent to the mixed solution in the previous step, and gently vortex to mix; after incubation for about 15 minutes, add 50 μl of the mixed solution per well into a 24-well plate, and observe for 24-72 hours result.

1.2.3 Screening and clone purification of transfected cells

After 48 hours of transfection, perform cell screening and clone purification according to the following steps: digest and count the transfected cells; calculate the total number of cells required according to the number of 1 cell per well; take a 96-well plate, Add 100 μl of medium containing G418 and 10% v/v FBS to make the number of cells per well 0.5-1; observe the number of cells under an inverted microscope after culturing for about 5 days, and record the cell colony that contains a single cell; The cells were identified by IFA to complete the screening and clone purification.

1.3 Experimental results

1.3.1 Construction of DC-SIGNR expression plasmid

After the pCAG-DC-SIGNR plasmid was identified by Sac I and Xho I double enzyme digestion, the result showed that an obvious target band appeared at 1218bp (Figure 1), and the pCAG-DC-SIGNR expression plasmid was successfully constructed.

1.3.2 IFA identification of transfected cells

The transfected cells were screened and cloned and purified for IFA detection. The screened cell clones expressed DC-SIGNR on the cytoplasm and cell membrane surface, while the control cells had no specific fluorescent signal. The results showed that after G418 screening and IFA identification, the present invention successfully screened out a cell line stably expressing DC-SIGNR, and the cell line was named BHK-DR cell.

Example 2: Construction of reporter Zika virus ZIKV-GFP expressing green fluorescent protein

2.1 Experimental materials

2.1.1 Main materials and reagents

HEK-293T, Vero, and BHK-21 cells were preserved by our laboratory; DMSO was purchased from Sigma; DMEM, fetal bovine serum, and Opti-MEM were purchased from Gibco; trypsin (TRYPSIN 0.25% EDTA) and penicillin-streptomycin mixed Polylysine was purchased from Sigma Company; Hoechst was purchased from Pierce Company; Triton X-100 was purchased from Sigma Company; infrared fluorescent-labeled anti-rabbit IgG antibody was purchased from KPL Company; FITC-labeled goat anti-small Mouse IgG was purchased from Zhongshan Jinqiao Company; QIAGEN Plasmid Extraction Kit was purchased from QIAGEN Company; TransIT-293Transfection Reagent was purchased from Mirus Company; Chloroquine (Chloroquin) and 6-Azauridine (6-Azauridine) were purchased from Sigma Company; ZIKV E protein Antibodies were purchased from GeneTex Company.

2.1.2 Experimental Instruments

High-content cell screening system (PerkinElmer); PCR instrument (Takara); nucleic acid electrophoresis instrument (Beijing Liuyi Instrument); nucleic acid gel imaging system (Bio-Rad); pure water instrument (Thermo Fisher); optical microscope (Olympus); Inverted fluorescence microscope (Life); electronic balance (Beijing Sartorius); CO2 incubator (Heal Force); standard pipette (Eppendorf); horizontal centrifuge (Eppendorf); protein electrophoresis (Bio-Rad); Dry protein transfer apparatus (Bio-Rad); protein gel imaging system (Odyssey).

2.2 Experimental method

2.2.1 Construction of ZIKV replicon and pC38GFP-CME-NS1 ₁₃₄ plasmid

According to the gene sequence of the popular ZIKV strain in Brazil in 2015 (Genbank accession number: KX280026), other genome sequences except the structural protein gene and the GFP reporter gene sequence gene of the structural protein gene position inserted or deleted were synthesized by segmented gene synthesis technology, The replicon recombinant plasmid pZIKVrepdCME was obtained by linking into the mammalian expression vector pCI-neo through multiple cloning restriction sites Sac I and Not I. The open reading frame of the replicon plasmid is "CMV promoter-SacI-5'UTR-the nucleotide sequence encoding the 20 amino acids at the N-terminal of the C protein-GFP-FMDV2A-the nucleotide sequence encoding the 22 amino acids at the C-terminal of the E protein Sequence-NS1-5 (non-structural protein 1-5 gene sequence)-3'UTR-HDVr (hepatitis delta virus ribozyme gene sequence)-Not I", the replicon plasmid construction process is shown in Figure 2. At the same time, according to the ZIKV gene sequence, the "5'UTR-C38 (nucleotide sequence encoding the 38 amino acids at the C-terminal of the ZIKV C protein)-GFP (green fluorescent protein gene sequence)-FMDV2A-C-prM was synthesized by gene synthesis technology. -E (ZIKV structural protein gene sequence)-NS1 ₁₃₄ (NS1 gene 1-134 nucleotide sequence)" clone the fragment into the pCI-neo vector to construct pC38GFP-CME through multiple cloning restriction sites XhoI and NotI - NS1 ₁₃₄ plasmid (Figure 2).

