CN102191223A - Preparation method and applications of H5N1 subtype bird flu pseudovirion - Google Patents
Preparation method and applications of H5N1 subtype bird flu pseudovirion Download PDFInfo
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Abstract
The invention relates to a preparation method and applications of an H5N1 subtype bird flu pseudovirion. Specifically, the application relates to a bird flu pseudovirion with packaging plasmid and the HA gene and NA gene of the highly virulent form of the bird flu virus and a preparation method and applications thereof; and the application also relates to a system for preparing the pseudovirion.
Description
Technical field
The present invention relates to the preparation method and its usage of H5N1 subtype avian influenza pseudovirus.
Background technology
(avian influenza AI) is the disease syndrome that a kind of poultry that caused by A type influenza virus and wild fowl infect in bird flu.(avian influenza virus AIV) belongs to orthomyxoviridae family to its cause of disease avian influenza virus, and Influenza Virus is the sub-thread minus-stranded rna virus.To A type influenza virus, antigenic different according to its surface glycoprotein hemagglutinin (HA) with neuraminidase (NA), again influenza virus is further divided into different hypotypes.At present, the A C-type virus C has 16 kinds of HA hypotypes (H1-H16) and 9 kinds of NA hypotypes (N1-N9), and these hypotypes all exist in avian influenza virus.
Avian influenza virus is divided into two classes again according to viral toxicity: Highly Pathogenic Avian Influenza Virus (HPAIV) (HPAI), and it can cause the deadly infection of general, kills birds in infection in back 24 hours the soonest, and is deadly in a week usually; Lowly Pathogenic Avian Influenza Virus (LPAI), it seldom can cause the outburst of serious disease, relevant morbidity and lethality rate are also low than HPAI.
In May, 1997, a strain H5N1 AIV separates in the three years old boy in Hong Kong and obtains, and boy finally dies from the serious grippe pneumonia that is caused by the REYE syndromes, and this shows that the birds of carrying the HPAI H5N1 virus can directly pass virus to the mankind.The most serious 10 countries once breaking into present 2003-2004 Asia relevant with HPAI H5N1 cause about 1.5 hundred million bird death.Although the outburst of HPAI virus is major limitation (mainly being the chicken class) in poultry in most countries, but exist potential may pass to the mankind, Vietnam, 53 examples take place and infect the dead case of HPAI virus in Thailand and Cambodia three countries altogether, and the lethality rate of current outburst is higher than that time in 1997 (54.6% pair 33.3%, sufferer numeral according to reports of official resources) far away.The outburst of bird flu has caused that world wide is interior to Highly Pathogenic Avian Influenza Virus (HPAIV) extensive infectious worry in no immunizing power crowd, presses for people Highly Pathogenic Avian Influenza Virus (HPAIV) is carried out systematic study.
Pseudovirus (pseudo type virus) is meant that a kind of virus can integrate the envelope glycoprotein of another different sorts virus, thus the coating that forms with exogenous virus, and genome is keeping the virus [4] of viral genomic characterization own.The pseudovirus The Application of Technology mainly is based on 2 points: promptly a kind of have its cells infected of virus of cyst membrane to be determined by its envelope protein, and secondly, a kind of coating of virus can be incorporated into the surface of another virion.
Pseudovirus infects system's easy handling, and it can only carry out the single cycle and duplicates in cell, and security is good; It simulates the process of natural viral infection cell under external environment, has host range widely, higher transfection efficiency, the attack that also has the opposing serum complement, advantages such as the efficient transfection resting cell in acellular cyclin dependent ground, thereby pseudovirus enters aspects such as process, acceptor evaluation, neutralizing antibody detection, vaccine evaluation in research virus and has vital role.Abroad, to some dangerous virus formulations a series of be the pseudovirus system of carrier with the retrovirus, as the human immunodeficiency virus, hepatitis C virus, Ebola virus, sars coronavirus etc.
