CN1587420A - Virus-like particle containing RNA virus nucleic acid and its preparing method and use - Google Patents

Abstract

The present invention belongs to the field of virus gene engineering, and relates to virus-like particle containing RNA virus nucleic acid and its preparation process and application. The virus-like particle is RNase resistant, and contains coat protein and inside recombinant gene sequence including partial MS2 bactereophage sequence, RNA virus nucleic acid sequence and partial RNA virus antigen. The virus-like particle of the present invention may be used as stable and reliable RNA quality controlling and standard article without biological infectious danger. The present invention provides also RT-PCR reagent kit amplification segment suitable for detecting virus RNA in several methods. The virus-like particle containing partial RNA virus antigen may be used as the mold for research of the interaction between virus and host cell, as antisense RNA tool for directional transportation of small molecule medicine, and in vaccine research.

Description

Include the virus-like particle and preparation method thereof and application of RNA viruses nucleic acid

[technical field]

The present invention relates to the viral genetic engineering field, specifically is a kind of virus-like particle that includes RNA viruses nucleic acid and preparation method thereof and application.

[background technology]

Hepatitis C virus (Hepatitis C virus, HCV), human immunodeficiency virus (humanimmunodeficiency virus, HIV), severe acute respiratory syndrome coronavirus (Severeacute respiratory syndrome coronavirus, SARS-CoV) etc. RNA viruses is the pathogenic agent that a class can cause human serious disease, at present, detection at these pathogenic agent mainly contains two class methods (Collins ML, et al., Nucleic Acids Res.1997,25 (15): 2979-84; MulderJ, et al., J Clin Microbiol.1994,32 (2): 292-300), a class is based on the method for immunity of antigen-antibody, mainly is to detect the antibody that virus antigen in the sample or body produce.Another kind of is the RNA that uses virus in the detection of nucleic acids patient specimen.Immunology detection is the ordinary method of diagnosis virus, and still, with respect to detection of nucleic acids, it is long that immunology detection exists window phase, and the shortcomings such as the situation of duplicating of person's body inner virus that can not reflect the virus infection.And detection of nucleic acids is because its susceptibility and specificity height, and the detection window phase is short, can directly reflect infectosome inner virus carrying capacity (vanGemen B, et al., J Virol Methods.1994 (2) again; 49:157-168), therefore, be widely used in blood screening, mother and baby's vertical infection diagnosis, all respects (Zhang M, Versalovic J such as patient's prognosis and the assessment of antiviral therapy curative effect, Am J Clin Pathol.2002,118Suppl:S26-32).

The method of multiple mensuration viral RNA (the Journot V that in clinical, is applied, etal., Control Clin Trials.2001,22 (6): 639-658), the amplification technique that relies on as competitive RT-polymerase chain reaction (RT-PCR), nucleotide sequence (nucleic acid sequence-basedamplification, NASBA), chain DNA (bDNA) signal amplifying system and Real-time quantitative fluorescent PCR etc.The process of these quantitative detecting methods is essentially: (1) sample collection, (2) nucleic acid extraction (3) reverse transcription (4) amplification (except the bDNA) (5) are detected.In the pattern detection process, above-mentioned technology is subjected to following influence of various factors (Yam WC easily, et al., J Cloin Microbiol.2003,41 (10): 4521-4524): the many materials in (1) sample, as cholate in the stool sample and polysaccharide, the urea in protoheme in the blood and the urine, can produce restraining effect to the PCR reaction, mechanism may be to disturb the polymerization of Taq enzyme.(2) some factor in the sample process process also can the suppression of amplification reaction as extraction reagent residual in the sepn process such as ethanol, phenol, SDS etc.The error of manual operation can cause losing of sample nucleic acid, purpose nucleic acid amount is reduced and causes occurring false negative result.(3) influence factor of reaction system: the enzyme in the reverse transcription process active on the low side, the purpose segment degradation of detection that inappropriate holding conditions causes, design of primers is unreasonable, and the thermal cycling process does not reach factors such as perfect condition also can cause the inaccurate of detected result.

Therefore, the European standard council (The European Standards Committee) and International Standards Organization (International Standard Organization) suggestion, when doing the nucleic acid amplification experiment, except with each the step in the erroneous effects factor drop to minimum, also need the strict quality measure, comprise the false positive Quality Control, the Quality Control of false negative Quality Control and detection by quantitative (Hoorfar J, et al., J Clin Microbiol.2003,41 (12): 5835).In addition, an outer marking quantitative measuring method also needs a standard series, is used for determining simultaneously treated clinical samples viral level.At present, the real time fluorescence quantifying PCR method of domestic normal use all is the outer marking quantitative method basically.

Because viral potential infects the unstable of danger and RNA nucleic acid thereof, the present Quality Control thing of using such as the virion of deactivation, cDNA, plasmid DNA and exposed RNA, all be difficult to reach this requirement, so the development lifeless matter infects dangerous and stable Quality Control thing and standard substance, not only the RT-PCR to viral RNA detects, and all significant to the evaluation of corresponding RT-PCR test kit.Someone uses bovine diarrhea virus (BVDV) as internal standard product (the van Gemen B that detects HCV, et al., J Virol Methods.1994,49 (2): 157-167), perhaps HCV standard substance (the Pickett GG that replaces WHO to provide as the standard substance that detect HCV RNA with the HCV virion, Peabody DS, Nucleic AcidsRes.1993,21 (19): 4621-4626), perhaps use the HIV virion as general viral RNA standard substance, but these technology can't overcome the virion infectivity, problems such as virion preparation transportation difficulty that is to say that virion is not the ideal standard substance.

The shell of known some RNA viruses such as MS2 phage, tobacco mosaic virus (TMV) (TMV) can make its rna gene group tolerance RNase.Therefore, as being wrapping to as the RNA sequence of standard substance in these virus coats, just can make specific RNA sequence avoid the degraded of RNase in the environment, simultaneously, the virolysis efficient in application process in all right simulated virus particle monitoring nucleic acid extraction process.In recent years, along with the development of Protocols in Molecular Biology, preparation includes RNA viruses sample particle becomes possibility.

The MS2 Phagus is in straight polarity single stranded RNA spherical virus, genome total length 3569nt, four kinds of protein molecules such as encoding mature zymoprotein, envelope protein, replicase protein and crack protein.Phage is made up of 180 envelope protein monomers, a part maturing enzyme albumen and a part geneome RNA.In the research of more early stage MS2 phage envelope protein, find that envelope protein and operon RNA sequence have specificity to interact, this effect can cause the assembling of phage ghost, simultaneously, phage genome RNA is packaged into (Yen-Lieberman B in the coating, et al., J ClinMicrobiol.1996,34 (11): 2695-2701).Therefore, if the cDNA of specific virus RNA is cloned in MS2 phage envelope protein gene sequence cDNA downstream, then terminator is inserted external source cDNA downstream, obtain having the specific virus rna transcription basis of phage operon RNA sequence after transcribing, operon RNA sequence just can cause the phage envelope protein and be assembled into shell, and the part phage genome that will carry specific virus RNA sequence is packaged in the coating formation phage virus-like particle.But, iff with the cDNA sequence clone of specific virus RNA in operon sequence cDNA downstream, express the phage envelope protein with double-plasmid expression system then after, the phage sample particle defectiveness of assembling promptly lacks ripe characteristic.This defective phage virus-like particle can not prevent Degradation (the Desire N of RNase to the specific RNA of interior bag, et al., J Clin Microbiol.2001,39 (4): 1303-1310), and it is colibacillary ribosome-RNA(rRNA) (rRNA) that assembling gained phage sample particle contains the host.Major cause is to contain in the host rRNA precursor and MS2 operon homologous sequence, intervening sequence between inner 16S of its rrnE transcript, rrnH transcript and rrnB transcript and the 23S RNA sequence etc. all has and MS2 operon RNA homologous sequence, and these homologous sequences can cause the packing of host rRNA.When making up plasmid expression vector, need phage maturing enzyme albumen, envelope protein gene sequence cDNA clonal expression is in expression vector promotor downstream, like this, through behind the 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 (Bagnarelli P to the expression process, et al., J Med Virol.1991,34 (2): 89-95), make transcribing of the expression of envelope protein and RNA harmonious, at this moment, assembling gained phage sample particle has ripe characteristic, thereby has the RNase resistance.Simultaneously, transcriptional expression coordinates to carry out specific recognition packing phage genome RNA, gets rid of the assembling of host rRNA.Therefore, consider in the process of phage assembling, except 21 poly-RNA stem ring specificitys in conjunction with, the envelope protein internal surface have arginine/Methionin enrichment region can with the RNA nonspecific action, promote the assembling of envelope protein.