2.2.2 Rescue of reporter ZIKV

Using the plasmid pC38GFP-CME-NS1 ₁₃₄ as a template, primers P1: 5'-GGCCTTTTGCTCACATGGCTCGACAG-3' and P2: 5'GACTGCTGCTGCCAATCTACGGGGG-3' were used for PCR amplification, and the PCR products were used to rescue the reporter virus.

Reporter ZIKV rescue: HEK-293T cells were cultured in 24-well plates with 10% v/v FBS DMEM medium to a density of 70%-90%, and the pC38GFP-CME-NS1 ₁₃₄ plasmid was transfected with TransIT-293Transfection Reagent The PCR product and the Xhol I linearized pZIKVrepdCME replicon plasmid were transfected at a ratio of 500ng per well. After 72 hours of transfection, the cells and supernatant were harvested and stored at -70°C. The rescued virus was named ZIKV-GFP.

2.2.3 Identification of recombinant virus

2.2.3.1 Electron microscope observation and identification

Normal passage BHK-DR cells in T25 cell flasks, when the cell density reached 70%-90%, ZIKV-GFP infected BHK-DR cells with MOI=1, and set BHK-DR cell blank control at 37°C, 5% CO2 Cultivate in an incubator for 72 hours, and after being treated with a fixative solution, slice samples are made, and virus particles are observed with an electron microscope.

2.2.3.2Western blot and IFA detection

The ZIKV-GFP constructed by the present invention was inoculated in BHK-DR cells at MOI=1, and a blank control of BHK-DR cells was set at the same time, and Western blot detection was carried out after 72 hours. The ZIKV-GFP constructed by the present invention was inoculated in BHK-DR cells at MOI=1, and a blank control of BHK-DR cells was set at the same time, and IFA detection was carried out after 48 hours.

2.2.3.3 Recombinant virus infected BHK-DR cells to form plaques

After BHK-21, Vero and BHK-DR cells were normally passaged in 24-well plates with 2% FBS 500ul medium, when the cell density reached 90%, the virus ZIKV-GFP constructed by the present invention was added at 10 ^-1 , 10 ^{- 2} , 10 ^-3 , 10 ^-4 , 10 ^-5 were serially diluted and added to a 24-well plate at 100ul per well. We added 600ul 3% methylcellulose (15g methylcellulose was sterilized and fully dissolved in 500mL DMEM solution containing 1% bovine serum) to cover the cells within 6-12h after infection. At the same time, uninfected BHK-DR cell wells were set as negative controls in the experiment. Discard the cover after 7 days, wash lightly with PBS, add 0.1% crystal violet 1mL per well, stain for 20-30 minutes, rinse gently with running water, invert to dry and observe and record plaque formation.

2.2.3.4 Determination of ZIKV-GFP growth kinetic curve

The ZIKV-GFP constructed in the present invention was inoculated in BHK-21 cells, Vero cells, and BHK-DR cells at MOI=0.1, respectively, and samples were taken continuously at 24h, 48h, 72h, 96h, 120h, and 144h after infection and stored at -20°C. The samples of ZIKV and ZIKV-GFP in each time period were collected for plaque counting experiments, and the samples of ZIKV-GFP collected in each time period were counted under an inverted fluorescence microscope to draw growth curves respectively.

2.3 Results

2.3.1 Construction of ZIKV replicon and pC38GFP-CME-NS1 ₁₃₄ plasmid

According to the ZIKV gene sequence, construct other genome sequences except the structural protein gene, and the GFP reporter gene sequence gene of the insertion-deleted structural protein gene position, and successfully construct the replicon recombinant plasmid pZIKVrepdCME after identification by Xhol I and Not I double enzyme digestion (FIG. 3A). According to the ZIKV gene sequence, the pC38GFP-CME-NS1 ₁₃₄ plasmid was constructed, and after identification by Sac I and Not I double digestion, the pC38GFP-CME-NS1 ₁₃₄ plasmid was successfully constructed (Fig. 3B).

2.3.2 Electron microscope identification of recombinant ZIKV-GFP

BHK-DR cells were infected with ZIKV-GFP at MOI=1. After being treated with fixative solution, sliced samples were made, and virus particles were observed by electron microscope. The results showed that BHK-DR cells infected by ZIKV-GFP could be in the endoplasmic reticulum Recombinant virus particles were found.