There are some kinds of pseudovirus preparation systems at present, common pseudovirus system as on murine leukemia virus (MuLV) basis, making up.For the MuLV system, mainly form by 3 plasmids, be respectively MuLV geneome plasmid (comprising packaging signal, marker gene LacZ), MuLV structural protein expression plasmid (gag, pol) and external source cyst membrane expression plasmid.When with three kinds of plasmid cotransfection HEK293T (human embryo kidney (HEK) 293T) cell, will produce virus-like particle in the middle of the supernatant, these replication-defective virus particles can infect many kinds of cell types.The present invention will adopt a kind of new pseudovirus systems produce method, and inquire into the security and the validity of this method.
Avian influenza virus surface antigen blood clotting poison (HA) and neuraminidase (NA) with virus pathogenic, duplicate and propagate in close relations.The HA surface antigen has mediated the release that combines and promoted in viral fusion process viral RNA of virus with the host cell surface acceptor.The precursor of HA molecule is slit into HA1 and HA2 subunit by the proteolytic cleavage in the host cell after translation, produced a blending inheritance zone at the N-terminal of HA2, has mediated the fusion of virus with cytoplasmic membrane.Therefore, the HA surface antigen plays the key player aspect virus pathogenic, be the prerequisite of virus infected cell.NA is the another kind of important antigen on avian influenza virus surface, and it further infects other host cell by the N-acetyl neuraminic acid of hydrolysis host cell surface specific receptors glycoprotein end thereby promote sophisticated virus to discharge from host cell.
Known H5N1 subtype highly pathogenic avian influenza virus has hyperinfection, and case fatality rate is higher, directly conduct a research and have sizable danger with the H5N1 subtype highly pathogenic avian influenza virus, and need harsh experiment condition, therefore develop the very necessary and urgent need of pseudovirus infection model of H5N1 subtype highly pathogenic avian influenza virus.
Summary of the invention
Pseudovirus of the present invention is with HIV virus packaging plasmid pNL4-3Luc
+Env
-Vpr
-Be the pseudovirus packaging plasmid, this plasmid can expressing luciferase as reporter gene, Vpr is the replicase protein gene, its defective causes virus to duplicate, the envelope glycoprotein of Env coding virus, its defective make this plasmid membranin that is beyond expression, disappearance in the time of Env and Vpr gene, the ability that makes this plasmid not have infection ability and further duplicate the virus that produces a new round guarantees biological safety.The present invention is by polymerase chain reaction (PCR) method, the HA and the NA full-length gene of H5N1 avian influenza virus are connected into an eukaryotic expression vector pBudCE4.1, compare two genes are connected into different carriers, this has guaranteed the consistence of gene expression efficiency, and this plasmid is packed out filial generation bird flu pseudovirion as external source target film protein gene with packaging plasmid cotransfection HEK293T cell.This pseudovirion has the function that infects avian influenza virus permissive cell system (as MDCK).Detect expressed luciferase behind the virus infected cell by vacation, just can reflect the infection conditions and and then the validity of judgement pseudovirus of pseudovirus pair cell quantitatively.Finish the present invention thus.
Therefore, the application provides a kind of bird flu pseudovirus, and this virus contains the HA gene and the NA gene of packaging plasmid and highly pathogenic avian influenza virus.
In a specific embodiment, described packaging plasmid is expressed reporter gene.
In a specific embodiment, the plain enzyme of described reporter gene coding fluorescence.
In a specific embodiment, described packaging plasmid is selected from pNL4-3Luc
+Env
-Vpr
-PHR ' CMV-Luc and pCMVR Δ 8.2.
In a specific embodiment, the HA gene of described highly pathogenic avian influenza virus and NA gene are selected from the HA gene and the NA gene of avian influenza virus H1 hypotype, H5 hypotype and H7 hypotype.
In a specific embodiment, described bird flu pseudovirus is that deposit number is the bird flu pseudovirus of CCTCC NO:V200917.
The application also provides a kind of method for preparing the bird flu pseudovirus, and this method comprises:
Use contains the HA gene of highly pathogenic avian influenza virus and the plasmid and the packaging plasmid cotransfection host cell of NA gene,
Cultivate this host cell and
From the substratum supernatant, collect the bird flu pseudovirus.
