CN102559616B - Norovirus RNA fragment-containing pseudoviral particle and preparation method thereof - Google Patents

Abstract

The invention discloses a norovirus RNA fragment-containing pseudoviral particle and a preparation method thereof. The pseudoviral particle is a spherical RNA-protein complex which is prepared by coating a norovirus RNA with an MS2 bacteriophage coat protein. The pseudoviral particle is prepared by the following steps: designing and manually synthesizing a primer and obtaining a target gene MS2 by a by PCR (polymerase chain reaction) method; connecting the target gene MS2 to a plasmid pET-28b(+) to obtain a recombinant plasmid pET-28b/MS2; connecting the recombinant plasmid pET-28b/MS2 with a NV fragment to obtain a recombinant plasmid pET-28b/MS2/NV; guiding the obtained recombinant plasmid pET-28b/MS2/NV into escherichia coli for prokaryotic expression; and precipitating a virus-like particle by a polyethylene glycol method, wherein the obtained virus-like particle is the norovirus RNA fragment-containing pseudoviral particle. The pseudoviral particle can be used as a standard product and a quality control product for RT-PCR detection, is non-infectious, safe, reliable, and good in stability, and has an RNase-resistant characteristic.

Description

A kind of pseudovirion containing the norovirus RNA fragment and preparation method thereof

technical field

The present invention relates to norovirus, is a kind of pseudovirion containing the norovirus RNA fragment and preparation method thereof specifically.

Background technology

Norovirus (Norovirus, NoV) be by American scholar Kapikin, at first adopted Iem Methods in 1972, from the stool sample that betides the gastro-enteritis patient that nineteen sixty-eight breaks out in the school in Ohio, USA Norwalk area, detect, and to find ground title called after norwalk group viruses (Norwalk-like virus, NLvS).Nineteen ninety-five, ICTV's the 6th subseries is formally listed norovirus in Caliciviridae in reporting, promise belongs to as poison.The diarrhoea that norovirus causes, have the characteristics such as morbidity is anxious, velocity of propagation is fast, coverage is wide.All can infect the whole year, often causes that collective breaks out, and infecting object is mainly school-ager and adult, and the major part of breaking out of this disease has seasonality, presents autumn and winter occurred frequently.Norovirus is one of most important cause of disease caused in global range acute human gastro-enteritis, can cause serious food safety and public health problem.It is relevant to the viral diarrhea that surpasses in the world 90% that norovirus infects.

The epidemic characteristic that norovirus infects: 1, low infective dose (<10 virus particle); 2, the longer toxin expelling time (viral shedding), even patient still can discharge virus after transference cure, and this has increased the risk that secondary is propagated; 3, still keep stable under higher chlorine solution and wider temperature (freezing-60 ℃) condition; 4 superinfections may be owing to lacking cross-protection between the norovirus strain and lacking long-acting immunity.

According to the difference of coat protein sequence, norovirus can be divided into 5 genomes (genogroups), and the virus strain that wherein can infect the people belongs to GI, GII and GIV, and on the whole, GII is the advantage strain.Wherein, GII-4 is preponderant genotype.Before the eighties in 20th century, the diagnostic method of norovirus mainly contains direct electron microscopy and immunoelectron microscopic method, radioimmunology (RIA), enzyme immunoassay (EIA) etc.Due to these method susceptibility with specificity is lower and material source is limited, be difficult to widespread use.After the nineties in 20th century, along with completing of the complete genome sequence of norovirus order-checking, in succession set up multiple sensitive, special diagnostic method.(1) ELISA method.Utilize gene engineering method to be expressed the recombinant capsid protein of norovirus strain, for detection and the seroepidemiology research of norovirus provides simple and feasible method.But due to not between homophyletic capsid protein antigen there is sizable variability, this has reduced specificity and the susceptibility of this method, and material source is limited, has limited the use of this method.(2) RT-PCR method.The method be at present for norovirus the most responsive, also be detection method the most widely.Because can check order after detection, so particularly useful at the molecule epidemic disease-ology research for determining the source of infection and the consecutive hours distinguished between similar breaking out.Recently, fluorescent quantitation RT-PCR is owing to having sensitive and quick advantage, is applied to the detection of gastro-enteritis a large amount of faecal samples while breaking out.But due to viral variation and restructuring, for primer and the probe of specific strain, can not detect all strains, usually cause undetected, so can detect the primer of institute's toxic strain and the design of probe just seems very crucial.Due to the unstable of the potential infectivity of norovirus and RNA thereof, so development is significant for the research and development of norovirus RT-PCR test kit without infectivity and stable quality control standard product.