2.3.3 Western blot and IFA detection of ZIKV structural protein

The ZIKV-GFP constructed in our laboratory was inoculated into BHK-DR cells at MOI=1, and the rabbit polyclonal antibody prepared in our laboratory was used for IFA and Western blot detection respectively, and the rabbit serum obtained after 3 times of immunization was used as a Anti-infrared fluorescence-labeled goat anti-rabbit IgG was used as the secondary antibody for western blot experiment, and ZIKV E protein-specific polyclonal antibody was used for detection. As a result, a specific band appeared at the relative molecular mass of 55kDa (Figure 4), which was consistent with the expected The results match. At the same time, the GFP was observed on the recombinant virus-infected cells. As a result, the infected cells produced bright green fluorescent signals, while the negative control did not produce fluorescent signals (Figure 5), indicating that the recombinant virus expresses GFP, which plays a significant role in the virus-infected cells. Good reporting and markup.

2.3.4 Formation of plaques in cells infected with recombinant virus ZIKV-GFP

The ZIKV-GFP virus constructed in the present invention was diluted in a gradient of 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 and added to BHK-21, Vero and in BHK-DR cells. We added 600ul 3% methylcellulose to cover the cells within 6-12h after infecting the cells. Discard the covering after 7 days, wash it lightly with PBS, add 0.1% crystal violet to each hole and stain for 20-30min, it is found that ZIKV-GFP can form virus plaques in BHK-DR cells and Vero cells, while in BHK-21 No plaques were seen in the cells (Figure 6).

2.3.5 Determination of growth kinetic curves of recombinant virus ZIKV and ZIKV-GFP

The ZIKV constructed by the present invention was inoculated into BHK-21 cells, Vero cells and BHK-DR cells respectively at MOI=0.1, and samples were taken continuously at 24h, 48h, 72h, 96h, 120h and 144h after infection for plaque counting experiment. At the same time, ZIKV-GFP was inoculated in Vero cells and BHK-DR cells at MOI=0.1, and the ZIKV-GFP samples collected in the above time periods were counted under an inverted fluorescence microscope to draw growth curves respectively. The results showed: Between 24h-72h, the virus titer of ZIKV and ZIKV-GFP shows an overall upward trend, and the virus titer reaches the maximum at 72h, and generally shows a downward trend at 96h-144h, so the best time to determine the virus collection is about 72h after infection (Figure 7).

Example 3 reporter virus system is used for antiviral drug screening

3.1 Antiviral drug inhibition experiment

Drugs: Drugs Ribavirin (Rib) and 6-Azauridine (6-Az) were dissolved in DMSO, chloroquine (Chloroquine, CQ) was dissolved in PBS, and the final concentration of DMSO in the culture medium was 1%. The %DMSO effect was used as a negative control. Drugs were diluted with DMSO and DMEM medium containing 2% fetal bovine serum (FBS) to different concentrations for use.

Drug treatment and virus infection: inoculate BHK-DR cells (3× ¹⁰⁴ cells/well) in black 96-well plate with 2% v/v FBS 100ul medium, and when the cell density reaches 90%, use 50ul containing 2% v The drugs diluted in DMEM medium of /v FBS acted for 1 hour, added 50 ul ZIKV-GFP virus (10 ⁶ TCID ₅₀ /ml) after 1 hour to infect, and cultured at 37° C., 5% CO ₂ incubator for 48 hours.

Statistical analysis of the high-content system: After the cells were treated with drugs and infected with the virus for 48 hours, the nuclei were stained with Hoechst (1:2000), the cell plate was placed in a high-content cell screening system (PerkinElmer) for observation, and the fluorescent channel of green fluorescent protein was used to stain the nucleus Fluorescent channel scanning analysis, counting the number of infected positive cells and the total number of cells in each well, calculating the virus infection rate, and calculating the inhibition rate of the drug on virus replication.

3.2 Inhibitory effect of drugs on viral replication

After ZIKV-GFP infected BHK-DR cells and was treated with drugs, the high-content cell screening system (PerkinElmer) was used to observe, record and analyze the results. The results showed that the antiviral drug Ribavirin had an inhibitory effect on Zika virus, with an IC50 of less than 5 μM. Fluorescent images showed When the drug concentration was 20 μM, there was almost no fluorescent signal. When the drug concentration was lower than 5 μM, the rate of virus-infected positive cells was significantly lower than that of the control group, and Ribavirin had no obvious toxicity to cells at these concentrations (Fig. 8A -C).