In a specific embodiment, described packaging plasmid is selected from pNL4-3Luc
+Env
-Vpr
-PHR ' CMV-Luc and pCMVR Δ 8.2.
In a specific embodiment, the HA gene of described highly pathogenic avian influenza virus and NA gene are selected from the HA gene and the NA gene of avian influenza virus H1 hypotype, H5 hypotype and H7 hypotype.
In an embodiment, described host cell is selected from HEK293T cell and 293FT cell.
In an embodiment, described HA gene is contained in the identical plasmid with the NA gene.
In a specific embodiment, use eukaryotic expression vector to make up the plasmid that contains HA gene and NA gene simultaneously.
In an embodiment, described eukaryotic expression vector is selected from pBudCE4.1, pcDNA3.1 and pCMV/R.
The application also provides the purposes of bird flu pseudovirus, is used to measure tiring or seeking avian influenza virus surface neutralizing antibody bonded epi-position of the intravital neutralizing antibody of the infected, perhaps is used for external drug screening or vaccine effect evaluation.
In an embodiment, described bird flu pseudovirus is that deposit number is the bird flu pseudovirus of CCTCC NO:V200917.
The application also provides a kind of bird flu pseudovirus preparation system, and this system comprises packaging plasmid and contains the HA gene of highly pathogenic avian influenza virus and the plasmid of NA gene.
In an embodiment, described HA gene and NA gene are in respectively independently in the plasmid.
In an embodiment, described HA gene and NA gene are in the same plasmid.
In an embodiment, described HA gene and NA gene are provided by pGEMT-NA and pGEMT-H plasmid respectively.
In an embodiment, described HA gene and NA gene are in the same eukaryotic expression vector.
In an embodiment, described eukaryotic expression vector is selected from pBudCE4.1, pcDNA3.1 and pCMV/R.
In an embodiment, described packaging plasmid is selected from pNL4-3Luc
+Env
-Vpr
-PHR ' CMV-Luc and pCMVR Δ 8.2.
In an embodiment, described system also comprises a specification sheets, is used for the described method for preparing the bird flu pseudovirus of conduct the application.
According to the application, H5N1 subtype avian influenza pseudovirus does not have replication, can not cause the specific cell pathology that the H5N1 subtype avian influenza virus causes yet, and therefore can at utmost reduce the various risks in the H5N1 subtype avian influenza virus research process.
And, because the biting property of parent of pseudovirus is identical with euvirus with course of infection, therefore the early process that can simulated virus infects, and carry reporter gene in the pseudovirus, can carry out various detections and analysis easily apace, therefore pseudovirus not only can be used for studying the relation of virus and host cell, the clone cell surface receptor, the more important thing is and to be used to measure tiring of the intravital neutralizing antibody of the infected, seek H5N1 subtype avian influenza virus surface neutralizing antibody bonded epi-position, estimating the effect of vaccine immunity, is the effective important channel of research deadly infectious disease virus safe.
In addition, thereby utilize approach that this model research H5N1 subtype avian influenza virus infects the whole process of host cell and this process of blocking-up for large-scale external drug screening, the primary dcreening operation of vaccine effect evaluation provides simple and safe method.
In addition, when with NA with the HA gene is connected in the same plasmid and then during with the packaging plasmid cotransfection, can be the application's providing convenience property of method, when preparing pseudovirus, transfection only need packaging plasmid and this carrier to get final product.And, farthest guaranteed the consistent of HA and NA protein translation efficient with a carrier as far as possible, help the successful packing of pseudovirus, according to contriver's experimental result demonstration, the infectious effect ratio that is connected into a carrier is with independently the carrier transfection is effective.In addition,, be connected into the cost that a carrier will prepare plasmid and just saved half, saved cost from the angle of extensive utilization.
Description of drawings
Fig. 1 shows HA and NA PCR product electrophorogram.Wherein, 1:100bp Marker; 2:HA PCR product; 3: blank; 4:NA PCR product; 5:1kb Marker.
Fig. 2 shows that the enzyme of recombinant plasmid cuts evaluation.Wherein, 1:Marker; 2:1# clone BamH I and XhoI enzyme are cut evaluation; 3:3# clone BamH I and Xho I enzyme are cut evaluation; 4:1# clone EcoR I and Xho I enzyme are cut evaluation; 5:3# clone EcoR I and Xho I enzyme are cut evaluation.
