CN103923887A - Pseudoviral particle containing hepatitis e virus RNA (Ribose Nucleic Acid) fragment and preparation method thereof - Google Patents
Pseudoviral particle containing hepatitis e virus RNA (Ribose Nucleic Acid) fragment and preparation method thereof Download PDFInfo
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Abstract
The invention provides a pseudoviral particle containing a hepatitis e virus (HEV) RNA (Ribose Nucleic Acid) fragment and a preparation method thereof. The pseudoviral particle is an RNA-protein complex formed by coating hepatitis e virus RNA by an MS2 bacteriophage coat protein, and the RNA-protein complex is spherical. The preparation method comprises the steps of designing and artificially synthesizing a primer to obtain a target gene MS2 through a PCR (Polymerase Chain Reaction) method, connecting the target gene MS2 to a plasmid pET (polyethylene glycol terephthalate)-28b(+) to obtain a recombinant plasmid pET-28b/MS2/HEV, guiding the recombinant plasmid into escherichia coli for prokaryotic expression, and settling a virus-like particle by adopting a polyethylene glycol method, wherein the virus-like particle is the pseudoviral particle containing the hepatitis e virus RNA fragment. The pseudoviral particle provided by the invention can be used as a standard substance and a quality control product of general HEV gene I-IV type RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection, and has the characteristics no infectivity, safety, reliability, high stability and resistance to ribonuclease.
Description
Technical field
The present invention relates to hepatitis E virus, is a kind of pseudovirion containing RNA of hepatitis E virus fragment and preparation method thereof specifically.
Background technology
Hepatitis E virus (Hepatitis E virus, HEV) is a kind of hepatitis virus of propagating through digestive tube, blood.This virus infection spectrum is very extensive, and current certified infection host, except people, also has other many animals, as pig, monkey, chicken etc.HEV has four genotype, and I type and IV type only limit to person-to-person propagation, and III type and IV type are to intersect and propagate between people and animals; People infects clinical patients and mostly is light medium-sized hepatitis, normal is self limiting, but suffering from the hepatitis E case fatality rate that is in a bad way, pregnant woman can reach 20%, therefore, HEV is defined as newfound a kind of infecting both domestic animals and human disease pathogen in this century by the World Health Organization (WHO), and its global public health security problem causing receives the concern of various countries day by day.The various HEV detection techniques of corresponding exploitation and method also become very necessary and urgent.
HEV be a kind of diameter be about 27nm without coating single strand plus RNA virus.The about 7.5kb of HEV genome total length, contains three open reading frame (ORF) 5 ' and 3 ' between non-coding region, is followed successively by ORF1, ORF2 and ORF3.Because the amino acid of viral nucleic acid and coding thereof has the features such as regional distribution widely and certain genetic heterogeneity having on the basis of high homology, one therefore, in molecular Biological Detection clinically usually taking ORF1, ORF2 and ORF3 as detecting target gene.
Taking HEV detection of nucleic acids as example, generally have following three key steps to need positive reference:
1, from sample, extract HEV RNA, comprising: outer virionic membrane, nucleocapsid are opened or be broken, and nucleic acid discharges.
2, HEV RNA is reversed record for cDNA.
3, cDNA mixes with PCR reaction solution and DNA polymerase, increases and obtain signal in PCR instrument.
Existing positive reference, mainly contains following several source: patient or infected animal positive serum; In body or in-vitro transcription RNA; Plasmid or chemosynthesis.
In all positive references, should be the natural sample (as the blood of patient or infected animal, ight soil treatment solution, cell culture fluid) containing this HEV virus the most accurately and reliably, homogeneity can fully ensure its positive reference as aforementioned three steps, but its defect is that natural sample source is limited, and the potential hazard that exists natural viral to disseminate.