The MS2 Phagus is in positivity single stranded RNA spherical virus, and genome consists of 3569 Nucleotide, four kinds of protein molecules such as coding assembly protein, envelope protein, replicase protein and crack protein.Phage is comprised of 180 envelope protein monomers, 1 maturing enzyme protein molecular and a geneome RNA.Research finds that MS2 phage envelope protein and operon RNA sequence have specificity to interact, and this effect can cause the assembling of phage ghost,, phage genome RNA is packaged in coating simultaneously.If the cDNA of a certain viral RNA is connected in to MS2 phage envelope protein gene cDNA downstream, to obtain the specific virus rna transcription basis with phage operon RNA sequence after transcribing, operon RNA sequence just can cause the phage envelope protein and be assembled into shell, and the phage genome that will carry a certain viral RNA sequence is packaged in coating, form phage virus-like particle.When the actual implementation plasmid expression vector, phage assembly protein, envelope protein gene sequence cDNA clone need be connected in to expression vector promotor downstream, like this, after abduction delivering, expressed envelope protein is not only as the constituent of virus-like particle, and can be used as the genetic expression regulatory factor and the pac signal is regulated the expression process, make transcribing of the expression of envelope protein and RNA harmonious, now, assembling gained phage sample particle has ripe characteristic, thereby has the rnase resistance.

Summary of the invention

The present invention is directed to the problems referred to above a kind of pseudovirion containing the norovirus RNA fragment and preparation method thereof is provided, this virion has the characteristics of anti-rnase, the standard substance and the quality control product that as norovirus RT-PCR, detect have good stability, be easy to storage and transport, the exploitation of the gene diagnosis kit of norovirus is had to stronger practical value.

A kind of pseudovirion containing the norovirus RNA fragment of the present invention, by a kind of RNA-protein complexes of MS2 bacteriophage coat protein parcel norovirus RNA; It contains RNA sequence in conservative gene district between norovirus ORF1 and ORF2, can express G 2 norovirus-4 type conserved region gene, has the characteristic of anti-rnase.

A kind of preparation method of the pseudovirion containing the norovirus RNA fragment, its concrete preparation process is:

The structure of step 1, pET-28b/MS2 recombinant plasmid

1) according to the MS2 phage gene sequence (NC_001417) in the GenBank database, the primer MS2A of design assembly protein and envelope protein gene and the MS2B pedestrian's work of going forward side by side is synthetic, and sequence is respectively: MS2A:5 ＇-AT cCATGGtCCTGCTCAACTTCCTGTCG-3 ＇, MS2B:5 ＇-GC gGATCCgTTAGTAGATGCCGGAGTTT-3 ＇, the underscore indication be the Nco I introduced respectively of upstream and downstream primer and bamthe H restriction enzyme site; Then the method by RT-PCR amplifies assembly protein and envelope protein gene (MS2 gene), and its base sequence is as SEQ ID NO:3;

2) adopt restriction enzyme ncoi and bamHi is double digestion assembly protein and envelope protein gene and plasmid pET-28b (+) respectively, after adopting glue to reclaim the test kit purifying, by the T4 DNA ligase, the two is connected, and obtains connecting product;