Drug 6-Azauridine has inhibitory effect on WNV and other flaviviruses in previous reports, and ZIKV in recent reports. Our test results also show that this drug has obvious inhibitory effect on ZIKV-GFP. After treatment of the cells with the drug at a concentration of 2 μM, the virus infection rate of the cells can be significantly reduced, and the toxicity of the drug to the cells is also small, and only when the concentration is 32 μM does it show a significant effect on the cell viability (Fig. 9A-C).

The test results show that Chloroquine also has a significant inhibitory effect on ZIKV-GFP. When the drug concentration is 4μM, the virus infection rate can be significantly reduced. When the drug concentration was 16 μM, the virus infection rate could be reduced by more than 80%, while the drug concentration of 2-16 μM showed no significant effect on cell viability ( FIG. 10A-C ).

sequence listing

<110> Harbin Institute of Veterinary Medicine, Chinese Academy of Agricultural Sciences (Harbin Branch of China Center for Animal Health and Epidemiology)

<120> A drug screening system based on recombinant Zika virus expressing green fluorescent protein and its application

<130> KLPI190266

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 1218

<212>DNA

<213> DC-SIGNR

<400> 1

gagctcgcca ccatgagtga ctccaaggaa ccaagggtgc agcagctggg cctcctggaa 60

gaagatccaa caaccagtgg catcagactt tttccaagag actttcaatt ccagcagata 120

catggccaca agagctctac agggtgtctt ggccatggcg ccctggtgct gcaactcctc 180

tccttcatgc tcttggctgg ggtcctggtg gccatccttg tccaagtgtc caaggtcccc 240

agctccctaa gtcaggaaca atccgagcaa gacgcaatct accagaacct gacccagctt 300

aaagctgcag tgggtgagct ctcagagaaa tccaagctgc aggagatcta ccaggagctg 360

acccagctga aggctgcagt gggtgagttg ccagagaaat ccaagctgca ggagatctac 420

caggagctga cccggctgaa ggctgcagtg ggtgagttgc cagagaaatc caagctgcag 480

gagatctacc aggagctgac ccggctgaag gctgcagtgg gtgagttgcc agagaaatcc 540

aagctgcagg agatctacca ggagctgacc cggctgaagg ctgcagtggg tgagttgcca 600

gagaaatcca agctgcagga gatctaccag gagctgacgg agctgaaggc tgcagtgggt 660

gagttgccag agaaatccaa gctgcaggag atctaccagg agctgaccca gctgaaggct 720

gcagtgggtg agttgccaga ccagtccaag cagcagcaaa tctatcaaga actgaccgat 780

ttgaagactg catttgaacg cctgtgccgc cactgtccca aggactggac attcttccaa 840

ggaaactgtt acttcatgtc taactcccag cggaactggc acgactccgt caccgcctgc 900

caggaagtga gggcccagct cgtcgtaatc aaaactgctg aggagcagaa cttcctacag 960

ctgcagactt ccaggagtaa ccgcttctcc tggatgggac tttcagacct aaatcaggaa 1020

ggcacgtggc aatgggtgga cggctcacct ctgtcaccca gcttccagcg gtactggaac 1080

agtggagaac ccaacaatag cgggaatgaa gactgtgcgg aatttagtgg cagtggctgg 1140

aacgacaatc gatgtgacgt tgacaattac tggatctgca aaaagcccgc agcctgcttc 1200

agagacgaat agctcgag 1218

Claims (6)

