CN101058803A - Virus-like particles containing O-type foot-and-mouth disease virus IRES RNA, preparation method and application - Google Patents

Abstract

A virus-like particle containing O-type foot-and-mouth disease virus IRES RNA, a preparation method and application thereof relate to a virus-like particle. Provide a safe, ribonuclease-resistant, stable, easy-to-preserve virus-like particle containing O-type foot-and-mouth disease virus IRES RNA that can be monitored throughout the process of RNA virus extraction, reverse transcription, amplification, and final detection, and its preparation method and application . Virus-like particles refer to an RNA-protein complex containing foot-and-mouth disease virus IRES RNA wrapped by MS ₂ bacteriophage coat protein. The RNA-protein complex is spherical in shape with a diameter of 24-28nm and has the characteristics of ribonuclease resistance. Preparation steps: construction of primary vector pET32a-CP; construction of final vector pET32a-CP-IRES; prokaryotic expression of virus-like particles; purification of virus-like particles.

Description

Contain virus-like particle and preparation method and the application of O type foot and mouth disease virus IRES RNA

Technical field

The present invention relates to a kind of virus-like particle, especially relate to a kind of virus-like particle that contains O type foot and mouth disease virus IRES RNA that can in RNA viruses detects, use and preparation method thereof.

Background technology

RNA viruses is to be the virus of genetic material with RNA, for example avian influenza virus, foot and mouth disease virus and SARS virus etc.Because these RNA viruses have serious hazardness,, it just seems most important so detecting fast and accurately.Detect RNA viruses method commonly used traditional viral separation test is arranged, hemagglutination-inhibition test, virus neutralization tests and enzyme linked immunosorbent assay (ELISA) etc., and amplification technique (NASBA) (the Romano J W of molecular Biological Detection technology such as nucleic acid dependence, Will-iams K G, Shurtliff R N, et al.NASBA technology:isothermal RNA amplification in qualitativeand quantitative diagnostics[J] .Immunol.Invest, 1997,26 (1-2): 15-28), conventional RT-PCR, real-time RT-PCR technology and PCR-ELISA technology etc.Wherein real-time fluorescence PCR is the new detection technique that grew up in recent years, advantage with aspects such as sensitivity for analysis are high and simple and efficient, (Spackman E with the obvious advantage in the RNA viruses quick diagnosis, Senne D A, Bulaga L L, et al.Development of real-time RT-PCR for the detection of avianinfluenza virus.Avian Dis, 2003,47 (3Suppl): 1079-1082; Heid C A, Stevens J, Livak J K, et al.Real time quantitative PCR[J] .Genome Res, 1996,6 (10): 986-994).

In the qualitative and detection by quantitative of RNA viruses on the PCR basis, influence factor is more, as test concentration and the efficient of reverse transcription and the problem of reagent etc. of residual, the target nucleic acid to be amplified of inhibition after the difference, sample nucleic acid extraction of temperature between random error in personnel's measurement operation, amplification instrument hole, these factors all may influence the efficient of pcr amplification, and then cause result's deviation, even false-negative result, quality control product and standard substance must be used so the PCR of RNA viruses measures, result's reliability could be guaranteed.The quality control product of traditional RNA viruses augmentation detection and standard substance have two kinds: a kind of RNA or provirus particle for exposing, another kind is in-vitro transcription cDNA.The Yeast Nucleic Acid enzyme liberating that the former is extensively existed easily and have infectivity, the latter can not response sample extract and the reverse transcription process to the influence of mensuration.