3) by above-mentioned steps 2) resulting connection product conversion importing intestinal bacteria, choose positive monoclonal and carry out PCR and double digestion evaluation, guarantee the exactness that recombinant plasmid pET-28b/MS2 builds ,-20 ^oc preserves and identifies correct recombinant plasmid pET-28b/MS2;

The structure of step 2, pET-28b/MS2/NV recombinant plasmid

1) the positive-sense strand NV1 of synthetic norovirus gene fragment and antisense strand NV2, its gene order is as sequence table sequence 4 and sequence 5; Respectively positive-sense strand 5 ' end adds the BamH1 restriction enzyme site, antisense strand 5 ' end adds hindthe III restriction enzyme site; Get the positive-sense strand NV1 of equimolar amount and the antisense strand NV2 renaturation of being annealed after synthetic, put 94 ℃ of water-bath 10min, be cooled to room temperature, make it be annealed into two strands, make the norovirus gene fragment, i.e. NV gene, its base sequence is as SEQ ID NO:6.

2) adopt restriction enzyme bamHi and hindiII is double digestion recombinant plasmid pET-28b/MS2 and above-mentioned steps 1 respectively) prepared norovirus gene fragment; After adopting glue to reclaim test kit difference purifying, the two is connected with the T4 DNA ligase, obtain connecting product;

3) above-mentioned steps 2) say that the connection product of preparation transforms the importing intestinal bacteria, choose positive single bacterium colony and extract recombinant plasmid, carry out PCR respectively and three enzymes are cut evaluation, to confirm that the pET-28b/MS2/NV recombinant plasmid correctly builds, and obtained the positive bacterium colony that contains the pET-28b/MS2/NV recombinant plasmid;

Step 3, abduction delivering and purifying

Above-mentioned steps two is identified to the positive bacterium colony that correctly contains the pET-28b/MS2/NV recombinant plasmid, recoat the LB solid medium flat board be distributed in containing kantlex, picking list colony inoculation, to containing in the LB liquid nutrient medium of kantlex, is cultured to A _600nmbe 0.5～1.0 o'clock, add IPTG to induce rear collection thalline; By the thalline Eddy diffusion after collecting in TE solution, after adding the N,O-Diacetylmuramidase dissolution of bacteria, add again rnase and deoxyribonuclease dissolving DNA and RNA, adopt polyethylene glycol precipitation to be operated, the virus-like particle thing extracted, be the pseudovirion of the present invention containing the norovirus RNA fragment, it is dissolved in to TE solution, be placed in 4 ℃ of storages.

beneficial effect

A kind of virus-like particle containing norovirus RNA of the present invention, include G 2 norovirus-4 type conserved region gene fragment, there is the following aspects through this virus-like particle of verification experimental verification: 1, without infectivity, safe and reliable, when preparation and use, personnel and environment are safe from danger; 2, good stability, the MS2 virus-like particle has the characteristics of the enzyme of anti-RNA, and the standard substance and the quality control product that as norovirus RT-PCR, detect have good stability, are easy to storage and transport; 3, RNA in pathogenic agent is had to good dummy activity; Because expression product is phage sample particle, there is the virion characteristic, can the simulated virus particle, omnidistance monitoring experiment process, avoid occurring false negative.A kind of virus-like particle containing norovirus RNA of the present invention can be used as quality control product and/or the standard substance that virus detects, and for the exploitation of the gene diagnosis kit of norovirus, has very strong practical value.

The accompanying drawing explanation

Fig. 1 is MS2 PCR product electrophorogram; Number in the figure: 1 is that MS2 pcr amplification product, M are DNA molecular amount standard.

Fig. 2 is pET-28b/MS2 plasmid double digestion electrophorogram; Number in the figure: 1 is that pET-28b/MS2 double digestion, M are DNA molecular amount standard.