Overcome natural viral and originate less and have communicable shortcoming, generally can solve by isolated culture, import suitable artificial culture host cell system by HEV, restructuring HEV particle can produce and secrete to culture supernatant from cell.But this virus infection is composed the low and crucial culturing step of narrow infection titer only by a few experiments chamber is had; On the other hand, this HEV mode of production may remain in some defects, as high in cost, viral load still lower, structure is not natural, poor repeatability etc.
The HEV RNA of in-vitro transcription, because it is the same with index to be measured in sample, is RNA, therefore can be used as the positive reference of reverse transcription; But, because it does not possess virus coat, be therefore not suitable as the positive reference of extracting viral RNA operation from sample.
With plasmid, as positive reference, its advantage is that to prepare purifying easy, but because plasmid is closed hoop superhelix double chain form, non-single linear, is not RNA, and therefore it can only serve as PCR reference, and cannot serve as, RNA extracts and the reference of reverse transcription.
Prepare the positive reference of RNA as for chemical synthesis, very high with current technical costs, also can easily degrade because there is no the protection of nucleocapsid, film, therefore up to the present there is no the report of practical application.
Artificial pseudovirus is that the particular sequence of object rna virus cdna is cloned on the expression vector that contains phage gene, produce the RNA segment being coated with by phage capsid protein by abduction delivering, this new RNA Quality Control technology of preparing claims again armoring RNA (Armored RNA) technology (Pasloske B L, et al.Armored RNA technology for production of ribonuclease-resistant viral RNA controls and standards[J] .J Clin Microbiol, 1998,36 (12): 3590-3594).This technology has solved many drawback problems that traditional RNA quality control product exists (poor stability exists Biosafety hidden danger or do not reach the object etc. of monitoring nucleic acid extraction and reverse transcription whole process).
The conventional pseudovirus carrier that builds at present pseudovirion is mainly made up of with the expression vector with high efficient expression the gene order of the MS2 bacteriophage coat protein of can encoding.MS2 phage is strand positivity RNA viruses, genome total length 3569 base pairs, four kinds of protein molecules such as encoding mature zymoprotein, envelope protein, replicase protein and crack protein.A phage virus particle is by 180 envelope protein monomers, a part maturing enzyme albumen, the icosahedron of parcel a part geneome RNA composition (Jia Panxing. phage molecular biology-ABC and technical ability [M]. Beijing: Science Press, 2001,2-5).In the research of MS2 bacteriophage coat protein, find that coat protein and phage replication enzyme 5 ' end 19 base loop-stem structure RNA sequences have specificity to interact, this effect can cause the assembling of phage ghost, again phage genome RNA is packaged in coating simultaneously.Researchist finds that after packaging site, introducing non-phage gene sequence also can cause packaging.Therefore, if by exogenous gene cloning in MS2 bacteriophage coat protein gene coded sequence downstream, and insert terminator in its downstream, after transcribing, obtain the foreign gene rna transcription basis with phage operon RNA sequence, protein coat is expressed and be assembled into abduction delivering process pnagus medius coat protein, and the phage genome that carries foreign gene is wrapping in coating, form phage virus-like particle.Because the coat protein assembling process of single expression is immature, can not obtain the peplos of resistance to RNase, investigator by the gene coded sequence of the maturing enzyme albumen of MS2 phage and coat protein with and the genome part corresponding cDNA sequence of 5 ' non-coding sequence that comprises gene regulatory elements sequence be all connected to expression vector promotor downstream, obtain maturing enzyme albumen and coat protein through abduction delivering, under the synergy of maturing enzyme and phage genome RNA, coat protein can be assembled into ripe pseudovirion shell, and there is characteristic (the Walker Peach C R of resistance to RNase effect, et a1.Ribonuclease resistant RNA controls (Armored RNA) for reverse transcription-PCR, branched DNA, and genotyping assays for hepatitis C virus[J] .Clin Chem, 1999, 45 (12): 2079-2085).So utilize the prepared artificial pseudovirus of armoring RNA (Armored RNA) technology being applied to detection of nucleic acids reagent positive control or nucleic acid standard substance, there is high specificity, highly sensitive, the advantage that stability is strong.