In recent years, people such as Pasloske have proposed a kind of new RNA quality control product technology of preparing, be armor RNA (armored RNA) technology (Pasloske B L, Walkerpeach C R, Obermoeller R D, et al.Armored RNA technology forproduction of ribonuclease-resistant viral RNA controls andstandards[J] .J Clin Microbiol, 1998,36 (12): 3590-3594; Walker Peach C R, Winkler M, DuBois D B, et al.Ribonuclease-resistantRNA controls (Armored RNA) for reverse transcription-PCR, branched DNA, and genotypingassays for hepatitis C virus[J] .Clin.Chem, 1999,45 (12): 2079-2085.Present this technology is applied to aspects such as detection (the Hietala S K of RNA viruses just gradually, Crossley B M.Armored RNA as Virus Surrogate in aReal-Time Reverse Transcriptase PCR Assay Proficiency Panel.J Clin.Microbiol.Jan.2006,67-70).Armor RNA technology is meant by MS ₂The technology of bacteriophage coat protein parcel recombinant RNA.Intestinal bacteria MS ₂Phagus is in straight polarity single stranded RNA spherical virus, genome total length 3659bp, 4 kinds of protein molecules such as encoding mature zymoprotein, coat protein, replicase protein and crack protein (Jia Panxing. phage molecular biology-ABC and technical ability [M]. Beijing: Science Press, 2001,2-5.).MS ₂Phage is made up of 180 coat protein monomers, a part maturing enzyme albumen and a part geneome RNA, at MS ₂Find in the research of bacteriophage coat protein, coat protein and operon RNA sequence have specificity to interact, this effect can be packaged into (Peabody DS.The RNA binding site of bacteriophage MS in the coating with phage genome RNA when causing the phage ghost assembling ₂Coat protein.The EMBO Journal, 1993,12 (2): 595-600; Peabody D S.Role of the coat protein-RNA interaction in the life cycle ofbacteriophage MS ₂.Mol Gen Genet, 1997,254:358-364).Discover MS ₂The maturing enzyme albumen of phage and the gene of coat protein and the gene clone of 5 ' non-coding sequence that comprises gene regulatory elements are in expression vector, obtain maturing enzyme albumen and coat protein through abduction delivering, and under maturing enzyme and the segmental synergy of phage genome RNA, coat protein can be assembled into sophisticated virus-like particle, and possesses the characteristic of anti-RNase effect.If with MS ₂These genes involveds and the external source segment of phage are cloned in the expression vector together, this carrier can be cloned segment with external source and is transcribed into RNA, coat protein can be assembled into it RNA-protein complex of spherical RNA viruses structure, i.e. armor RNA viruses sample particle (Peabody D S.Translational repression by bacteriophage MS simultaneously ₂Coat proteinexpressed from a plasmid.The journal of biological chemistry.1990,265 (10): 5684-5689; PickettG G, Peabody D S.Encapsidation of heterologous RNAs by bacteriophage MS ₂Coatprotein.Nucleic Acids Research, 1993,21 (19): 4621-4626).The present invention utilizes above principle with MS exactly ₂Phage portion gene and foot and mouth disease virus IRES gene clone are carried out the virus-like particle that contains foot and mouth disease virus IRES RNA that prokaryotic expression obtains anti-RNase to expression vector.

Summary of the invention

The objective of the invention is to infectivity that the quality control product that detects at traditional RNA viruses and standard substance exist, easily by the Yeast Nucleic Acid enzyme liberating, can not the complete monitoring RNA viruses problems such as detection, but provide a kind of safe, anti-rnase, good stability, easily preservation complete monitoring RNA viruses extraction, reverse transcription, amplification and the final virus-like particle and preparation method and the application thereof that contain O type foot and mouth disease virus IRES (internal ribosoma entry site, internal ribosome entry site) RNA that detects.

The virus-like particle of the O of containing type foot and mouth disease virus IRES RNA of the present invention is meant by MS ₂The bacteriophage coat protein parcel contains a kind of RNA-protein complexes of foot and mouth disease virus IRES RNA, and being shaped as of RNA-protein complexes is spherical, and diameter is 24～28nm, has the characteristic of anti-rnase.

The preparation method of the virus-like particle of the O of containing type foot and mouth disease virus IRES RNA of the present invention may further comprise the steps:

1. the first structure of carrier pET32a-CP:

1) according to MS ₂The phage gene sequence, design primer PCR amplification MS ₂The CP gene of phage, amplification MS ₂The primer sequence of the CP gene of phage is:

Upstream primer: 5 '-CGCGGTACCCCCTTTCGGGGTCCTGCTC-3 ',

Downstream primer: 5 '-CGCGGATCCCGAGAGAAAGATCGCGAGGAAG-3 '.