(J Clin Microbiol.1998 such as Pasloske, 36 (12): 3590-3594) utilize the MS2 phage rna can tolerate the RNase degraded and under general physico chemical factor, keep stable properties, study the RNA quality control product or the standard substance of preparation good stability at occurring in nature.They are inserted into purpose fragment (Quality Control RNA) in the MS2 phage genome at first, are built into to contain reRNA, activated MS2 phage.But this class reRNA can lose the insertion fragment because MS2 recovers wild-type because of in activated MS2 phage; The reorganization MS2 can also breed, thereby the amount of causing be difficult to determine that the propagation of MS2 also causes breadboard pollution easily.And the replicative enzyme of MS2 is a polysaccharase that fidelity is very poor, is easy to mix wrong base in the rna replicon process, thereby causes the instability of Quality Control sequence.So in research afterwards, they have adopted plasmid expression system to express the HIV-1 virus-like particle.This expression vector contains following sequence: maturing enzyme encoding sequence, maturing enzyme ribosome bind site, capsid protein encoding sequence, packaging site, the part initiation site of replicative enzyme, and the aim sequence (142bp) used of HIV-1 Quality Control.But the aim sequence that the HIV-1 Quality Control is used in the expressed virus-like particle of this expression vector is too little, is not suitable for several different methods or contains the application of the RT-PCR test kit of multiple detection viral RNA; Virus-like particle is not very stable simultaneously, and can not produce the virus-like particle with HIV-1 incomplete antigen, and its application surface is less.

In sum, in the RT-PCR of RNA viruses detects, because it is more to influence the factor of experimentation, as test residual, the concentration of 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, the efficient of reverse transcription and the problem of reagent etc., these factors all might influence the efficient of pcr amplification, and then cause result's deviation.Therefore, PCR measures and must use quality control product to carry out the reliability that strict indoor quality control could guarantee the result.In addition, for carrying out quantitative assay, also need quantitative standards product or interior mark, interior mark can also play the effect of the instant Quality Control of single tube.A reliable and stable and dangerous RNA quality control product and the standard substance of lifeless matter infection, high-quality detection has important value for the RT-PCR of RNA viruses.In this, exposed RNA or virion and viral substitute all are difficult to as ideal quality control product and standard substance, the segmental phage virus-like particle of specific virus RNA that includes that adopts molecular cloning and prokaryotic expression method to make up then preferably resolves these problems, the stdn that it not only detects for RNA viruses RT-PCR, and the potential using value is all arranged for the detection of specific mRNA etc.

[summary of the invention]

Technical problem to be solved by this invention just provide a kind of can be reliable and stable and lifeless matter infect the dangerous RNA quality control product and the virus-like particle of anti-RNase that includes RNA viruses nucleic acid of standard substance.

Another technical problem to be solved of the present invention provides a kind of virus-like particle of anti-RNase that includes RNA viruses nucleic acid that is applicable to several different methods or contains the RT-PCR test kit institute amplified fragments of multiple detection viral RNA.

At present, studies show that (Elbeik T, et al., Expert Rev Mol Diagn, 2002; 2 (3): 275-285) have jejune characteristic in the envelope protein assembling process according to single expression, can not obtain the peplos of anti-RNase, so with MS ₂The maturing enzyme albumen of phage and the gene coded sequence of envelope protein with and the genome corresponding cDNA sequence of 5 ' non-coding sequence that partly comprises the gene regulatory elements sequence all be connected to expression vector such as pET28b T ₇The promotor downstream obtains maturing enzyme albumen and envelope protein through abduction delivering, and under maturing enzyme and the segmental synergy of phage genome RNA, envelope protein can be assembled into sophisticated virus-like particle, and possesses the characteristic of anti-RNase effect.In view of the above, the present invention has successfully made up and has included MS ₂The maturing enzyme albumen of phage and the gene coded sequence of envelope protein with and genome partly comprise the expression vector of 5 ' non-coding sequence of gene regulatory elements sequence, and behind prokaryotic expression, the virus-like particle that obtains has the characteristic of anti-RNase.Then with the MS of cDNA sequence construct in this plasmid vector of HCV RNA, HIVRNA and SARS-CoV RNA etc. ₂Phage 1.7kb genome downstream after transform bacteria is expressed, can obtain having the virus-like particle that includes specific RNA of the characteristic of anti-RNase, and this particle can be used as stable standard substance and the quality control product that the RT-PCR of specific RNA measures, and does not have infectivity.

MS2 phage rna genome sequence total length is 3.6kb, removes the MS2 sequence of the 1.7kb of unlap, the fragment that can also pack 1.9kb in theory.But discover at present, when the length of packing nucleic acid fragment surpasses 500bp, the packaging efficiency of the virus-like particle (CindyR that can descend greatly, et al., Clin Chem 1999,45 (12): 2079-2085), and the general way of guarding that adopts of existing like this technology, only pack the aim sequence (142bp) that the HIV-1 Quality Control is used as Pasloske etc., be only applicable to the detection kit of Roche company.The present invention breaks through the nucleotide sequence limitation of length that includes RNA viruses, provides to contain the required primer complementary sequence of multiple detection method, is with a wide range of applications.The nucleotide sequence length of RNA viruses provided by the invention is 150～1000bp, and as the about 1000bp of the fragment length of packaging, the virus-like particle of finding to obtain is in dilution 10 ⁵Doubly, after the RT-PCR amplification, still can detect, its concentration is 10 ¹⁰Level has satisfied practical application.

Simultaneously, in a large amount of experiments, find to have summed up another technical problem to be solved, just provide the virus-like particle that contains RNA viruses nucleic acid within a kind of incomplete antigen with the RNA viruses of including in the present invention.The RNA viruses nucleic acid that includes in this virus-like particle is conservative encoding sequence, and sequence length is at 500～1000bp; Preferred sequence is for containing HIV-1 virus gag region sequence among the present invention.

The preparation method of above-mentioned virus-like particle, the method includes the steps of: make up plasmid; Designated rna virus primer sequence; Pcr amplification, enzyme are cut and are obtained the purpose fragment; Be connected with plasmid, transform; The inducing and expressing of virus-like particle.

PNCCL1 construction process and sequence are referring to (Chinese laboratory medicine magazine, 2003,26 (2): the 86-88) structure of the virus-like particle of anti-rnase the and express a literary composition such as Li Jinming; Needs according to constructed carrier, restriction enzyme HindIII restriction enzyme site (underscore part) and protectiveness base are introduced in upstream at primer, Tm value and other the amplification parameter of operation to mate each primer on Primer 5.0 softwares designs synthetic primer then; Then carry out the segmental amplification purification of purpose; Enzyme is cut and is obtained the purpose fragment and be connected with plasmid, transforms; The inducing and expressing of virus-like particle.