Summary of the invention
Based on above-mentioned weak point, the object of the present invention is to provide a kind of pseudovirion containing RNA of hepatitis E virus fragment and preparation method thereof.
The object of the invention is to realize by the following method:
1, contain a pseudovirion for RNA of hepatitis E virus fragment, as shown in sequence table SEQ ID NO.1;
2, contain a preparation method for the pseudovirion of RNA of hepatitis E virus fragment, concrete preparation process is as follows:
(1) structure of pET-28b/MS2 recombinant plasmid
(1) structure of pET-28b/MS2 recombinant plasmid
(1.1) primer pair of the MS2 gene of design pcr amplification MS2 phage,
Upper primer Pri-MS2F: as shown in sequence table Seq ID No.2,
Lower primer Pri-MS2R: as shown in sequence table Seq ID No.3;
Then amplify MS2 gene clone by the method for RT-PCR, as shown in sequence table SEQ ID NO.4;
(1.2) adopt restriction enzyme Nco I and BamH I double digestion MS2 gene and plasmid pET-28b (+) respectively, after purifying, connect, obtain connecting product;
(1.3) connection product step (1.2) being obtained is transformed into intestinal bacteria competence DH5 α, extracts plasmid performing PCR and the double digestion qualification of going forward side by side, by recombinant plasmid correct qualification in-20 DEG C of preservations;
(2) structure of pET-28b/MS2/NV recombinant plasmid
(2.1) the pcr amplification primer pair of synthetic hepatitis E virus HEV ORF2/3 gene fragment,
Upper primer Pri-ORF2/3F: as shown in sequence table SEQ ID NO.5,
Lower primer Pri-ORF2/3R: as shown in sequence table SEQ ID NO.6,
Then amplify part ORF2/3 gene clone by the method for RT-PCR, as shown in sequence table SEQ ID NO.7;
(2.2) adopt restriction enzyme BamH I and HindIII double digestion ORF2/3 gene clone plasmid and plasmid pET-28b/MS2 respectively, after purifying, connect, obtain connecting product;
(2.3) connection product prepared step (2.2) is transformed and import intestinal bacteria competence DH5 α, extract plasmid and carry out single double digestion qualification, by recombinant plasmid called after pET-28b/MS2/HEV correct qualification;
(3) abduction delivering and purifying
By the positive bacterium colony of recombinant plasmid pET-28b/MS2/HEV correct qualification in step (2), be inoculated into containing in the LB liquid nutrient medium of kantlex, while being cultured to OD600 value for 0.5-1.0, add inductor IPTG to final concentration be 0.5mmol/L, continue to cultivate 5h, centrifugal collection thalline; Be resuspended in appropriate TE solution, adding N,O-Diacetylmuramidase to final concentration is 10mg/L, mixes rear 37 DEG C of water-bath effect 30min, and centrifugal removal precipitation adds rnase and deoxyribonuclease I to final concentration to be 1mg/L, 37 DEG C of water-bath effect 1h in supernatant liquor.Then adopt polyethylene glycol precipitation concentrating virus sample particulate matter, be the pseudovirion of the present invention containing RNA of hepatitis E virus fragment, be dissolved in TE damping fluid 4 DEG C of preservations;
3, described above a kind of preparation method of the pseudovirion containing RNA of hepatitis E virus fragment, described MS2 gene is effable MS2 phage maturing enzyme albumen and the gene of envelope protein and the gene coded sequence of part crack protein and replicative enzyme.
4, described above a kind of preparation method of the pseudovirion containing RNA of hepatitis E virus fragment, the sequence of described HEV gene fragment is the overlapping 5225-5510 base place in the most conservative ORF2 district of HEV G1-4 type and ORF3 district, as shown in sequence table SEQ ID NO.6.