2) Kpn I and BamH I double digestion CP segment and plasmid pET32a connect behind the purifying.

3) connect the product transformed into escherichia coli, choose that positive monoclonal carries out PCR and enzyme is cut evaluation, after the sequencing result comparison to confirm the correct carrier pET32a-CP just that makes up.

2. the structure of whole carrier pET32a-CP-IRES (being called for short pCPES):

1) according to the IRES gene order, the gene segment of design primer PCR amplification foot and mouth disease virus IRES, the sequence of foot and mouth disease virus IRES is:

CGGTCACCTATTCAGGCGTAGAAGCTTTTTAAACTGGGCACTTTTAAAGAAGTCCCAATCCCCTTCTCAGATCCCGAGT

GTCCCGTGTTACCTCGGGGTACCTGAAGGGCATCCTTAG

The primer sequence of amplification foot and mouth disease virus IRES gene segment is:

Upstream primer: 5 '-TCGAGCGGCCGCGGTC-3 ',

Downstream primer: 5 '-CTCGGATCCCTAAGGATGCCC-3 '.

2) Not I and BamH I double digestion IRES segment and plasmid pET32a-CP connect behind the purifying.

3) connect the product transformed into escherichia coli, choose positive monoclonal and carry out PCR and identify, after the sequencing result comparison to confirm correctly to make up whole carrier pET32a-CP-IRES, i.e. pCPES.

3. the prokaryotic expression of virus-like particle:

Plasmid pCPES is transformed expressive host bacterium BL21 E.Coli, the bacterium of positive colony is seeded in the LB liquid nutrient medium that contains penbritin, shaking culture, to the OD value be 0.6～0.8, under 28～37 ℃ of conditions, add isopropylthio-(IPTG), making its final concentration is 0.8～1.5mmol/L, abduction delivering 4～8h.With the centrifugal collection bacterial sediment of the bacterium liquid after inducing, the resuspended bacterial sediment of supersound process damping fluid is with the broken bacterium of ultrasonic cell disintegration instrument.After the fragmentation fully that the bacteria breaking thing is centrifugal, get supernatant and transfer to another clean centrifuge tube, obtain desired expression product.

4. the purifying of virus-like particle:

4～6 kinds of solution of the different gradients of preparation sucrose density add in the centrifuge tube, obtain saccharose gradient liquid.In the expression product solution of ultrasonication, add excessive RNaseA and DNase I, to remove degerm own RNA and DNA.SYBRGreen I added in the expression product that two enzymes handled dye, then sample is added to the top of centrifuge tube, centrifugal on ultracentrifuge, the sample segment that green fluorescence is arranged is taken out, PBS solution is dialysed, and obtains the virus-like particle that contains O type foot and mouth disease virus IRESRNA of purifying.

In the purification step of virus-like particle, 4 kinds of solution of the different gradients of preparation sucrose density, sucrose density preferably is respectively 15%, 25%, 35%, 45% solution.

The virus-like particle of the O of containing type foot and mouth disease virus IRES RNA of the present invention can be used for the detection of RNA viruses.

The present invention has the following advantages:

1. this virus-like particle does not have infectivity, safe and reliable, can not work the mischief to the experimenter in the preparation.

2. this virus-like particle has anti-rnase characteristic, good stability, easily preserves, transports storage conveniently.

3. this virus-like particle has and the RNA viruses analog structure, promptly all is the nucleic acid of albumen parcel, therefore can add it in the viral material, experience nucleic acid extraction together, reverse transcription, pcr amplification, complete monitoring is carried out in detection to RNA viruses, also avoids occurring false negative result.

4. the preparation method is simple, and is with low cost, is beneficial to and promotes the use of.

Description of drawings

Fig. 1 is the RNaseA of expression product after the ultrasonication and two enzymic digestion results of DNase I.In Fig. 1,1 for adding RNaseA digestion; 2 for adding DNase I digestion; 3 for adding RNaseA and DNase I digestion; 4 are not enzyme-added digestion product; M representation DNA marker DL2000; The nucleic acid fragment of DNA marker DL2000 is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp from top to bottom.