1. A drug screening system based on the recombinant Zika virus (Zika virus, ZIKV) expressing green fluorescent protein (GFP), characterized in that, the drug screening system is composed of recombinant Zika virus expressing green fluorescent protein and expression BHK-21 cell line composition of DC-SIGNR; Wherein, the recombinant Zika virus expressing green fluorescent protein is prepared by the following method: 1) According to the Zika virus gene sequence, other genomic sequences except the structural protein gene and the GFP reporter gene sequence gene of the structural protein gene position of the insertion and deletion were synthesized by segmented gene synthesis technology, and connected into the mammalian through the multiple cloning restriction site Animal expression vector, obtained replicon recombinant plasmid, the open reading frame of the replicon recombinant plasmid is "CMV promoter-restriction site-5'UTR-nucleotide sequence encoding 20 amino acids at the N-terminal of C protein-GFP- FMDV2A-the nucleotide sequence encoding the 22 amino acids at the C-terminus of the E protein-NS1～5-3′UTR-HDVr-enzyme cleavage site”; 2) According to the Zika virus gene sequence, the structural protein gene sequence and the GFP sequence were synthesized by gene synthesis technology, and the connection sequence of each element was 5'UTR-the nucleotide sequence encoding the 38 amino acids at the C-terminal of the ZIKV C protein-GFP- FMDV2A-C-prM-E-NS1 gene 1-134 nucleotide sequence, the fragment is cloned into a mammalian expression vector through multiple cloning restriction sites, and a plasmid containing ZIKV structural gene and green fluorescent protein is obtained; 3) Rescue of reporter ZIKV Using the plasmid obtained in step 2) as a template, perform PCR amplification, and amplify to obtain the nucleotide sequence containing CMV promoter and 5'UTR-coding 38 amino acids at the C-terminal of ZIKV C protein-GFP-FMDV2A-C-prM - The PCR product of the 1-134 nucleotide sequence of the E-NS1 gene, which is used for the rescue of the reporter virus; Rescue of reporter ZIKV: HEK-293T cells were cultured in 24-well plates with 10% v/v FBS DMEM medium to a density of 70%-90%, and the obtained PCR products and linear The transfected replicon recombinant plasmids were respectively transfected. After 72 hours of transfection, the cells and supernatant were harvested and stored at -70°C to obtain recombinant Zika virus expressing green fluorescent protein; Wherein, the Zika virus gene sequence is the gene sequence of the ZIKV strain popular in Brazil in 2015, and the Genbank accession number is: KX280026; Wherein, the BHK-21 cell line expressing DC-SIGNR is constructed by the following method: 1) Construct an expression plasmid containing DC-SIGNR: The coding nucleotide sequence of DC-SIGNR is shown in SEQ ID NO.1, and the synthesized DC-SIGNR sequence is cloned into the pCAGneo vector to obtain an expression plasmid containing DC-SIGNR; 2) Linearize the prepared expression plasmid containing DC-SIGNR, recover from gel, and transfect BHK-21 cells; 3) Screening to obtain a BHK-21 cell line stably expressing DC-SIGNR. 2. drug screening system as claimed in claim 1, is characterized in that, in the step 1) that the recombinant Zika virus preparation of described expression green fluorescent protein is to link in by polyclonal enzyme cutting site Sac I and Not I The mammalian expression vector pCI-neo obtains the replicon recombinant plasmid, and the open reading frame of the replicon recombinant plasmid is CMV promoter-Sac I-5'UTR-the nucleotide sequence encoding the N-terminal 20 amino acids of the C protein-GFP - FMDV2A - the nucleotide sequence encoding the 22 amino acids at the C-terminal of the E protein - NS1-5-3'UTR-HDVr-Not I. 3. The drug screening system according to claim 1, characterized in that, in the step 2) of the preparation of the recombinant Zika virus expressing green fluorescent protein, the 5'UTR is cut by multiple cloning restriction sites XhoI and NotI -The nucleotide sequence encoding the 38 amino acids at the C-terminal of the ZIKV C protein-GFP-FMDV2A-C-prM-E-NS1 gene 1-134 nucleotide sequence was cloned onto the pCI-neo vector. 4. The drug screening system according to claim 1, wherein the step 3) of preparing the recombinant Zika virus expressing green fluorescent protein uses the plasmid obtained in step 2) as a template, and uses primer P1:5 '-GGCCTTTTGCTCACATGGCTCGACAG-3' and P2: 5'GACTGCTGCTGCCAATCTACGGGGG-3' were amplified by PCR, and the PCR products were used to rescue the reporter virus. 5. Application of the drug screening system described in any one of claims 1-4 in screening anti-Zika virus drugs. 6. A method for screening anti-Zika virus drugs using the drug screening system described in any one of claims 1-4, characterized in that, comprising the following steps: 1) Drug treatment and virus infection: Inoculate the BHK-21 cell line expressing DC-SIGNR in a black 96-well plate with 2% v/v FBS 100 ul medium, and when the cell density reaches 90%, use 50 ul containing 2% v/v FBS v The drug to be tested diluted in DMEM medium of FBS was acted for 1 hour, after 1 hour, the recombinant Zika virus expressing green fluorescent protein was added to infect, and cultured in a 5% CO ₂ incubator at 37°C for 48 hours; 2) Statistical analysis of the high-content system: After the cells were treated with drugs and infected with the virus for 48 hours, the nuclei were stained with Hoechst, and the cell plate was placed in a high-content cell screening system for observation, and the green fluorescent protein fluorescent channel and the nuclear staining fluorescent channel were used for scanning analysis. The number of infected positive cells and the total number of cells in each well were used to calculate the virus infection rate and the inhibitory rate of the drug to be tested on virus replication.

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