5, described above a kind of preparation method of the pseudovirion containing RNA of hepatitis E virus fragment, the sequence of described HEV gene fragment is the overlapping 5225-5510 base of the most conservative ORF2 district of HEV G1-4 type and ORF3, and manually design and introduced 24 T bases in its afterbody, the PolyA producing while transcribing can be used as the special complementary land of universal primer Oligo (dT).
6, described above a kind of preparation method of the pseudovirion containing RNA of hepatitis E virus fragment is a kind of RNA-protein complexes by a MS2 phage glutelin parcel hepatitis virus RNA containing the pseudovirion of RNA of hepatitis E virus fragment; It contains HEV gene G1-4 type ORF2 conserved sequence district RNA sequence, and this RNA-protein complexes has the characteristic of resistance to rnase.
Pseudovirus carrier pET-28b/MS2/HEV provided by the invention has following features for the preparation of HEV pseudovirion:
1, versatility is good, is the overlapping part of the common conserved regions of nucleic acid, i.e. ORF2 and the ORF3 of HEV genotype I-IV, and therefore, this HEV pseudovirion can be as general positive quality control reference in the time detecting the HEV of genotype I-IV; In addition, manually-injected 24 the Poly-A poly-A tails of HEV gene order afterbody of HEV pseudovirion parcel, can simulate the special complementary land of RNA viruses cDNA Oligo (dT) primer when synthetic, therefore, in the time carrying out RNA viruses reverse transcription with Oligo (dT) primer, there is good versatility.
2, good stability, resistance to nuclease, easily preservation.Due within HEV ORF2 geneome RNA is wrapped in the coat protein of pseudovirus, so can resist the effect of extraneous nuclease, be difficult for being degraded.Can preserve one month at ambient temperature, the lower shelf time of cold condition is longer, has solved RNA quality control product stability problem in use.
3, emulation greatly, in the complete monitoring process of nucleic acid preparation and qualification, the RNA-protein complexes structure of HEV pseudovirion is similar to natural viral, therefore virion etc. can be all to viral material, together through RNA extraction, the laggard performing PCR amplification of reverse transcription, well the testing process of target RNA viruses is carried out to omnidistance quality monitoring, ensured the reliability of testing data.
4, safe, HEV pseudovirion only has similar virus nucleocapsid, and parcel nucleic acid RNA does not have infectivity, lose viral the of self-replication capacity, so HEV pseudovirion, without infectivity, can not form injury to experimenter, also can not produce pollution effect by holy environment.
5, be easy to purifying, to be HEV RNA-protein complex at its product of protokaryon expression in escherichia coli due to pseudovirion, in the time of bacterial body cracking, can be discharged into rapidly in supernatant liquor, simple centrifugal bacterial chip can being removed afterwards, has improved the purification efficiency of pseudovirion.
Brief description of the drawings
Fig. 1 is that the enzyme of HEV pseudovirus carrier pET-28b/MS2/HEV is cut qualification result,
Wherein: 1 is DNA Marker1kbp Ladder; 2 is the single endonuclease digestion result of pET-28b/MS2/HEV recombinant plasmid; 3 is the double digestion result of pET-28b/MS2/HEV recombinant plasmid Nco I, HindIII; 4 is pET-28b/MS2/HEV recombinant plasmid BamH I, HindIII double digestion result; 5 is pET-28b/MS2/HEV recombinant plasmid Nco I, BamH I double digestion result; 6 is pET-28b (+) plasmid single endonuclease digestion result.
Fig. 2 is HEV pseudovirus carrier pET-28b/MS2/HEV structural pattern figure, represents respectively with Nco I/BamH I double digestion and be inserted into the MS2 gene order in pET-28b carrier and add 24 Poly-A tails with the HEV ORF2/3 conserved sequence of BamH I/HindIII double digestion in rectangle frame from left to right.