Fig. 3 shows combined vaccine immune serum neutralization test result.
Fig. 4 shows in the HA1 three strain monoclonal antibodies and active detected result.
Embodiment
Therefore, the application's first aspect provides a kind of bird flu pseudovirus, and this virus contains the HA gene and the NA gene of packaging plasmid and highly pathogenic avian influenza virus.
In specific embodiment, this plasmid expression reporter gene of described packaging plasmid.In a specific embodiment, the plain enzyme of described reporter gene coding fluorescence.
The application's second aspect provides a kind of preparation method of bird flu pseudovirus, this method comprises that use contains the HA gene of highly pathogenic avian influenza virus and the plasmid and the packaging plasmid cotransfection host cell of NA gene, and collects the bird flu pseudovirus from the substratum supernatant.
The application's third aspect provides a kind of purposes of bird flu pseudovirus, is used to measure tiring or seeking avian influenza virus surface neutralizing antibody bonded epi-position of the intravital neutralizing antibody of the infected.
The application's fourth aspect provides a kind of purposes of bird flu pseudovirus, is used for external drug screening or vaccine effect evaluation.
The application the 5th aspect provides a kind of bird flu pseudovirus preparation system, and this system comprises packaging plasmid and contains the HA gene of highly pathogenic avian influenza virus and the plasmid of NA gene.
In concrete embodiment, the application's bird flu pseudovirus preparation system can comprise (1) packaging plasmid; (2) contain the HA gene of highly pathogenic avian influenza virus and the plasmid of NA gene simultaneously; Perhaps comprise (1) packaging plasmid; (2) contain the plasmid of HA gene and NA gene separately; (3) be used to be connected into the empty carrier of HA gene and NA gene.
The application's bird flu pseudovirus preparation system also can contain other relevant reagent, for example primer, enzyme, conversion reagent etc.
The application the 6th aspect provides a kind of culture, and this culture contains the application's bird flu pseudovirus and host cell.This culture also can contain the substratum that is suitable for this host cell growth.
Herein, highly pathogenic avian influenza virus comprises various H1 hypotypes, H5 hypotype and H7 hypotype, includes but not limited to H5N1 hypotype etc.Therefore, bird flu pseudovirus of the present invention can comprise the HA gene and the NA gene of H5 hypotype or H7 hypotype, for example contains the HA gene and the NA gene of H5N1 hypotype.
The HA gene of highly pathogenic avian influenza virus and NA gene are that prior art is known; its nucleotide sequence can obtain from Genebank; the plasmid that comprises HA gene or NA gene that also can buy from the market obtains, and this class plasmid includes but not limited to pGEMT-HA plasmid and pGEMT-NA plasmid.
The HA gene and the NA gene that are used for the application comprise its varient, as long as this varient still can keep the function of wild-type HA gene and NA gene.For example, the application comprises having certain sequence homogeny with the HA gene of H5N1 hypotype and NA gene and possess the HA gene of H5N1 hypotype and the varient of the biologic activity of NA gene.Described homogeny is at least, for example 80%, 85%, 90%, 95%, 97%, more than 99%.
Usually, " homogeny " refers on two polynucleotide or the peptide sequence that accurately nucleotide pair Nucleotide or amino acid are to the amino acid correspondence.Sequence by arranging two molecules is their sequence information relatively directly, calculates the accurate quantity that mates between the sequence of two arrangements, and it divided by the length of short sequence, be multiply by 100 then, thereby can obtain homogeny percentage ratio.