Fig. 3 is recombinant plasmid pET-28b/MS2/NV tri-restriction enzyme digestion and electrophoresis figure; Number in the figure: 1 cut for recombinant plasmid three enzymes, M is DNA molecular amount standard.

Fig. 4 is the qualitative test figure as a result of virus-like particle; Number in the figure: M is DNA molecular amount standard, and 1 negative contrast, 2 positive contrasts, 3,4 are that after reverse transcription, amplified production, 5,6 is without the direct amplified production of reverse transcription.

Fig. 5 is virus-like particle rnase challenge trial figure as a result.

Fig. 6 is virus-like particle stability test figure as a result.

embodiment

The embodiment of the present invention relates to following bacterial classification, reagent and instrument:

Intestinal bacteria MS2 phage (ATCC 15597B1) is purchased from U.S. ATCC company; PET-28b (+) plasmid/e. coli bl21 (DE3) bacterial strain is purchased from U.S. Novagen company; Intestinal bacteria TGl bacterial strain is purchased from Stratagen company; RNA extraction test kit, plasmid extraction purification kit, DNA gel reclaim test kit, ThermoScript II M-MLV is U.S. Promega company product; Restriction endonuclease bamHi, ncoi and hindiII is purchased from sky, Beijing root biochemical technology company purchased from Dalian precious biotinylated biomolecule engineering corporation, T4 DNA ligase purchased from Shanghai Sheng Gong bio-engineering corporation, Taq archaeal dna polymerase; Primer and type G 2 norovirus conserved regions oligonucleotide fragment are synthetic by Shanghai Sheng Gong bio-engineering corporation, the American AB I PE7700 of company type quantitative real time PCR Instrument is used in the pcr amplification operation.

A kind of preparation method of the pseudovirion containing the norovirus RNA fragment, its concrete preparation process is:

The structure of step 1, pET-28b/MS2 recombinant plasmid

1) according to the MS2 phage gene sequence (NC_001417) in the GenBank database, the primer MS2A of design assembly protein and envelope protein gene and the MS2B pedestrian's work of going forward side by side is synthetic, and sequence is respectively: MS2A:5 ＇-AT cCATGGtCCTGCTCAACTTCCTGTCG-3 ＇, MS2B:5 ＇-GC gGATCCgTTAGTAGATGCCGGAGTTT-3 ＇, the underscore indication be the Nco I introduced respectively of upstream and downstream primer and bamthe H restriction enzyme site; Then the method by RT-PCR amplifies assembly protein and envelope protein gene (MS2 gene), and its base sequence is as SEQ ID NO:3; The method amplification assembly protein of RT-PCR and the concrete grammar of envelope protein gene are, at first prepare inverse transcription reaction liquid, contain 1 * reverse transcription damping fluid, 50U RNA enzyme inhibitors, 100U MLV ThermoScript II, 50mmol N6 random primer, get the MS2 phage rna (after adopting RNA extraction test kit to be extracted according to its specification sheets, adding aseptic ultrapure water is that 100:1 is diluted according to the volume ratio of water and RNA) 1 μ L is as template, add in inverse transcription reaction liquid, mix, hatch after 1h for 37 ℃ and get 2.0 μ l and carry out pcr amplification.The PCR reaction system is containing 1.5mmol/L MgCl ₂, 100 μ mol/L dNTP, 3U Taq archaeal dna polymerase, 10 μ L reverse transcription products and primer MS2A and each 10pmol of MS2B, reaction parameter is 94 ℃ of denaturation 300s, 94 ℃ of 60s then, 55 ℃ of 60s, 72 ℃ of 180s, 300s are extended in latter 72 ℃ of 35 circulations.The PCR product reclaims purifying after 1% agarose gel electrophoresis, obtains and the goal gene (as shown in Figure 1) of expecting that clip size (1.7kb) conforms to;