Fig. 3 is the RT-PCR qualification result of HEV pseudovirion,
Wherein: M is DNA Marker100bp Ladder; 1-7 is respectively 10
4to 10
10doubly HEV pseudovirion sample RT-PCR detected result after the purifying of dilution; 8-9 is respectively ddH
2o is RT-PCR and the PCR control test result of template; 10-16 is respectively 10
4to 10
10doubly after the purifying of dilution HEV pseudovirion sample without reverse transcription Direct PCR detected result.
Fig. 4 is rnase (RNase A) the resistance detected result of HEV pseudovirion,
Wherein: M is DNA Marker100bp Ladder; 1-4 is respectively the RT-PCR detected result of the HEV pseudovirion sample of processing through RNase A1 μ L/5 μ L/10 μ L/15 μ L; The 5 HEV pseudovirion sample RT-PCR detection contrasts of processing for not adding RNase A.
Fig. 5 is the susceptibility qualification result of HEV pseudovirion to temperature,
Wherein, M is DNA Marker100bp Ladder; 1-6 road is respectively 10 of every part of 250 μ L
4the HEV pseudovirion sample of dilution after purifying is placed in lower following condition: the result of carrying out RT-PCR detection after 60 DEG C/1h, 37 DEG C/10h, 25 DEG C/10d, 4 DEG C/30d ,-20 DEG C/90d and-80 DEG C/180d.
Embodiment
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit of the invention, the amendment that the inventive method, step or condition are done or replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art, in embodiment, biochemical reagents used are commercially available purchase and obtain.
The structure of pseudovirus carrier pET-28b/MS2 before embodiment 1HEV
1, the MS2 phage gene sequence (NC_001417) in synthetic GenBank database by synthetic sequences Design assembly protein and primer pair Pri-MS2F, the Pri-MS2R of envelope protein gene, and it is carried out to synthetic.
Pri-MS2F:5 '-GGT
cCATGGcCTTTCGGGGTCCTGCTCAACTT-3 ' (underscore represents that the restriction enzyme site of introducing is Nco I)
Pri-MS2R:5 '-GGT
gGATCCgCTGAGGGAATCGGGTTTCCATCTT-3 ' (underscore represents that the restriction enzyme site of introducing is BamH I)
2, pcr amplification, PCR product is connected to clone T carrier pMD18-T, builds pMD18-T/MS2 plasmid.
3, with fast enzyme Nco I and BamH I double digestion recombinant plasmid pMD18-T/MS2 and pET28b (+) expression vector (being this laboratory preserves) respectively.It is as follows that enzyme is cut system:
37 DEG C connect 15min.Respectively enzyme is cut to target product and carried out glue recovery purifying, product is called after MS2-T (N/B), pET28b (N/B) respectively.
4, the enzyme of recovery is cut to product MS2-T (N/B), pET28b (N/B) Ligation kit ligase enzyme test kit connects, linked system is:
16 DEG C connect 30min, then ice bath immediately.
5, will connect product and transform bacillus coli DH 5 alpha competent cell, coated plate, the qualification of picking mono-clonal bacterium colony, the bacterial strain name pET-28b/MS2 that checks order correct.
The structure of embodiment 2HEV pseudovirus carrier pET-28b/MS2/HEV
1, with reference to HEV (DQ279091.2) gene IV type ORF2 conserved regions sequence, direct gene is synthesized the overlapping ORF3 conserved regions of HEV ORF2 sequence.To Pri-ORF2/3F, Pri-ORF2/3R, make segment afterbody contain manually-injected 24 Poly-A tails according to above-mentioned synthetic gene order design synthetic primer.
Pri-ORF2/3F:5 '-
gGATCCcCTATGCTGCCCGCGCCACCG-3 ' (underscore represents that the restriction enzyme site of introducing is BamH I)
Pri-ORF2/3R:5'-
aAGCTTtTTTTTTTTTTTTTTTTTTTTTTTACGGCGAAGCCCCAGCT-3 ' (underscore is that the restriction enzyme site of introducing is HindIII).
2, pcr amplification, PCR product is connected to clone T carrier pMD18-T, builds DMD18-T/ORF2/3 plasmid.