In homology and homogeny analysis, can assist and use the computer program that is easy to obtain, (Atlas of Protein Sequence and Structure, M.O.Dayhoff edit as ALIGH, Dayhoff, M.O., 5 Suppl., 3:353-358, National Biomedical Research Foundation, Washington, DC), it is applicable to that Smith and Waterman analyze local homology's algorithm (Advances in Appl.Math., 2:482-489,1981) that peptide is used.Can be from Wisconsin Sequence Analysis Package (the 8th edition, from Genetics Computer Group, Madison, the WI acquisition) program of nucleotide sequence homology is measured in acquisition, for example, BESTFIT, FASTA and GAP program, these programs also depend on Smith and Waterman algorithm.That uses producer's suggestion can easily use these programs with the described default parameters of above-mentioned Wisconsin Sequence Analysis Package.For example, can use the nucleotide sequence that the interval point penalty (gap penalty) of the acquiescence score-sheet of homology algorithm of Smith and Warerman and 6 nucleotide positions measures and the homology percentage ratio of reference sequence.
The other method that the present invention sets up homology percentage ratio be to use copyright belong to the Edinburgh University, by John F.Collins and Shane S.Sturrok exploitation, by IntelliGenetics, Inc. (Mountain View, CA) Fa Hang MPSRCH routine package.The Smith-Waterman algorithm can overlap in the routine package at this and use, and wherein, uses default parameters (for example, point penalty=1 is extended, at interval=6 at interval at interval open point penalty=12) in score-sheet." coupling " value that produces from this batch data reflects " sequence homology ".Homogeny percentage ratio between the sequence of calculation or other suitable procedure of similarity percentage ratio generally all are known in the art, and for example, another kind of alignment problem is BLAST, use default parameters.For example, can use the BLASTN and the BLASTP of following default parameters: genes encoding=standard; Filtration=nothing; Chain=two; Hold back=60; Expected value=10; Matrix=BLOSUM62;=50 sequences are described; Ordering=HIGH SCORE; Database=irredundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+Swiss albumen+Spupdate+PIR.On http://www.ncbi.nim.gov/cgi-bin/BLAST network address, can find the detailed description of these programs.
Perhaps, under the condition that forms stable two strands between the homology zone, carry out multi-nucleotide hybrid,, measure the segmental size of digestion then then with the enzymic digestion of strand specific nucleic acid, thus the homology of measuring.In as the Southern cross experiment that carries out under (defined) stringent condition, can differentiate basic homologous dna sequence dna to concrete system.Determine that suitable hybridization conditions is within the knowledge that those skilled in the art grasped.For example, referring to Sambrook etc., the same; DNA Cloning, the same; Nucleic Acid Hybridization, the same.
Can adopt conventional method to obtain to be used for the application's HA gene and NA gene.Then it is cloned into respectively in the identical plasmid.For example; in the application's a embodiment; obtain NA gene and HA gene from pGEMT-NA plasmid and pGEMT-HA plasmid respectively; then earlier with the NA gene clone in the pBudCE4.1 carrier; obtain the pBudCE4.1-NA plasmid; and then with the HA gene clone in the pBudCE4.1-NA plasmid, thereby obtain to contain simultaneously the plasmid of HA gene and NA gene.
The carrier that is used to be connected into NA gene and HA gene comprises various eukaryotic expression vectors, includes but not limited to pBudCE4.1 (American I nvitrogen company product), pcDNA3.1 (American I nvitrogen company product), pCMV/R (U.S. Promega company product).
The carrier that the present invention can be made up is transformed into escherichia coli DH5 α according to a conventional method, thereby carries out the amplification and the purifying of plasmid, and plasmid is checked order.The available DNA STAR of dna sequencing result software analysis, with Influenza A virus among the gene bank (A/Chicken/Henan/12/2004 (H5N1)) sequence alignment, whether correct in order to judge vector construction.
Make up thus the expression plasmid obtain can with the packaging plasmid cotransfection host cell.The preferable reporter gene that contains of packaging plasmid, for example luciferase gene.Packaging plasmid commonly used for example is pNL4-3Luc+Env-Vpr-.(two plasmids use together, construction process reference Naldini et al., 1996 can also to use pHR ' CMV-Luc and pCMVR Δ 8.2; Zufferey et al.1997; Naldini, L., Blomer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F.H., Verma, I.M., Trono, D., 1996.In vivogene delivery and stable transduction of nondividing cells by alentiviralvector.Science 272,263-267; Zufferey, R., Nagy, D., Mandel, R.J., Naldini, L., Trono, D., 1997.Multiply attenuated lentiviral vectorachieves efficient gene delivery in vivo.Nat.Biotechnol.15,871-875).