2) adopt restriction enzyme Nco I and BamH I double digestion above-mentioned steps 1 respectively) the MS2 gene and the plasmid pET-28b (+) that obtain, after adopting glue to reclaim the test kit purifying, by the T4 DNA ligase, the two is spent the night in 16 ℃ of connections, obtain connecting product;

3) by above-mentioned steps 2) resulting connection product importing intestinal bacteriathe TG1 competent cell, the LB flat board of coating kalamycin resistance, picking positive colony after 37 ℃ of overnight incubation, through bacterium colony PCR identify with ncoi, bamHthe I double digestion is identified, obtains recombinant plasmid pET-28b/MS2.After MS2 gene and carrier pET-28b (+) restructuring, recombinant plasmid pET-28b/MS2 warp ncoi and bamHthe I double digestion, expection band (as shown in Figure 2) appears in electrophoresis detection.

The structure of step 2, pET-28b/MS2/NV recombinant plasmid

1) the positive-sense strand NV1 of synthetic norovirus gene fragment and antisense strand NV2, its base sequence is as SEQ ID NO:4 and SEQ ID NO:5; Respectively positive-sense strand 5 ' end adds bamH1 restriction enzyme site, antisense strand 5 ' end adds hindthe III restriction enzyme site; With distilled water, by type G 2 norovirus conserved region gene fragment NV1 and NV2 dilution, be 100 μ mol/L, after getting the positive-sense strand NV1 and antisense strand NV2 mixing of equimolar amount, put 94 ℃ of water-bath 10min, then slowly cool to room temperature, make it be annealed into two strands, make the norovirus gene fragment, i.e. NV gene, its base sequence is as SEQ ID NO:6.

2) pET-28b ∕ MS2 is through restriction enzyme bamHcarry out 1% agarose gel electrophoresis after I, Hind III double digestion, reclaim large fragment, be connected acquisition pET-28b/MS2 recombinant plasmid by the T4 ligase enzyme with the NV gene and be connected product with the NV gene.

3) above-mentioned steps 2) prepared connection product conversion e. coli bl21 (DE3) competent cell, coating kalamycin resistance LB flat board, 37 ℃ of overnight incubation, picking list colony inoculation spends the night to 37 ℃ of shaking culture in 3mL LB substratum, extract recombinant plasmid dna, pET-28b/MS2/NV recombinant plasmid warp bamHi, ncoi and hindafter III three enzymes are cut, cut out the NV fragment (Fig. 3) of MS2 and 131bp.

Step 3, abduction delivering and purifying

Bacterium by the above-mentioned evaluation positive, recoat the LB solid medium flat board be distributed in containing kantlex, 37 ℃ of overnight incubation, picking list colony inoculation spends the night to 37 ℃ of shaking culture in 3mL LB substratum, then the 1L that transfers is containing in the LB liquid nutrient medium of kantlex, and 37 ℃ of shaking culture are to A _600nmbe 0.5～1.0 o'clock, adding IPTG is 0.4mmol/L to final concentration, and 5h, centrifugal collection thalline are cultivated in continuation.Thalline is suspended in TE solution, add N,O-Diacetylmuramidase to 10mg/L, room temperature is placed 20min, centrifugal removal precipitation, add rnase and deoxyribonuclease I to supernatant liquor, make both be 1mg/L by final concentration, after 37 ℃ of incubation 30min according to molecular cloning experiment guide (third edition) [M]. Science Press, polyethylene glycol precipitation in 2002 editions is operated, the virus-like particle thing extracted, be the pseudovirion of the present invention containing the norovirus RNA fragment, it is dissolved in TE solution, be placed in 4 ℃ of storages.