3, with fast enzyme BamH I and HindIII double digestion recombinant plasmid pMD18-T/ORF2/3 and the front pseudovirus carrier pET-28b/MS2 of HEV respectively.It is as follows that enzyme is cut system:
37 DEG C connect 15min.Respectively enzyme is cut to target product and carried out glue recovery purifying, product is called after ORF2/3-T (E/X), pET-28b/MS2 (E/X) respectively.
4, the enzyme of recovery is cut to product MS2-T (N/B), pET28b (N/B) Ligation kit ligase enzyme test kit connects, linked system is:
16 DEG C connect 30min, then ice bath immediately.
5, will connect product and transform bacillus coli DH 5 alpha competent cell, coated plate, picking mono-clonal bacterium colony upgrading grain carries out enzyme with Nco I, HindIII and BamH I restriction enzyme afterwards and cuts qualification, and enzyme is cut result as shown in Figure 1.Enzyme is cut to the correct plasmid of qualification and check order, by plasmid called after pET-28b/MS2/HEV correct order-checking, recombinant plasmid structure as shown in Figure 2.
The preparation of embodiment 3HEV pseudovirus pET-28b/MS2/HEV.
1, the recombinant bacterium DH5 α/pET-28b/MS2/HEV that 100mL OD600 value is about to 0.5-1.0 carries out abduction delivering, and IPTG final concentration is 0.5mol/L, and 37 DEG C, 200r/m, 4h; Centrifugal 12000r/m, 5min, abandons supernatant, collects bacterial sediment.
2,10mL lysate (50mM Tris-HCl, pH8.5～9.0,2mM EDTA, 100mM NaCl, 0.5%Triton X-100, N,O-Diacetylmuramidase 10mg/L) resuspended bacterial sediment, 37 DEG C of water-bath effect 30min, ultrasonic disruption at 4 DEG C, condition is 400W, ultrasonic 5s, intermittently 10s, ultrasonication 5min altogether, after bacterium liquid is transparent 4 DEG C, the centrifugal 10min of 10000r/m, collects supernatant liquor.Add rnase and deoxyribonuclease I to final concentration to be 1mg/L, 37 DEG C of water-bath effect 1h, 4 DEG C, the centrifugal 10min of 10000r/m, collects supernatant liquor.Add NaCl to final concentration 0.5mol/L, add isopyknic 10%PEG6000, mix latter 4 DEG C and spend the night or place for some time; The centrifugal 30min of 8000r/m, collecting precipitation, precipitation is concentrated HEV pseudovirus pET-28b/MS2/HEV particulate samples.
The qualification of embodiment 4HEV pseudovirus pET-28b/MS2/HEV particle characteristics
1, following three aspects: qualification test RT-PCR primers designed used is: with reference to synthetic 1 the downstream primer Pri-ORF2R2 of HEV (DQ279091.2) Genotype IV ORF2 conserved regions sequences Design, as shown in sequence table SEQ ID NO.8, with Pri-ORF2F composition qualification primer pair, reverse transcription primer is universal primer OligoDT (15), as shown in sequence table SEQ ID NO.9.
Pri-ORF2R2：5'-AAGCTTACGGCGAAGCCCCAGCT-3’。
The system of following three aspects: qualification test RT-PCR qualification used is:
Reaction conditions is: 45 DEG C of 30min of reverse transcription; 95 DEG C of 2min of denaturation; 94 DEG C of 30s of sex change, the 62 DEG C of 30s that anneal, extend 72 DEG C of 30s, totally 35 circulations; Extend eventually 72 DEG C of 5min.