Fig. 2 is the electron microscopic observation result of the virus-like particle of purifying.

Fig. 3 is for extracting the RT-PCR detected result of RNA in the virus-like particle.In Fig. 3, M representation DNA marker DL2000; 1 is PCR result after the RNA reverse transcription; 2 is not PCR result after the reverse transcription of RNA; 3 positive contrasts; 4 negative contrasts.The nucleic acid fragment of DNA marker DL2000 is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom.

Fig. 4 is a RNA single stage method reverse transcription real-time fluorescence PCR detected result in the virus-like particle.In Fig. 4, X-coordinate is cycle number cycle, and ordinate zou is fluorescence intensity fluorescence.Curve A is virus-like particle RNA one step reverse transcription fluorescent PCR result, the negative contrast of curve B.

Fig. 5 detects avian influenza virus RNA fluorescence RT-PCR result for Two Colour Fluorescence.In Fig. 5, X-coordinate is cycle number cycle, and ordinate zou is fluorescence intensity fluorescence.Curve A, B, C are respectively the fluorescence result of 3 pipe avian influenza virus RNA, the negative contrast of curve D.

Fig. 6 detects the result of virus-like particle RNA for Two Colour Fluorescence.In Fig. 6, X-coordinate is cycle number cycle, and ordinate zou is fluorescence intensity fluorescence.Curve A, B, C are respectively the fluorescence result of 3 pipe virus-like particle RNA, the negative contrast of curve D.

Embodiment

Following examples are to further specify of the present invention.

Embodiment 1: the preparation method who contains the virus-like particle of O type foot and mouth disease virus IRES RNA

The preparation method who contains the virus-like particle of O type foot and mouth disease virus IRES RNA may further comprise the steps:

(1) structure of first carrier pET32a-CP:

With pMS ₂7 plasmids are template amplification CP segment, and the PCR system is: 10 * PCR buffer, 2 μ l, TaqDNA Polymerase0.2 μ l (5u/ μ l), dNTP 0.5 μ l (10mmol/L), template 1 μ l, each 0.5 μ l (10 μ mol/L) of upstream and downstream primer, distilled water is supplied 20 μ l.The PCR reaction conditions is: 94 ℃ of sex change 5min, and 94 ℃ of 30s then, 60 ℃ of 30s, 72 ℃ of 2min, 28 circulations, last 72 ℃ are extended 7min.Amplified production is carried out agarose gel electrophoresis, reclaim the test kit purifying with glue and reclaim the CP segment.The CP segment and the plasmid pET32a that reclaim are cut with Kpn I and BamH I enzyme respectively, glue reclaims CP gene and the carrier pET32a that test kit reclaims double digestion, spends the night according to 16 ℃ of connections of following system then: purpose fragment CP 5 μ l, plasmid pET32a1.5 μ l, connect damping fluid 1 μ l, T ₄Dna ligase 0.5 μ l, RNase 0.5 μ l (10mg/ml), ddH ₂O supplies 10 μ l.To connect product transformed into escherichia coli DH5 α, bed board is cultivated 12h, and the picking mono-clonal carries out PCR and enzyme is cut evaluation, positive recombinant plasmid called after pET32a-CP.Sequencing result and known sequences comparison, presentation of results CP gene correctly is cloned on the carrier pET32a.

(2) structure of whole carrier pET32a-CP-IRES (being called for short pCPES):

With the pcDNA3-IRES plasmid is template amplification IRES segment, and the PCR reaction system is identical with the system condition of CP amplification with condition.Amplified production is carried out agarose gel electrophoresis, reclaim test kit with glue and reclaim the IRES segment.Utilize the same method of carrier structure just, the IRES segment of Not I and BamH I digested plasmid pET32a-CP and recovery, glue reclaims the purpose product that enzyme is cut, and the IRES segment is connected with first carrier pET32a-CP.To connect product transformed into escherichia coli DH5 α, the picking mono-clonal carries out PCR identifies that positive recombinant plasmid called after pET32a-CP-IRES is called for short pCPES.Sequencing result and known IRES sequence alignment, homology is 100%.