The virus-like particle that includes RNA viruses nucleic acid that the present invention obtains can be used as structure and prepares the RNA viruses nucleic acid standards of anti-RNase and the platform of quality control product.As the outer marking quantitative standard of can be directly measuring as the positive quality control thing in the RNA viruses nucleic acid determination and outer marking quantitative; Can infect dangerous sample for quality evalution between RNA viruses nucleic acid RT-PCR detection kit and chamber, laboratory provides lifeless matter.If the purpose fragment of the RNA viruses detection of nucleic acids in the virus-like particle that includes RNA viruses nucleic acid is lacked, inserts and modification such as sudden change, the virus-like particle that is obtained can be used as interior mark standard and the RNA viruses nucleic acid RT-PCR interior mark Quality Control thing measured of RNA viruses nucleic acid quantification in measuring.In the present invention, the albumen of the conservative coding region of RNA viruses nucleic acid also is proved and is expressed on the virus-like particle.Expression has the virus-like particle of corresponding antigens can be used as the instrument of directed transportation small-molecule drug, sense-rna as research virus and the interactional model of host cell.In addition, there is the corresponding antigen epi-position can be used for studying vaccine at the virus-like particle surface expression.

Use includes the virus-like particle of specific RNA as quality control product and standard substance that RNA viruses detects, and have the advantage of following several aspects: (1) lifeless matter infects dangerous.(2) standard substance and the quality control product that detects as RNA RT-PCR has good stability.Because this virus-like particle has anti-RNase, therefore can avoid being degraded by immanent RNase, and this degraded to detect for the RT-PCR of RNA viruses be the problem that needs are paid close attention to.(3) RNA in the pathogenic agent there is good dummy activity.Because expression product is phage sample particle, have good virion analog functuion, therefore, in the nucleic acid extraction process, good similarity is arranged with the leaching process of real viral nucleic acid in the sample.

[description of drawings]

Fig. 1, carrier pNCCL1 clone/expression site;

Fig. 2, HIV TA clone is through the HindIII restriction enzyme mapping;

Fig. 3, the pNCCL1-HIV collection of illustrative plates after NcoI and HindIII enzyme are cut

Fig. 4, expression product carry out reverse transcription respectively and react without the laggard performing PCR of reverse transcription after RNA extracts electrophoretogram; 1,2,3,4,5,6 are respectively the electrophoretogram of expression product PCR behind reverse transcription of dilution in 1: 10 to 1: 106, and the 1-5 pipe can both increase and obtain the band of 1000bp.7,8,9,9,10,11 directly carry out the reacted electrophoresis of PCR for the 1-5 sample without reverse transcription;

Fig. 5, the electrophoretogram 1,2 of expression product after DNaseI and the single enzyme of RNaseA or two enzymic digestion are that expression product is preserved 16h for 4 ℃; 3:37 ℃ does not add enzymic digestion; 4:RNaseA digestion; 5:DNaseI digestion; The two enzymic digestions of 6:DNaseI and RNaseA;

Fig. 6, virus-like particle place 45 ℃, the electrophoretogram behind the 3d;

The clone of Fig. 7, pNCCL1-HIV carrier/expression site;

Fig. 8, P24 antigen are confirmed experiment:

1,2,3,4 is the band of expression product behind western-blot, and comparison molecular weight of albumen standard is positioned near the 97KD molecular weight;

Fig. 9, the checking of intact virus sample particulate;

1,2,3,4,5,6 band lines are respectively former times to 1: 105 dilution series of expression product, and RNA extracts after reverse transcription carries out the electrophoretogram behind the PCR; 7,8 be respectively the P24 antigenic reagent box with negative serum, positive quality control serum.The 9-16 band is respectively the 1-8 sample, and RNA extracts PCR is just carried out in the back without reverse transcription electrophoretogram;

Figure 10, SARS-Cov TA cloning vector are through HindIII restriction enzyme digestion and electrophoresis collection of illustrative plates;

Figure 11, expression vector PNCCL1-SARS-Cov be through HindIII, the electrophoretogram after the NcoI enzyme is cut;

Figure 12, PNCCL1-SARS be through NcoI, HindIII, the collection of illustrative plates after the XhoI enzyme is cut;

Figure 13, the electrophoretogram of expression product behind DnaseI and the single enzyme of RNaseA or two enzymic digestion 16h;

The electrophoretogram of Figure 14, the experiment of expression product patience;

The electromicroscopic photograph of Figure 15, virus-like particle, display result is normal.

[embodiment]

1, the key instrument of selecting for use in the embodiment of the invention is as follows:

The PE2400 PE 5700 ABI company U.S.

Line-Gene fluorescence quantitative PCR detection system Dahe Thermomagnetic Electronic Co., Ltd., Hangzhou China

Ultraviolet projection analyser Beijing Yi Cheng scientific ﹠ technical corporation China

The cytoclasis instrument Branson sonic power Co. U.S.

Table model high speed centrifuge Oserode Heraeus Insrument Germany

The desk type high speed refrigerated centrifuge Beckman I Instruments U.S.

D-type ultrapure water machine USF ^ELGAThe RESERVOIR 75L U.S.

Ultraviolet spectrophotometer island proper Tianjin on the 1601st company Japan

The microplate reader BIO-RAD company U.S.

The automatic enzyme of DEM-III type mark is washed trigger Beijing and is opened up general analytical instrument company China

HW-8B type trace thermostatted Shanghai Pujiang analytical instrument factory China

The Sartorius BP 310S electronic balance U.S.

2, the main raw of selecting for use in the embodiment of the invention is as follows:

2.1 biomaterial:

Bacterial strain: DH5 α E.Coli.BL21-DE3E.Coli. (Invitrogen Corp., U.S.A)

Plasmid: pNCCL1 construction process and sequence are referring to (Chinese laboratory medicine magazine, 2003,26 (2): the 86-88) structure of the virus-like particle of anti-rnase the and express a literary composition such as Li Jinming; HIV gag plasmid is so kind as to give by Chinese Center for Disease Control std aids center, and the gag gene on this plasmid is by the gained that directly increases in the positive peripheral blood of uncultivated HIV-1, and this strain is China's AIDS poison E, B hypotype representative strains; P24 monoclonal antibody (magnificent company buys).

SARS-CoV RNA is so kind as to give by middle mountain Da An company.Derive from SARS burst period patient's sample.

The novel coronavirus nucleic acid amplification fluorescent detection kit that SARS is relevant: Zhongshan University reaches peace gene diagnosis center to be provided

2.2 toolenzyme

Taq archaeal dna polymerase, T ₄Dna ligase, RQ1 deoxyribonuclease 1, rnase (RNaseA), restriction enzyme XhoI, HindIII are available from Promega company (U.S.); NcoI is available from NewEngland company (U.S.);

2.3 microbial culture and bacterial classification are preserved

2.3.1 microbial culture

(1) solid medium is cultivated: get bacterial classification with getting collarium, carry out streak inoculation on solid medium, be inverted overnight incubation for 37 ℃ in the CO2gas incubator.

(2) liquid nutrient medium is cultivated: with getting the single colony inoculation of collarium picking in the 5ml liquid nutrient medium, put 37 ℃ of shaking tables, 140-180rpm shakes bacterium and spends the night.

2.3.2 bacterial classification is preserved

(1) the solid medium bacterial classification is preserved: single bacterium colony can be preserved the several months on solid medium.

(2) the liquid nutrient medium bacterial classification is preserved: fresh bacterium liquid is added 15% glycerine, place the medium-term and long-term preservation of-70 ℃ of refrigerators or liquid nitrogen container.