2, the RT-PCR qualification test of HEV pseudovirion
Get the HEV pseudovirion expression product 1mL after concentrated, purifying, carry out 10
4to 10
10doubly dilution is respectively as sample to be tested A group and B group; A group sample carries out normal RT-PCR qualification, and B group sample directly carries out PCR qualification without reverse transcription, and agarose electrophoresis detects amplification.A organizes all positive (Fig. 3,1-7 road/10 of electrophoresis detection result
4-10
10), show that HEV virus O RF2/3 conserved region gene fragment and T base tailer sequence (To Template sequence) have been reconstituted among MS2 pseudovirus sample particle, and To Template sequence exists with rna form; B organizes pattern detection result negative (Fig. 3,10-16 road/10
4-10
10), in the HEV pseudovirion sample liquid that shows to prepare, pollute without DNA profiling.
3, the rnase of HEV pseudovirion (RNase A) resistant proof
Get 10
4doubly 5 parts, dilution HEV pseudovirion sample, adds respectively RNase A (1mg/mL) 1 μ L/5 μ L/10 μ L/15 μ L, in 37 DEG C of water-bath effect 30min, sets up simultaneously and does not add the control group that RNase A processes.By parallel all samples carry out RNA extracting go forward side by side RT-PCR detect, detected result is as shown in Figure 4.Consistent with undressed check sample detected result through the HEV of rnase processing pseudovirion sample, show the HEV pseudovirion Degradation that can be good at resisting rnase of preparation.
4, the temperature sensitivity qualification test of HEV pseudovirion
Get 10
4doubly 6 parts, dilution HEV pseudovirion sample, is placed in respectively following condition: 60 DEG C/1h, 37 DEG C/10h, 25 DEG C/10d, 4 DEG C/30d ,-20 DEG C/90d and-80 DEG C/180d.Afterwards by the parallel all samples RNA of carrying out extracting and carry out RT-PCR detection.Detected result as shown in Figure 5.The HEV pseudovirion that shows preparation exists: 60 DEG C/1h, 37 DEG C/10h, 25 DEG C/10d, 4 DEG C/30d ,-20 DEG C/90d and-80 DEG C/180d, stability is better.
Claims (6)
1. containing a pseudovirion for RNA of hepatitis E virus fragment, it is characterized in that, as shown in sequence table SEQ ID NO.1;
2. a preparation method who contains the pseudovirion of RNA of hepatitis E virus fragment, is characterized in that, concrete preparation process is as follows:
(1) structure of pET-28b/MS2 recombinant plasmid
(1.1) primer pair of the MS2 gene of design pcr amplification MS2 phage,
Upper primer Pri-MS2F: as shown in sequence table Seq ID No.2,
Lower primer Pri-MS2R: as shown in sequence table Seq ID No.3;
Then amplify MS2 gene clone by the method for RT-PCR, as shown in sequence table SEQ ID NO.4;
(1.2) adopt restriction enzyme Nco I and BamH I double digestion MS2 gene and plasmid pET-28b (+) respectively, after purifying, connect, obtain connecting product;
(1.3) connection product step (1.2) being obtained is transformed into intestinal bacteria competence DH5 α, extracts plasmid performing PCR and the double digestion qualification of going forward side by side, by recombinant plasmid correct qualification in-20 DEG C of preservations;
(2) structure of pET-28b/MS2/NV recombinant plasmid
(2.1) the pcr amplification primer pair of synthetic hepatitis E virus HEV ORF2/3 gene fragment,
Upper primer Pri-ORF2/3F: as shown in sequence table SEQ ID NO.5,
Lower primer Pri-ORF2/3R: as shown in sequence table SEQ ID NO.6,
Then amplify part ORF2/3 gene clone by the method for RT-PCR, as shown in sequence table SEQ ID NO.7;
(2.2) adopt restriction enzyme BamH I and HindIII double digestion ORF2/3 gene clone plasmid and plasmid pET-28b/MS2 respectively, after purifying, connect, obtain connecting product;
(2.3) connection product prepared step (2.2) is transformed and import intestinal bacteria competence DH5 α, extract plasmid and carry out single double digestion qualification, by recombinant plasmid called after pET-28b/MS2/HEV correct qualification;
(3) abduction delivering and purifying
By the positive bacterium colony of recombinant plasmid pET-28b/MS2/HEV correct qualification in step (2), be inoculated into containing in the LB liquid nutrient medium of kantlex, while being cultured to OD600 value for 0.5-1.0, add inductor IPTG to final concentration be 0.5mmol/L, continue to cultivate 5h, centrifugal collection thalline; Be resuspended in appropriate TE solution, adding N,O-Diacetylmuramidase to final concentration is 10mg/L, mixes rear 37 DEG C of water-bath effect 30min, and centrifugal removal precipitation adds rnase and deoxyribonuclease I to final concentration to be 1mg/L, 37 DEG C of water-bath effect 1h in supernatant liquor.Then adopt polyethylene glycol precipitation concentrating virus sample particulate matter, be the pseudovirion of the present invention containing RNA of hepatitis E virus fragment, be dissolved in TE damping fluid 4 DEG C of preservations.