(3) prokaryotic expression of virus-like particle:

Plasmid pCPES is transformed expressive host bacterium BL21 E.Coli, and picking positive monoclonal bacterium is seeded in incubated overnight in the LB liquid nutrient medium that contains penbritin.The bacterium liquid of getting the 2ml incubated overnight is inoculated into 200ml and contains in the LB liquid nutrient medium of penbritin, and 200r/min shaking culture 6h to OD value is 0.8.Adding final concentration is the isopropylthio-(IPTG) of 1mmol/L, at 28 ℃, and abduction delivering 16h under the 200r/min condition.With the centrifugal 10min of bacterium liquid 4000r/min, collect bacterial sediment, use the STE washed twice.Again be suspended from the 5ml ultrasonication damping fluid, ultrasonic on ice: ultrasonic time 20s, pitch time 15s, ultrasonic number of times 20 times, twice of re-treatment.With the fragmentation centrifugal 10min of expression product 4000r/min completely, the sedimentation cell fragment is collected supernatant and is transferred in another clean centrifuge tube, obtains containing the expression product of virus-like particle.

(4) purifying of virus-like particle:

The preparation sucrose density is respectively 15%, 25%, 35%, 45% solution, adds in the centrifuge tube, obtains saccharose gradient liquid.In the expression product solution of ultrasonication, add excessive RNaseA and DNase I, to remove degerm own RNA and DNA.SYBR Green I added in the expression product that two enzymes handled dye, then sample is added to the top of centrifuge tube, centrifugal on ultracentrifuge, the sample segment that green fluorescence is arranged is taken out, PBS solution is dialysed, and obtains the virus-like particle that contains O type foot and mouth disease virus IRES RNA of purifying.

Embodiment 2: two enzymic digestions of the RNaseA of expression product and DNase I are identified after the ultrasonication

With the expression product after the ultrasonication, carry out single enzyme and two enzymic digestion 1h with RNaseA and DNase I respectively, as shown in Figure 1: expression product can be seen a fluorescence striped about 1500bp after using the two enzymic digestions of RNaseA and DNase I; Only can see the existence of bacteria RNA after the DNase I digestion; Only after the RNaseA digestion, bacteria RNA is degraded.Expression product carries out enzymic digestion respectively again after room temperature is placed 20d, electrophoresis result is the existence of visible expression product all, illustrates that virus-like particle has the characteristic of anti-rnase and has good stability.

Embodiment 3: the morphologic observation of purified virus sample particulate

The virus-like particle of sucrose density gradient centrifugation purifying acetic acid uranium dyeing 5min, the JM2100 electron microscopic observation can see that as shown in Figure 2 diameter is approximately the circular granular of 26nm, the virus-like particle that Here it is expresses (viral likeparticles, VLPs).

Embodiment 4: the extraction of RNA and RT-PCR detect in the virus-like particle

The virus-like particle of purifying is extracted RNA with the Trizol method, divide two parts, portion carries out reverse transcription, and portion does not carry out reverse transcription in addition, carries out pcr amplification then, and electrophoresis is identified.The RNA of Ti Quing has the purpose band of FMDVIRES after pcr amplification is carried out in reverse transcription as shown in Figure 3; And the RNA that extracts without the direct pcr amplification of reverse transcription after, the purpose band does not appear.Illustrate this virus-like particle coat protein parcel be the RNA segment that contains foot and mouth disease virus IRES.