2.3.3 plasmid extracts

A small amount of alkaline lysis method of extracting plasmid (generally be used for enzyme and cut checking)

(penbritin concentration is: 50 μ g/ml, kantlex concentration is: 30 μ g/ml), 37 ℃ are shaken bacterium and spend the night in 5ml contains suitable antibiotic LB substratum with single colony inoculation.Get the 1.5ml culture and change in the Eppendorf tube, 12, the centrifugal 30s of 000g, precipitum, supernatant discarded adds the STE mixing as far as possible, and the washing bacterium is to remove opsonigenous substances, 4 ℃, 12, the centrifugal 30s of 000g.Concrete grammar is referring to " molecular cloning ", and final product adds TE damping fluid dissolving plasmid DNA, can standing storage in-20 ℃.

2.3.4 the digestion of RNA in the plasmid DNA

RNA tentatively digests: in the plasmid leaching process, before plasmid DNA precipitated, adding 37 ℃ of temperature of 10-20 μ g/ml RNaseA bathed 15-20 minute, the plasmid DNA that the most of RNA in the plasmid DNA can be digested this thick digestion has multiple use, such as: the preliminary digestion of restriction enzyme, identify segmental amplification of purpose or the like.

RNA digestion: in dna solution, add 20 μ g/ml RNaseA and bathed 15-30 minute 37 ℃ of temperature.Also can add certain density RNaseA in the TE damping fluid, the concentration that adopts in this research is 10-20 μ g/ml.

2.3.5 determining of plasmid concentration

Plasmid DNA after utilizing the ultraviolet spectrophotometer colorimetric to purifying is quantitative, measures 260nm, and the absorbancy during 280nm is also calculated both ratio.

Can calculate the purity of DNA in the resulting dna sample according to the ratio of OD260/OD280, the ratio of purity should be greater than or equal to 1.8.

Extrapolate plasmid DNA concentration according to 260nm absorbancy (thickness of cuvette is 1.0cm), used formula is: the concentration value of A * 50=DNA (μ g/ml).

Embodiment 1 includes the preparation of the virus-like particle of HIV-1 viral nucleic acid 956bp

The contained HIV nucleic acid fragment of virus-like particle that the present invention is prepared contains 10 kinds and detects the fragment that HIV RT-PCR test kit is increased, and is the zone that quantitative PCR increased at HIV, and the present invention finds that fragment concentrates on HIV genome gag district 1-956nt.As:

Primer 1:5 '-GGGCAAATGGTACATCAGGCCATATCAC

3’-AGCCGAGCAAGCTTCACAGGAGGTAAAA

The length that is increased is 525bp

The HIV gag plasmid that the present invention is used is so kind as to give by Chinese Center for Disease Control std aids center, and the gag gene on this plasmid is by the gained that directly increases in the positive peripheral blood of uncultivated HIV-1, and this strain is China's AIDS poison E, B hypotype representative strains CN54 strain; Search for this strain sequence on the net at MEDLINE, utilize primer-design software Primer 5.0, design amplification HIV genome gag district 1-956nt comprises needed purpose fragment, also contains the initiator codon Met of HIV gag district proteins encoded simultaneously.Primer is respectively:

Sense primer: 5 '-AG AAGCTTATGGGTGCGAGAGCGT-3 ';

Antisense primer: 5 '-AT AAGCCTTGGACCAACAAGGTGTCTGTC-3 '.

And according to the needs of constructed carrier; restriction enzyme HindIII restriction enzyme site (underscore part) and protectiveness base are introduced in upstream at primer; on Primer 5.0 softwares, move Tm value and other amplification parameter then to mate each primer; and it is synthetic to match hundred victory companies by Beijing; synthetic primer is a lyophilized dna, with aseptic deionized water (ddH ₂O) be dissolved to working concentration 10umol/l ,-20 ℃ of preservations.

1.1 the segmental amplification of purpose

Reaction system is as follows:

Gag plasmid 1.0 μ l

DNTP miscellany 1.0 μ l

10 * amplification buffer, 5.0 μ l

10umol/l upstream primer 1.0 μ l

10umol/l downstream primer 1.0 μ l

Taq polysaccharase 0.4 μ l

25mmol/lMg ²⁺ 3.0μl

Add ddH ₂O complements to 50 μ l

Reaction conditions: 94 ℃, the pre-sex change of 3min; 94 ℃ of 45s; 56 ℃ of 45s; 72 ℃ of 2.0min; 30 circulations are extended 5min for back 72 ℃.Get 5 μ lPCR products and carry out the observation of 1.4% agarose gel electrophoresis.

1.2PCR the purifying of product

(1) the PCR product is carried out electrophoresis, cut the blob of viscose that contains the purpose band with clean blade, blob of viscose will be tried one's best little and be contained the purpose fragment as much as possible, puts into the 1.5ml centrifuge tube.The weight of this glue of weighing is no more than 150mg as far as possible.The adding ddH of not enough 100mg ₂O mends to 100mg.

(2) be equivalent to 1 μ l by 1mg and convert, with sol solutions: the glue weight ratio is that 3: 1 ratio adds sol solutions.Put 50 ℃ of water-bath 10min, every 2min jolting once melts glue fully.Mixed solution is transferred in the centrifugal post that is enclosed within on the clean adapter, 12,000g, centrifugal 1min.

(3) discard liquid in the adapter, insert centrifugal post again.On centrifugal post, add and wash post liquid 750 μ l, 12,000g, centrifugal 30s.

(4) add and wash post liquid 250 μ l, repeating step 3.

(5) centrifugal post is transferred on the clean adapter, void column is centrifugal 12,000g, 2min.

(6) repeating step 5.

(7) centrifugal post is transferred on the clean adapter, added the ddH of 60 ℃ of-70 ℃ of preheatings ₂O 30 μ l room temperatures are placed 1min, centrifugal 1min.The dna solution of gained solution for reclaiming.

1.3 restriction enzyme is to the digestion of plasmid DNA

The present invention need be to TA cloning vector pBS-T, and expression vector pNCCL1 carries out enzyme and cuts, and wherein the clone of expression vector pNCCL1/the expression site as shown in Figure 1.

The used restriction enzyme of the present invention is HindIII, three kinds of enzymes of XhoI and NcoI, and each enzyme tangent condition such as table 1, shown in 2:

Table 1, the HindIII of Promega company and XhoI enzyme are cut the activity in different damping fluids

A B C D E MULTI-CORE ^TM

HindIII 25-50％ 100％ 75-100％ 10-25％ 100％ 50-75％

XhoI 25-50％ 75-100％ 75-100％ 100％ 10-25％

The activity of the NcoI of table 2, NEB company in different damping fluids

NEB?buffer 1 2 3 4

％Activity 100％ 100％ 100％ 100％

According to the condition of above-mentioned enzyme cutting buffering liquid, empirical tests, when adopting single endonuclease digestion, the damping fluid that each enzyme is selected for use is respectively: HindIII is Buffer E, XhoI is Buffer D; Work as HindIII, Nco1 double digestion or HindIII, XhoI, when Nco1 three enzymes are cut, employing be the Buffer B Buffer B of the packing of magnificent company (separately available from); The above-mentioned enzyme system of cutting is 40 μ l, and is as follows: HindIII:Buffer E 4.0 μ l, HindIII 4.0 μ l, BSA 0.4 μ l, substrate DNA 31.6 μ l; XhoI:Buffer D 4.0 μ l, XhoI 4.0 μ l, BSA 0.4 μ l, substrate DNA 31.6 μ l; HindIII, NcoI double digestion: Buffer B 4.0 μ l, HindIII 2.0 μ l, XhoI 2.0 μ l, BSA 0.4 μ l, substrate DNA 31.6 μ l;

HindIII, XhoI, NcoI three enzymes are cut: Buffer B 4.0 μ l, HindIII 2.0 μ l, XhoI 2.0 μ l, NcoI BSA 0.4 μ l, substrate DNA 29.6 μ l;

Enzyme is cut the time according to the purpose difference, and is also different in the actually operating: cut the purpose fragment if obtain enzyme from the TA cloning vector, need enzyme to cut and spend the night; Cut evaluation if only the carrier behind the clone is carried out enzyme, enzyme is cut and was got final product in 2-4 hour; Connecting needs enzyme to cut with expression vector pNCCL1 to spend the night.The enzyme Qie Wendu of above-mentioned each system is 37 ℃.