3. the preparation method of a kind of pseudovirion containing RNA of hepatitis E virus fragment as claimed in claim 2, is characterized in that: described MS2 gene is effable MS2 phage maturing enzyme albumen and the gene of envelope protein and the gene coded sequence of part crack protein and replicative enzyme.
4. the preparation method of a kind of pseudovirion containing RNA of hepatitis E virus fragment as claimed in claim 2, it is characterized in that: the sequence of described HEV gene fragment is the overlapping 5225-5510 base place in the most conservative ORF2 district of HEV G1-4 type and ORF3 district, as shown in sequence table SEQ ID NO.6.
5. a kind of preparation method of the pseudovirion containing RNA of hepatitis E virus fragment as claimed in claim 2, it is characterized in that, the sequence of described HEV gene fragment is the overlapping 5225-5510 base of the most conservative ORF2 district of HEV G1-4 type and ORF3, and manually design and introduced 24 T bases in its afterbody, the Poly A producing while transcribing is as the special complementary land of universal primer Oligo (dT).
6. a kind of preparation method of the pseudovirion containing RNA of hepatitis E virus fragment as claimed in claim 2, it is characterized in that, be a kind of RNA-protein complexes by a MS2 phage glutelin parcel hepatitis virus RNA containing the pseudovirion of RNA of hepatitis E virus fragment; It contains HEV gene G1-4 type ORF2 conserved sequence district RNA sequence, and this RNA-protein complexes has the characteristic of resistance to rnase.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105734046A (en) * | 2014-12-12 | 2016-07-06 | 中国检验检疫科学研究院 | Method of preparing Armored RNA with non-cellular expression system |
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| CN105734046A (en) * | 2014-12-12 | 2016-07-06 | 中国检验检疫科学研究院 | Method of preparing Armored RNA with non-cellular expression system |
| CN105734046B (en) * | 2014-12-12 | 2019-04-26 | 中国检验检疫科学研究院 | The method that Cell free expression system prepares Armored RNA |
| CN105907727A (en) * | 2016-05-03 | 2016-08-31 | 中国水产科学研究院黄海水产研究所 | Pseudovirus particle comprising three food-borne virus nucleic acid fragments and preparation method of pseudovirus particle |
| CN105907726A (en) * | 2016-05-03 | 2016-08-31 | 中国水产科学研究院黄海水产研究所 | Pseudovirus particle comprising hepatitis-A-virus nucleic acid fragment and preparation method of pseudovirus particle |
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| CN106011078A (en) * | 2016-05-03 | 2016-10-12 | 中国水产科学研究院黄海水产研究所 | Pseudovirion containing rotavirus nucleic acid fragments and preparation method of pseudovirion |
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| CN112877366A (en) * | 2020-03-10 | 2021-06-01 | 广州复能基因有限公司 | Incorporatable reference standards for detecting sample targets from DNA or RNA organisms |
| CN112625141A (en) * | 2020-12-28 | 2021-04-09 | 昆明海关技术中心 | Protein standard substance of tomato spotted wilt virus and application thereof |
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