Embodiment 5: RNA single stage method reverse transcription real-time fluorescence PCR detects in the virus-like particle

With the RNA in the extraction virus-like particle is template, carries out one-step method real-time fluorescent RT-CPR and detects.Designed the probe that is used for detecting virus-like particle goal gene IRES, sequence is as follows:

Fluorescence chain: ROX-5 '-TCCCCTTCTCAGATCCCGAGTGTC-3 ' phosphorylation

The cancellation chain: 5 '-TCGGGATCTGAGAAGGGGA-3 ' Dabcyl

In 20 μ l reaction solutions, contain 4.0mmol/L Mg ²⁺10mmol/LTris-HCl, 50mmol/LKCl, 0.25mmol/LdNTP, 0.4 μ mol/L upstream primer, 0.4 μ mol/L downstream primer, 1.8U Taq archaeal dna polymerase, 0.3U AMV (ThermoScript II), 0.12U RNase inhibitor, 0.2 μ mol/L fluorescence chain, 0.25 μ mol/L cancellation chain, RNA template 1 μ l.Reaction conditions is: it is cDNA that 42 ℃ of reverse transcription 30min make the template ribonucleic acid reverse transcription, and 94 ℃ of pre-sex change 3min enter circulation then, and cycling program is 94 ℃ of sex change 15s, 55 ℃ of annealing 20s, and 72 ℃ are extended 20s, totally 40 circulations.Gather ROX fluorescence when annealing, Rotorgene3000 real-time fluorescence PCR instrument detects at every turn.Template ribonucleic acid fluorescence after the reverse transcription PCR amplification has obvious rise as shown in Figure 4.

Embodiment 6: Two Colour Fluorescence detects the result of avian influenza virus RNA and virus-like particle RNA reverse transcription PCR

As interior mark, is example to detect avian influenza virus HA gene with virus-like particle, has designed isometric double-chain probe and has detected the HA gene, and sequence is as follows:

Fluorescence chain: FAM-5 '-CCTCTCTTTTTTCTTCTTCTCTCTC-3 ' phosphorylation

The cancellation chain: 5 '-CTCTCAGAAGAAGAAAAAAGAGAGG-3 ' Dabcyl

The primer sequence of HA gene is:

HAS-1：5′-CATTCCACAACATACACCC-3’

HAS-2：5′-CAACCATCTACCATTCCCT-3’

The viral material of bird flu and the virus-like particle of purifying are extracted RNA together, carry out the reverse transcription PCR amplification, and carry out whole process with Two Colour Fluorescence and detect.In the 20ul reaction solution, contain 4.0mmol/LMg ²⁺, 10mmol/LTris-HCl, 50mmol/LKCl, 0.25mmol/L dNTP, 3.0U the Taq archaeal dna polymerase, 0.3U AMV (ThermoScript II), 0.12U RNase inhibitor, two couples of each 0.4 μ mol/L of primer, two couples of each 0.2 μ mol/L of probe, each 1 μ l of template repeats 3 pipes and detects.Reaction conditions is: 42 ℃ of reverse transcription 45min, and 94 ℃ of pre-sex change 5min enter circulation then, and cycling program is 94 ℃ of sex change 15s, 55 ℃ of annealing 20s, 72 ℃ are extended 20s, totally 40 circulations.Gather FAM and ROX fluorescence when annealing, Rotorgene3000 real-time fluorescence PCR instrument detects at every turn.As illustrated in Figures 5 and 6, the fluorescence of FAM and ROX passage all has rise, illustrates that the amplification of avian influenza virus and virus-like particle RNA reverse transcription PCR is normal.

Sequence table

Amplification MS ₂The primer sequence of the CP gene of phage is:

Upstream primer: 5 '-CGC GGTACCCCCTTTCGGGGTCCTGCTC-3 ',

Downstream primer: 5 '-CGC GGATCCCGAGAGAAAGATCGCGAGGAAG-3 '

The sequence of foot and mouth disease virus IRES is:

CGGTCACCTATTCAGGCGTAGAAGCTTTTTAAACTGGGCACTTTTAAAGAAGTCCCAATCCCCTTCTCAGATCCCGAGT

GTCCCGTGTTACCTCGGGGTACCTGAAGGGCATCCTTAG

The primer sequence of the gene segment of amplification foot and mouth disease virus IRES is:

Upstream primer: 5 '-TCGA GCGGCCGCGGTC-3 ',

Downstream primer: 5 '-CTC GGATCCCTAAGGATGCCC-3 '

Claims (4)