1.4 purpose sheet segment DNA is connected with TA cloning vector pBS-T or expression vector pNCCL1's, transforms and identifies.

1.4.1 purpose sheet segment DNA is connected with TA cloning vector pBS-T's, transforms and identifies:

Connect: it is the TA clone test kit in epoch that the present invention adopts the sky, and linked system is as follows:

PBS-T carrier 1.0 μ l, purpose fragment 7.0 μ l, T4 ligase enzyme 1.0 μ l, T4 ligase enzyme damping fluid 1.0 μ l.Connect 1h.

Transform: after from-70 ℃ of refrigerators, taking out cloning host competence bacteria DH5a, be placed on ice bath 10min with holding.

(1) dna fragmentation that will transform joins (50 μ l competent cells need 25ngDNA) in the pipe that competent cell is housed, and volume should not surpass 5% of competent cell, rotates mixing content several times gently, ice bath 30min.

(2) pipe is put into heat in advance and placed 90 seconds, do not shake centrifuge tube to 42 ℃ of water-baths.

(3) fast pipe is transferred in the ice bath, made cell cooling 1-2min.

(4) every pipe adds 400 μ l SOC substratum, places water bath substratum is heated to 37 ℃, then pipe is transferred on 37 ℃ of shaking tables incubation 45min, the antibiotics resistance marker gene that bacteria resuscitation and expression plasmid are encoded.

(5) competent cell that has transformed of proper volume is transferred to contained on the antibiotic flat board of Amp.Dull and stereotyped before being coated with bacterium liquid, need to add 100 μ l X-gal, 20 μ l IPTG are applied on the flat board behind the mixing fully.

(6) place room temperature to be absorbed fully flat board until liquid.

(7) be inverted plate, cultivated 12-16 hour in 37 ℃.

Flat board after the conversion, blue hickie ratio generally need be between 1: 5 to 2: 1.

Identify: the picking hickie is inoculated in the culture tube of 50ml of 5ml Amp+LB substratum, 37 ℃, 250rpm/min, cultivate 6-8h after, extract plasmid.Carry out enzyme with HindIII and cut, cut the rear electrophoresis collection of illustrative plates according to enzyme and identify, the purpose fragment band of visible 1000bp behind the electrophoresis.The restriction enzyme digestion and electrophoresis collection of illustrative plates as shown in Figure 2.

1.4.2 purpose sheet segment DNA and expression vector pNCCL1 are connected, and transform and identify:

The purpose fragment comes from 1.2, extracts plasmid after the positive bacterium colony after cloning TA successfully increases bacterium, carries out enzyme with HindIII and cuts, and cuts glue after the agarose electrophoresis and reclaims the purpose fragment, and the gained dna fragmentation is the purpose fragment of HindIII complete degestion.

The enzyme of expression vector pNCCL1 is cut: according to carrying out purifying shown in the step 1.4.

Before connection, expression vector pNCCL1 electrophoresis after purpose sheet segment DNA after enzyme cut and enzyme are cut carries out quantitatively rough, when two band brightness are suitable, connect by following system: purpose fragment 7.0 μ l, plasmid PNCCL1 1.0 μ l, T4DNA ligase enzyme 1.0 μ l, T4 ligase enzyme damping fluid 1.0 μ l.4 ℃ of connections are spent the night.The 2.6.1 operation transforms expressive host bacterium BL21-DE3 set by step.Do not adopt the screening of blue hickie, choose bacterium and carry out the PCR primary dcreening operation, and establish positive bacteria and contrast but use the reaction conditions the same with amplification purpose fragment.Occur consistent with the purpose clip size, and the purpose band very bright be decided to be the primary dcreening operation positive, get plasmid and use restriction enzyme HindIII, NcoI that plasmid is carried out enzyme and cut, obtain 1000bp, 1700bp reaches the band of 5200bp, as shown in Figure 3.The positive recombinant sequencing result is online through PubMed, finds behind the Blast, and the frameshit phenomenon of MS2 single open reading frame does not appear in the insertion fragment in the recombinant plasmid consistent with the gene order of MS2 and HIV (concordance rate 100%), thereby determines final positive bacteria.

1.5 the inducing and expressing of virus-like particle

The positive bacteria of gained in 1.4 is inoculated in 2ml contains in the LB liquid nutrient medium of Kana+ resistance, 37 ℃, 130-180rpm/min shakes the bacterium incubated overnight.The bacterium liquid (being in logarithmic phase) of getting 100 μ l incubated overnight then is inoculated in the LB liquid nutrient medium that 10ml contains the Kana+ resistance, 37 ℃, 200rpm/min shakes bacterium 1.5h, adds isopropylthio-(IPTG), and the final concentration that makes IPTG is 1mM/ml.Abduction delivering 16 hours, carry out ultrasonic broken bacterium and handle: get 1.5ml bacterium liquid in 4 ℃ 14 with the 1.5ml centrifuge tube, the centrifugal 2min of 000r/min collects bacterium, after PBS washs three times, adds the resuspended bacterial precipitation of 0.25ml supersound process damping fluid.Handle the broken bacterium of probe with ultrasonic cell disintegration instrument miniature ultrasonic, then the bacteria breaking thing is put ice bath 1min.Repeat above-mentioned shattering process 5-6 time.With 4 ℃ of bacteria breaking things, 14, the centrifugal 2min of 000r/min gets supernatant and transfers in another clean centrifuge tube, obtains the virus-like particle of the HIV-1 of containing viral nucleic acid of the present invention.

Embodiment 2, include the virus-like particle preparation of HIV-1 viral nucleic acid 500bp

The contained HIV nucleic acid fragment of virus-like particle that the present invention is prepared contains 5 kinds and detects the fragment that HIV RT-PCR test kit is increased, for the zone that quantitative PCR increased at HIV is HIV genome gag district 1-500nt.Other steps are with embodiment 1.

Embodiment 3, virus-like particle include the evaluation of viral RNA

The embodiment products therefrom is through reverse transcription and do not do the laggard performing PCR reaction of reverse transcription electrophoresis result as shown in Figure 4; Experimental result shows, through the dilution series of reverse transcription process, can amplify the purpose fragment; And directly carry out the dilution series of pcr amplification without reverse transcription, and fail to amplify the purpose fragment, the nucleic acid that proves the VLPs internal packing is RNA but not DNA.

The evaluation of the anti-RNase of virus-like particle of embodiment 4, HIV-1 viral nucleic acid

PNCCL1-HIV among the embodiment 1 is expressed the phage sample particle that also supersound process obtained through transforming the host bacterium, carry out RNaseA (100U) digestion respectively, DNaseI (20U) digestion, RNaseA (100U) and the two enzymes of DNaseI (20U) are united digestion, do not add 37 ℃ of DNaseI and RNaseA digestion and 4 ℃ of preservations without two kinds of enzymic digestions, then with three kinds of enzymic digestion products and the expression product that do not carry out enzymic digestion electrophoresis together, electrophoresis result (Fig. 5) shows that three kinds of enzymic digestion product electrophoretograms have nothing in common with each other, and two enzymic digestion after product electrophoresis have only a band line about 1000bp.