The identification experiment of step 4, virus-like particle

1) PCR identification experiment

Get the 12ml virus-like particle suspension of expression, purifying, carry out 10 ⁶, 10 ⁷, 10 ⁸, 10 ⁹doubly dilution, one group is directly carried out the PCR reaction without reverse transcription; After another group sample reverse transcription, then carry out the PCR amplification, the electrophoresis detection amplification.After sample dilution, wherein one group is directly carried out the PCR reaction without reverse transcription, and detected result is negative (the 5th, 6 swimming lanes in Fig. 4) all, show to prepare in virus-like particle suspension and virus-like particle in, all do not have the DNA template to pollute; After another group sample reverse transcription, then carry out the PCR amplification, detected result positive (the 3rd, 4 swimming lanes in Fig. 4), show that type G 2 norovirus conserved region gene fragment is reconstituted in virus-like particle, and template exists with the RNA form.

2) virus-like particle rnase challenge trial

Get virus-like particle 10 ⁶20 parts of times extent of dilution samples, each 50 μ l, every 10 parts one group.One group is directly carried out sample preparation, reverse transcription and fluorescent quantitative PCR.Another group adds the rnase of 1mg/L, and room temperature is placed 60min and carried out sample preparation, reverse transcription and fluorescence quantitative PCR detection again, relatively the Ct value of two groups.Result as shown in Figure 5, process virus-like particle with rnase, with untreated control group (each 10 parts), parallel RNA extraction, reverse transcription and the fluorescence quantitative PCR detection of carrying out, two groups of detected results are basically identical, show the virus-like particle Degradation that can resist rnase of preparation.

3) virus-like particle stability experiment

Get virus-like particle 10 ⁶times extent of dilution sample, place 5d, 10d, 15d, 20d, 25d, 30d respectively at 37 ℃, then carries out sample preparation, reverse transcription and fluorescence quantitative PCR detection, relatively the variation of its Ct value.As shown in Figure 6,37 ℃ of Ct value variation tendencies of placing after different time show the stability experiment result of virus-like particle, and the virus-like particle of preparation at least can store 30 days at 37 ℃, and stability is better.

Intestinal bacteria MS2 phage of the present invention, for Bacillus coli communis MS2 phage, can buy and obtain by market, such as receiving and create purchases such as joining Bioteknologisk Institut from U.S. ATCC company, Beijing North;

Described pET-28b (+) plasmid is prokaryotic expression carrier, can buy and obtain by market;

Described e. coli bl21 (DE3) bacterial strain, intestinal bacteria TGl bacterial strain is Bacillus coli communis, all can buy and obtain by market;

The composition of described TE solution: the Tutofusin tris-hydrochloric acid that contains 10mmol in every liter of Tutofusin tris-edta solution, the ethylenediamine tetraacetic acid (EDTA) of 5mmol, surplus is water, the pH value is 7.5;

The composition of described LB solid medium: contain by weight percentage 1% Tryptones, 0.5% yeast extract, 1% sodium-chlor in the LB liquid nutrient medium, 1.8% agar, surplus is water;

The composition of described LB liquid nutrient medium: contain by weight percentage 1% Tryptones, 0.5% yeast extract, 3% sodium-chlor in the LB liquid nutrient medium, surplus is water.

SEQUENCE LISTING

<110 > University Of Science and Technology Of He'nan

<120 > a kind of pseudovirion containing the norovirus RNA fragment and preparation method thereof

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Claims (4)

1. the preparation method containing the pseudovirion of norovirus RNA fragment, it is characterized in that: its concrete preparation process is:

The structure of step 1, pET-28b/MS2 recombinant plasmid:

1) primer of the MS2 gene of design pcr amplification MS2 phage, the pedestrian's work of going forward side by side is synthetic, and its sequence is respectively: upstream primer: 5 '-AT cCATGGtCCTGCTCAACTTCCTGTCG-3 ',

Downstream primer: 5 '-GC gGATCCgTTAGTAGATGCCGGAGTTT-3 ', the underscore indication is that the upstream and downstream primer is introduced respectively ncoi and bamHrestriction enzyme site; Then the method by RT-PCR amplifies the MS2 gene, and its base sequence is as SEQ ID NO:3;