1. The virus-like particle containing O-type foot-and-mouth disease virus IRES RNA is characterized in that it refers to a kind of RNA-protein complex containing foot-and-mouth disease virus IRES RNA wrapped by MS ₂ bacteriophage coat protein, and the shape of RNA-protein complex is spherical, The diameter is 24-28nm, and it has the characteristics of resistance to ribonuclease. 2. the preparation method of the virus-like particle containing O type foot-and-mouth disease virus IRES RNA as claimed in claim 1, is characterized in that comprising the following steps: (1) Construction of the initial vector pET32a-CP: 1) According to the MS ₂ phage gene sequence, design primer PCR to amplify the CP gene of MS ₂ phage, and the primer sequence for amplifying the CP gene of MS ₂ phage is: Upstream primer: 5′-CGCGGTACCCCCCTTTCGGGGTCCTGCTC-3′, Downstream primer: 5′-CGCGGATCCCGAGAGAAAGATCGCGAGGAAG-3′; 2) Kpn I and BamH I double digest the CP fragment and plasmid pET32a, and connect after purification; 3) The ligation product was transformed into Escherichia coli, and positive single clones were selected for PCR and enzyme digestion identification, and finally the sequencing results were compared to confirm the correct construction of the initial vector pET32a-CP; (2) Construction of the final vector pET32a-CP-IRES: 1) according to IRES gene sequence, design the gene segment of primer PCR amplification foot-and-mouth disease virus IRES, the sequence of foot-and-mouth disease virus IRES is: CGGTCACCTATTCAGGCGTAGAAGCAGCTTTTTAAACTGGGCACTTTTAAAGAAGTCCCAATCCCCTTCTCAGATCCCGAGT GTCCCGTGTTACCTCGGGGTACCTGAAGGGCATCCTTAG The primer sequence of amplifying foot-and-mouth disease virus IRES gene fragment is: Upstream primer: 5′-TCGAGCGGCCGCGGTC-3′, Downstream primer: 5′-CTCGGATCCCTAAGGATGCCC-3′; 2) IRES fragment and plasmid pET32a-CP were double digested with Not I and BamH I, and ligated after purification; 3) The ligation product was transformed into Escherichia coli, positive single clones were selected for PCR identification, and finally the sequencing results were compared to confirm the correct construction of the final vector pET32a-CP-IRES, namely pCPES; (3) Prokaryotic expression of virus-like particles: Transform the plasmid pCPES into the expression host strain BL21 E.Coli, inoculate the positively cloned strains in LB liquid medium containing ampicillin, culture with shaking until the OD value is 0.6-0.8, and add isopropanol at 28-37°C thio-β-D-galactoside, so that the final concentration is 0.8 ~ 1.5mmol/L, induced expression for 4 ~ 8h; centrifuge the induced bacterial liquid to collect the bacterial pellet, and resuspend the bacterial cell in the ultrasonic treatment buffer Precipitate, crush the bacteria with an ultrasonic cell disruptor; centrifuge the crushed bacteria after complete crushing, and transfer the supernatant to another clean centrifuge tube to obtain the desired expression product; (4) Purification of virus-like particles: Prepare 4 to 6 kinds of solutions with different gradients of sucrose density, add them to centrifuge tubes to obtain sucrose gradient solution, add excess RNaseA and DNase I to the expression product solution of ultrasonic crushing, to remove the RNA and DNA of the bacteria itself, and SYBRGreen I Add the double-enzyme-treated expression product for staining, then add the sample to the top of the centrifuge tube, centrifuge on an ultracentrifuge, take out the part of the sample with green fluorescence, dialyze with PBS solution, and obtain the purified O-type foot-and-mouth disease virus IRESRNA virus-like particles. 3. the preparation method of the virus-like particle that contains O-type foot-and-mouth disease virus IRES RNA as claimed in claim 2 is characterized in that in the purification step of virus-like particle, prepares 4 kinds of solutions of different gradients of sucrose density, and sucrose density is respectively 15%, 25%, 35%, 45% solutions. 4. the application of the virus-like particle containing O-type foot-and-mouth disease virus IRES RNA as claimed in claim 1 in RNA virus detection.

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