The evaluation of the virus-like particle stability of embodiment 5, HIV-1 viral nucleic acid

Get the expression product 400 μ l that obtain among the step embodiment 1, add PEG600/NaCl 100 μ l (v/v 20%), 4 ℃, the centrifugal 1h of 15000rpm/min adds normal people's negative serum 500 μ l.Respectively get 50 μ l and place four Eppendorf pipes, 45 ℃, 3d respectively.The stability of virus-like particle in the electrophoresis observation expression product.

To the resulting expression product of ultrasonic broken bacterium, by 1: 10,1: 100,1: 1000,1: 10000,1: 100000, after ratio was diluted in 1: 1000000, each pipe adds the two enzymes of RNaseA and DNaseI respectively unite digestion 24h after, add 4M isothiocyanic acid guanidinesalt, 1% inositol solution stops endonuclease reaction.

Extract the method for RNA according to the isothiocyanic acid guanidinesalt and carry out the sample rna extraction, method is as follows:

(1) adds 150 μ l lysates (4mol/L GuSCN, 25mmol/l Trisodium Citrate pH7.0,0.5% inositol, 100mmol/L beta-mercaptoethanol), add 50 μ l samples, add 50 μ l chloroforms behind the mixing.

(2) 4 ℃, the centrifugal 15min of 13000rpm.

(3) get with step (1) in the 0.5ml sterilization centrifuge tube of equal amts, add 100 μ l Virahols, perform mark.The upper phase of drawing in the step (2) is transferred to (do not run into the middle layer, this layer is rich in DNA and protein) in the corresponding pipe, puts upside down mixing.

(4) 4 ℃, the centrifugal 15min of 13000rpm.Remove supernatant gently, be inverted on the thieving paper, be stained with dry liquids; Add 300 μ l, 75% ethanol, put upside down washing.

(5) 4 ℃, the centrifugal 15min of 13000rpm.Remove supernatant gently, be inverted on the thieving paper, be stained with dry liquids;

(6) centrifugal 5sec will manage the bottom small amount of liquid and blot with micro sample adding appliance.65 ℃ of oven for drying, but avoid too dry as far as possible.

(7) add 25 μ l ddH ₂O dissolves RNA ,-20 ℃ of preservations.

Carry out RT-PCR then: with primer 5 '-ATAAGCTTGGACCAACAAGGTGTCTGTC-3 ' is reverse transcriptase primer, carries out reverse transcription by following system: get the RNA solution 15.0 μ l in the above-mentioned steps (7); AMV buffer, 4.0 μ l; Four kinds of dNTP mixing solutionss of 10mmol/l, 2.5 μ l; The 10umol/l primer, 0.01 μ l; 9U/ μ l reversed transcriptive enzyme (AMV), 2.0 μ l; 25mmol/l Mg ²⁺, 1.0 μ l.Behind 42 ℃ of incubation 1h, 93 ℃ of 3min deactivation reversed transcriptive enzymes.Amplification condition in 3 increases set by step again.For the checking amplified reaction really is due to the RNA, the reaction tubes that directly carries out pcr amplification not carry out reverse transcription in contrast.1.4% agarose horizontal strip electrophoresis checking amplified production.

Electrophoretogram is still found the bright band of 1000bp as shown in Figure 6.The proof virus-like particle has good stability.

Embodiment 6, the virus-like particle protein-contg evaluation in surface

The cloning site of the expression vector pNCCL1-HIV that newly successfully constructs is as shown in Figure 7:

As shown above: occurring initiator codon ATG among the restriction endonuclease Nco1, is the proteic initiator codon of MS2 just.HindIII site before and after the MS2 sequence of inserting adds, 1690nt altogether.The 3rd amino acid coding before the HindIII site just is MS ₂The initiator codon ATG of phage replication enzyme, the initiator codon ATG in the gag district that distance is inserted is 15 bases just, so there is not phase shift mutation.Institute is so that gag albumen obtains expressing.In HIV-1 virus, gag albumen is P7, P17, the precursor protein of P24.Therefore as long as any albumen in the checking gag albumen exists, promptly provable gag albumen does not have phase shift mutation and obtains expressed intact.

6.1P24 proteic preliminary evaluation:

Use Murex HIV Antigen Mab VK86 8E77-01 (P24 antigen detecting agent box) and carry out preliminary evaluation.The resulting virus-like particle of step 2.8 was pressed 1: 10 respectively, 1: 50,1: 100,1: 200,1: 400,1: 800; Series was diluted in 1: 1600, then the dilution series sample was identified by the test kit specification sheets

6.1.2P24 proteic preliminary evaluation step:

(1) every hole adds 100 μ l working fluids 1, adds 100 μ l samples or yin and yang attribute Quality Control more respectively.

(2) lucifuge is hatched 60min for 37 ℃; Wash plate 5 times.

(3) add connection 200 μ l working fluids 2.

(4) lucifuge is hatched 30min for 37 ℃; Wash plate 5 times.

(5) every hole adds 200 μ l substrate solutions.

(6) room temperature (15 ℃-30 ℃) is hatched 30min.

(7) every hole add 50 μ l 1-2mol/l sulphuric acid soln with termination reaction, and shake up gently.

(8) in the 5-15min, under the 450nm wavelength, read absorbancy.

6.1.3 the electromicroscopic photograph of virus-like particle (Figure 15) display structure is normal.

6.1.4P24 proteic preliminary evaluation result:

Result such as table 3,4:

Do the P24 preliminary evaluation behind table 3, the expression product doubling dilution

The positive negative empty bacterium of sample contrasts empty bacterium and contrasts 1: 50 1: 10 former times

OD value 0.989 0.025 0.006 0.589 0.600 Over Over Over

Do the P24 preliminary evaluation behind table 4, the expression product doubling dilution

The positive negative empty bacterium contrast of sample 1: 1,600 1: 800 1: 400 1: 200 1: 100

OD value 0.507 0.005 0.005 0.293 0.558 0.985 1.162 1.816

By above table as can be seen, after expression product is done dilution in 1: 800, still be positive, the result is positive for HIV-1 P24 antigen preliminary evaluation, and expression amount is more than μ g/ml level.

6.2 do the antigenic affirmation experimental identification of P24 by Western-blot

(1) obtains expression product according to the ultrasonic broken bacterium of step 3.1.

(2) proteinic SDS-polyacrylamide gel electrophoresis

Sheet glass is installed, is prepared 12% separation gel, carefully inject in the sheet glass gap overlying water layer, air-isolation, polymerized at room temperature 1-2h.The coverture that inclines, thieving paper blots residual liquid, and preparation 5% concentrates glue, injects the separation gel upper end, carefully inserts comb, avoids bubble to produce polymerized at room temperature 30min.The sample that the last step was extracted mixes with sample-loading buffer, 100 ℃ of heating 3min, and last sample, 100V constant voltage electrophoresis to bromjophenol blue arrive at the separation gel bottom.

(3) change film

Distilled water cleans graphite cake, after drying, wears gloves, cuts 6 3M filter paper and 1 nitrocellulose filter.The size of filter paper and film will equate with the size of gel or fully less than gel.Nitrocellulose filter is floated on the water surface of a dish deionized water, film is immersed in the water fully after borrowing wicking action to make film wetting from the bottom up.Soak 5min to remove bubble residual on the filter membrane and aquation.

In a plate, add a spot of transfering buffering liquid, 6 filter paper are soaked in wherein.Keeping flat the base (+) of Graphite Electrodes, place 3 filter paper that soaked through transfering buffering liquid on graphite, rolls on the filter paper face with a glass stick in the alignment back, to get rid of bubble.Then nitrocellulose filter is placed on the filter paper, guarantees that accurately alignment is avoided leaving bubble between filter paper and nitrocellulose filter.Gel is moved in the deionized water, and rinsing once accurately lies against on the nitrocellulose filter then slightly, removes bubble side by side.3 filter paper of another group are placed on gel top, guarantee that equally each layer accurately aligns and avoid bubble.