2) adopt restriction enzyme ncoi and bamHi is double digestion MS2 gene and plasmid pET-28b (+) respectively, after purifying, is connected, and obtains connecting product;

3) by above-mentioned steps 2) resulting connection product conversion importing intestinal bacteria, choose positive single bacterium colony and carry out PCR and double digestion evaluation, guarantee the exactness that recombinant plasmid pET-28b/MS2 builds, preserve the recombinant plasmid pET-28b/MS2 that evaluation is correct for-20 ℃;

The structure of step 2, pET-28b/MS2/NV recombinant plasmid:

1) the positive-sense strand NV1 of synthetic norovirus NV gene fragment and antisense strand NV2, its base sequence is as SEQ ID NO:4 and SEQ ID NO:5; 5 ' the end at positive-sense strand adds respectively bamHthe I restriction enzyme site, hold and add at 5 ' of antisense strand hindthe III restriction enzyme site; The positive-sense strand NV1 and the antisense strand NV2 that get after equimolar amount synthetic add same PCR pipe, mix and are placed on 94 ℃ of water-bath 10min, are cooled to room temperature, make it be annealed into two strands, have synthesized the NV gene fragment;

2) adopt restriction enzyme bamHi and hindiII is double digestion recombinant plasmid pET-28b/MS2 and above-mentioned steps 1 respectively) prepared NV gene fragment, after purifying, connected, obtain connecting product;

3) above-mentioned steps 2) prepared connection product conversion importing intestinal bacteria, choose positive single bacterium colony and extract recombinant plasmid, carry out respectively PCR evaluation and three enzymes and cut evaluation, to confirm that the pET-28b/MS2/NV recombinant plasmid correctly builds, and obtain the positive bacterium colony that contains the pET-28b/MS2/NV recombinant plasmid;

Step 3, abduction delivering and purifying

Above-mentioned steps two is identified to the positive bacterium colony that correctly contains the pET-28b/MS2/NV recombinant plasmid, recoat the LB flat board be distributed in containing kantlex, after cultivating, picking list colony inoculation, to containing in the LB liquid nutrient medium of kantlex, is cultured to A _600nmdetected value be 0.5～1.0 o'clock, add sec.-propyl-β-D-sulfo-galactopyranoside, making its final concentration is 0.4mmol/L, continue to cultivate 5h, centrifugal collection thalline; Thalline is suspended in TE solution, adds N,O-Diacetylmuramidase, making its final concentration is 10mg/L, and room temperature is placed 20min, and centrifugal removal precipitation, add rnase and deoxyribonuclease I to supernatant liquor, makes both be 1mg/L by final concentration, 37 ^o1 hour dissolving DNA of C water-bath and RNA, then adopt polyethylene glycol precipitation to be operated, and the virus-like particle thing extracted is the pseudovirion of the present invention containing the norovirus RNA fragment, and it is dissolved in to the TE damping fluid, is placed in 4 ℃ of storages.

2. as claim 1 preparation method containing the pseudovirion of norovirus RNA fragment, it is characterized in that: described MS2 gene is for expressing the gene of MS2 phage assembly protein and envelope protein.

3. as claim 1 preparation method containing the pseudovirion of norovirus RNA fragment, it is characterized in that: 131 bases of the junction region that the sequence of described NV gene fragment is the most conservative ORF1 of G II-4 type norovirus gene order and ORF2, its base sequence is as SEQ ID NO:6.

One kind by the described method of claim 1, make containing the pseudovirion of norovirus RNA fragment, it is characterized in that: the pseudovirion containing the norovirus RNA fragment is a kind of RNA-protein complexes by MS2 bacteriophage coat protein parcel norovirus RNA; It contains RNA sequence in conservative gene district between G II-4 type norovirus ORF1 and ORF2, can express G 2 norovirus-4 type conserved region gene, has the characteristic of anti-rnase.

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