The electrophoretic blotting groove upper cap is anchored on Graphite Electrodes-transfer film glue complex body.Connect power supply, 0.6-1.0mA/cm ², making current, Constant Electric Current is changeed 2h.

(4) antigen antibody reaction

A. will carry out nitrocellulose filter after the transfer printing of mark after the PBST rinsing, and add in the suitable confining liquid, 4 ℃ are spent the night.

B. nitrocellulose filter is transferred to confining liquid and be diluted in the diluent of the polyvalent antibody of proper concn or odd contradictive hydroperitoneum room temperature vibration 1h.

C. wash film 5 times with PBST, add HRP-sheep anti mouse or anti-rabbit igg, room temperature vibration 1h with the dilution in 1: 30000 of PBST damping fluid.PBST washes film 5 times, each 15min, and then wash film 4 times, every 5min fully washes film and will reduce background.

D. excess liquid control on the film is done, be placed on the preservative film, isopyknic luminal/enhancer solution and stable peroxide solution are mixed, be poured on the protein powder of film incubated at room 5min.

E. control detection reagent unnecessary on the dry film, wrap with preservative film, with membranin towards on be placed in the film magazine, compressing tablet, the exposure 1min, develop a film, determine the compressing tablet time of next slice, thin piece on this basis.

(5) result

Use the P24 monoclonal antibody and do one anti-ly, enzyme mark goat anti-human igg is two anti-.Result such as Fig. 8: find that with molecular weight of albumen Marker comparison back this band is positioned near the 97KD molecular weight of albumen marker.Insert fragment and HIV (956) insertion fragment sum because the fragment nucleic acid number that we insert is MS2 (1690), estimate to express proteic molecular weight: 110 * (1690+956)/3=97,020 dalton according to formula.The MS2 phage capsid protein on experimental result proof virus-like particle surface, maturing enzyme albumen, the gag albumen of HIV-1 merges.

6.3 capsid protein, gag albumen and nucleic acid RNA form intact virus sample particulate and identify

Principle: the enzyme plate among the Murex HIV Antigen Mab VK86 8E77-01 (P24 antigen detecting agent box) is through HIV-1 P24 antigen polyclonal antibody bag quilt, with expression product and the warm altogether 1h that bathes of polyclonal antibody, the P24 polyclonal antibody is combined with the P24 antigen on virus-like particle surface.Wash plate and remove other materials 10 times, add the combination of glycine (pH3.0) the wash-out antigen-antibody of 100mmol/l.The method that adopts " guanidinium isothiocyanate is in conjunction with phenol-chloroform method " to extract RNA is then extracted the nucleic acid RNA of virus-like particle.Through RT-PCR reaction, detect the RNA whether the HIV-1gag district is arranged in the nucleic acid, do contrast with reverse transcription pipe not.

6.3.1 concrete steps:

(1) expression product was pressed 1: 10,1: 100,1: 1000,1: 10000,1: 100000, after diluting, be labeled as the 2-6 pipe respectively, former times of expression product is labeled as the negative control in the pipe Murex HIVAntigen Mab VK86 8E77-01 test kit No. 1, and positive control is labeled as respectively No. 7, No. 8.

(2) in every hole, add 100 μ l working fluids 1, add 100 μ l samples or yin and yang attribute Quality Control more respectively.

(3) lucifuge is hatched 60min for 37 ℃; Wash plate 10 times.

(4) glycine (pH3.0) the wash-out 1min of adding 100mmol/l.

(5) add PEG6000,4 ℃ of precipitation 3h, 4 ℃, 12,000rpm is centrifugal, and 15min abandons supernatant.

(6) add 50 μ l TE (pH8.0), with resolution of precipitate.

(7) with reference to guanidinium isothiocyanate among the step 2.9.1 in conjunction with phenol-chloroform method " method of extracting RNA extracts the nucleic acid RNA of virus-like particle.

(8) reverse transcription system and condition are with reference to step embodiment 3.

(9) pcr amplification, condition and system be with reference to embodiment 1, and every increment is originally established not the reverse transcription pipe and done contrast.

(10) amplified production is carried out 1.4% agarose gel electrophoresis.

6.3.2 result:

Result such as Fig. 9 owing to wash plate through ten times behind immunosorption, if P24 merges on virus-like particle but free, then can not be adsorbed on virus-like particle on 96 orifice plates.Therefore, prove and contain P24 antigen in the virus-like particle.

Embodiment 7, include the construction and expression of the virus-like particle of sars coronavirus nucleic acid

1. design of primers: amplification SARS-CoV rna gene group partial sequence (CHUK10, primer 18026-18327) is right, seven pairs of primers (PCR primers for SARS developed by WHO networklaboratories.Geneva:World Health.Organization that this is announced for WHO the primer amplification zone, 2003.Accessed one of place two big main region April17 at http://www.who.int/csr/sars/primers/en/), at present both at home and abroad the fragment of test kit augmentation detection many in this zone (Wu Xinwei, Cheng Gang, Di Biao etc., China's laboratory medicine magazine, 2003,26 (5): 300-302).Introduce HindIII restriction enzyme site (underscore part), the primer of anamorphic zone restriction enzyme site respectively at upstream and downstream primer 5 ' end:

Upstream primer, 5 '-AG AAGCTTACCTACACACCTCAGCGTTG-3 ';

Downstream primer, 5 '-AT AAGCTTTAACCAGTCGGTACAGCTAC-3 ';

Then, on Primer 5.0 softwares, move Tm value and other amplification parameter, and synthesize by Beijing hundred victory companies that match to mate each primer.

2.SARS the segmental pcr amplification of genome purpose, TA clone and enzyme are cut product purification with embodiment 1: utilize downstream primer reverse transcription SARS-Cov RNA, the warm start method is carried out pcr amplification, 94 ℃ of 45s; 53 ℃ of 45s; 72 ℃ of 1.0min; Extend 5min after 30 circulations.2.0% agarose gel electrophoresis is observed amplification.Cut the purpose band, carry out TA after glue reclaims and clone, blue hickie screening back is extracted plasmid and is cut with restriction enzyme HindIII enzyme, cuts the glue recovery behind 1.6% agarose gel electrophoresis again and obtains the purpose fragment.Select the LB substratum of hickie inoculation culture Amp+, the extraction plasmid carries out the HindIII enzyme to be cut, and the purpose fragment band of visible 300bp behind the electrophoresis is as Figure 10.

3. the structure of expression vector PNCCL1-SARS-CoV plasmid: with HindIII the PNCCL1 plasmid is carried out enzyme and cut, resulting enzyme is cut product and is directly carried out purifying with the DNA purification kit.Agarose gel electrophoresis is done quantitatively rough.Connect by following linked system: purpose fragment 7 μ l, plasmid 1 μ l, T4DNA ligase enzyme 1 μ l, T4 ligase enzyme damping fluid 1 μ l.4 ℃ of connections are spent the night.Transform bacteria BL21-DE3E.Coli chooses bacterium and cultivates, and extracts plasmid, cuts with the XhoI enzyme, downcuts the segmental positive colony that is decided to be of 1000bp.Positive recombinant is served Hai Shenggong company check order, and determine to insert segmental along in the other direction.The clone of expression vector PNCCL1/the expression site as shown in Figure 1.With the expression vector PNCCL1-SARS-CoV transform bacteria overnight incubation that makes up, extract plasmid and use restriction enzyme HindIII, the Nco1 enzyme is cut, and electrophoretogram is as shown in figure 11.Use restriction enzyme Xho1, HindIII, Nco1 carry out enzyme to plasmid and cut, and obtain 300bp, 721bp, and the electrophoretic band of 970bp and 5200bp, as shown in figure 12.The positive recombinant sequencing result shows behind Pubmed Blast, the insertion fragment in the recombinant plasmid consistent with the gene order of MS2 and SARS-CoV (concordance rate 100%).

4. include the prokaryotic expression and the acquisition of virus-like particle of the anti-RNase of SARS-CoV part RNA sequence: inoculation 1ml contains the LB liquid nutrient medium of kantlex, the 200rm/min shaking culture is spent the night, get nutrient solution 50 μ l inoculation 5ml kalamycin resistance substratum, 200rm/min shaking culture 1.5 hours, add isopropylthio-(IPTG), make the final concentration of IPTG reach 1mM/ml, abduction delivering 16 hours carries out ultrasonic broken bacterium.Promptly get 1.5ml bacterium liquid with the 1.5ml centrifuge tube, 4 ℃, 14, the centrifugal 2min of 000r/min collects bacterium, with the resuspended bacterial precipitation of 0.25ml supersound process damping fluid.Handle the broken bacterium of probe with ultrasonic cell disintegration instrument miniature ultrasonic, then the bacteria breaking thing is put ice bath 1min.Repeat above-mentioned shattering process 5-6 time.With 4 ℃ of bacteria breaking things, 14, the centrifugal 2min of 000r/min gets supernatant and transfers in another clean centrifuge tube.PNCCL1-SARS-Cov is through transforming the phage sample particle that the host bacterium is expressed and supersound process obtained, unite digestion through the two enzymes of RNaseA digestion, DNaseI digestion, RNaseA and DNaseI respectively, do not add 37 ℃ of DNaseI and RNaseA and hatch and do not add the phage sample particle of 4 ℃ of preservations of two kinds of enzymes, then with the digestion product of three kinds of enzymes with without the expression product of enzymic digestion electrophoresis together, electrophoresis result shows that three kinds of enzymic digestion product electrophoretograms have nothing in common with each other, two enzymic digestion samples have only a band line about 1000bp, as shown in figure 13.

5. the evaluation of expression product: get 3 part of 20 μ l expression product and carry out 100U RNaseA and the single enzyme of 2U DNaseI and 37 ℃ of digestion of two enzymes 40min respectively, with the expression product of digestion and indigested expression product electrophoresis respectively, observe the electrophoretogram after more digestion, each single enzymic digestion and the two enzymic digestion.Simultaneously expression product is carried out the patience experiment: get four each 20 μ l of pipe expression product, add the DNaseI of 100-200U, 100URNaseA, 37 ℃ of incubation 4d; 4 ℃ of preservations, expression product two pipes of 4d; Room temperature is placed the pipe of 4d; And the sample of the following processing of three pipe processes: each 20 μ l of expression product, add DNaseI 100-200U, RNaseA 100U, 37 ℃ of incubation 24h, through 95 ℃, the 5min thermo-cracking is destroyed coating and is discharged RNA.Carrying out electrophoresis simultaneously with above-mentioned several samples immediately after the thermo-cracking. after expression product is united digestion 24h with the two enzymes of RNaseA digestion, DNaseI digestion, RNaseA and DNaseI, add 4M isothiocyanic acid guanidinesalt, 1% inositol solution stops endonuclease reaction, SARS-Cov RNA fluorescence RT-PCR test kit with the exploitation of Da An company detects, and operation is undertaken by the test kit specification sheets.This test kit is the real-time fluorescence quantitative PCR test kit, and primer is: PF, 5 '-CGGCAAAATGAAAGAGCTCA-3 '; PR, 5 '-CGCCGTAGGGAAGTGAAGCT-3 '; Probe sequence is: 5 '-CCCAGATGGTACTTCTATTACCTA-3 ', and reaction conditions is: 93 ℃ of pre-sex change of 2min, 93 ℃ of 15s then, 55 ℃ of 15s, 72 ℃ of 20s, 10 circulations; 93 ℃ of 15s then, 58 ℃ of 30s, 30 circulations.Extension increasing sequence 84bp (18164-18247, CHUK-10).For the checking amplified reaction really is due to the RNA, the reaction tubes that directly carries out pcr amplification not carry out reverse transcription in contrast.Expression product patience experiment: get four each 20 μ l of pipe expression product, add the DNaseI of 100-200U, 100URNaseA, 37 ℃ of incubation 4d.The electrophoretogram of expression product patience experiment is as shown in figure 14:

Verify that with reaching the peace test kit result all is positive, the concentration of virus-like particle is greater than 10 ¹⁰Copy/ml.After the one pipe expression product of anti-RNase process DNaseI and RNaseA enzyme are cut, be divided into two parts, carrying out RNA respectively extracts, a through the laggard performing PCR amplification of reverse transcription reaction, another part is then directly carried out pcr amplification without reverse transcription reaction, the result shows: do not carry out the reverse transcription reaction pipe, amplification is negative; And carrying out the reverse transcription reaction pipe, amplification is strong positive.Illustrate that expression product really contains SARS-CoV RNA.

Claims (10)

1. include the virus-like particle of RNA viruses nucleic acid, it is characterized in that the anti-RNase of this virus-like particle, contain coat protein and be included in interior recombination sequence: the initiation site gene order of the maturing enzyme protein gene sequence of MS2 phage, capsid protein gene sequence, packaging site gene order, crack protein and replicative enzyme, RNA viruses nucleotide sequence.

2. virus-like particle as claimed in claim 1, wherein the RNA viruses nucleotide sequence length in the recombination sequence that contains of this virus-like particle is 150～1000bp.

3. virus-like particle as claimed in claim 1 or 2, wherein the RNA viruses nucleotide sequence in the recombination sequence that contains of this virus-like particle is the nucleotide sequence of hepatitis C virus, human immunodeficiency virus or severe acute respiratory syndrome coronavirus.

4. virus-like particle as claimed in claim 1 is characterized in that this virus-like particle contains coat protein, includes the incomplete antigen of RNA viruses and is included in interior recombination sequence: the initiation site gene order of the maturing enzyme protein gene sequence of MS2 phage, capsid protein gene sequence, packaging site gene order, crack protein and replicative enzyme, the conservative encoding sequence of RNA viruses nucleic acid.

5. virus-like particle as claimed in claim 4 is characterized in that this virus-like particle contains the incomplete antigen of HIV-1 virus, and the conservative encoding sequence of the RNA viruses nucleic acid in the recombination sequence in being included in is 500～1000bp, and contains the gag region sequence.

6. as the application of arbitrary described virus-like particle of claim 1～5 application of the outer marking quantitative standard series measured as the positive quality control thing in the RNA viruses nucleic acid determination and RNA viruses outer marking quantitative.

7. the application as arbitrary described virus-like particle of claim 1～5 is to provide lifeless matter to infect the application of dangerous sample as quality evalution between RNA viruses RT-PCR detection kit and chamber, laboratory.

8. as the application of arbitrary described virus-like particle of claim 1～5 application of the interior mark Quality Control thing measured as the interior mark standard in the RNA viruses quantitative assay and RNA viruses RT-PCR.

9. the application of virus-like particle as claimed in claim 5 is as the viral and interactional model of host cell as research, be used as the instrument of directed transportation small-molecule drug, sense-rna, have the corresponding antigen epi-position to be used for studying the application of vaccine at the virus-like particle surface expression.

10. as the preparation method of arbitrary described virus-like particle of claim 1～5, the method includes the steps of: make up plasmid; Designated rna virus primer sequence; Pcr amplification, enzyme are cut and are obtained the purpose fragment; Be connected with plasmid, transform; The inducing and expressing of virus-like particle.

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