Host cell is selected from the HEK293T cell usually, can also use 293FT (the 293FT cell be a kind of on the 293F cell base improved clone, have very high growth multiplication rate).
The transfection method that is used for the application is conventional, for example can be referring to " molecular cloning experiment guide " (third edition, Science Press).
Can under the condition that is suitable for this host cell growth, cultivate the host cell of this transfection, and can adopt the pseudovirus in the conventional method collection host cell culture supernatant, for example, adopt conventional centrifugal method to get final product.
The pseudovirus that is obtained can adopt conventional method validation, for example, verifies by detecting entrained reporter protein of this pseudovirus such as luciferase.Also can be by in infection experiment and the antibody and verify the infectivity and the neutralizing antibody activity of pseudovirus.
In one embodiment, adopt the application's preparation method to prepare H5N1 subtype avian influenza pseudovirus ABRC (pseudo-type H5N1 avian influenza virus-ABRC), this pseudovirus has been preserved in Chinese typical culture collection center (CCTCC on December 27th, 2009, China Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University preservation center, 430072), deposit number is CCTCC NO:V200917.
Therefore, the preparation method of the application's bird flu pseudovirus can comprise:
(1) provides highly pathogenic avian influenza virus HA gene and NA gene;
(2) connect into described HA gene and NA gene in the eukaryotic expression vector altogether;
(3) step (2) gained is comprised the eukaryotic expression vector and the packaging plasmid cotransfection host cell of HA gene and NA gene; With
(4) cultivate the host cell of transfection and from cell conditioned medium, reclaim pseudovirus.
Can from the plasmid that contains virulence avian influenza virus HA gene and NA gene, provide described HA gene and NA gene respectively, then it be connected in the eukaryotic expression vector altogether.
Enforcement of the present invention will be used chemistry well known by persons skilled in the art, biological chemistry, recombinant DNA technology and immunologic ordinary method unless otherwise indicated.These technology have complete explanation in the literature.Referring to, as " basic immunology " (Fundamental Virology), second edition, I and II volume (B.N.Fields and D.M.Knipe compile); " experiment immunization is learned handbook (Handbook of Experimental Immunology), I-IV volume (D.M.Weir and C.C.Blackwell compile, Blackwell ScientificPublications); T.E.Creighton, " protein: structure and molecular characterization " (Proteins:Structures and Molecular properties) (W.H.Freeman and Company, 1993); A.L.Lehninger, " biological chemistry " be (Worth Publishers, Inc. latest edition) (Biochemistry); Sambrook etc., " molecular cloning: laboratory manual " (Molecular Cloning:aLaboratory Manual), second edition, 1989; " Enzymology method " (Methods in Engymology) (S.Colowick and N.Kaplan compile, Academic Press, Inc.).
The clone of embodiment 1:H5N1 subtype highly pathogenic avian influenza virus HA and NA protein gene
1.1 amplify the dna sequence dna of NA by PCR method
The PCR design of primers of clone NA gene, according to the pGEMT-NA gene that has checked order [Influenza Avirus (A/Chicken/Henan/12/2004 (H5N1))] sequence, according to the design of primers principle, use Primer premier5.0 software design primer, primer is given birth to worker Bioisystech Co., Ltd by Shanghai and is synthesized.Sequence is respectively:
Sp 5’-CCC
AAGCTTATGAATCCAAATCAGAAG-3’(SEQ ID NO：1)
As 5’-GC
TCTAGACTACTTGTCAATGGTGAATGG-3’(SEQ ID NO：2)
X underscore in the primer sequence is represented restriction enzyme HindIII and Xba I site respectively.
1.2 the PCR product is connected to pBudCE4.1 carrier (available from Invitrogen company)
Be inserted into the pBudCE4.1 carrier of cutting through same enzyme after cutting with restriction enzyme HindIII and Xba I enzyme, be positioned at the CMV promotor after, obtain pBudCE4.1-NA.
1.3 amplify the dna sequence dna of HA by PCR method
The PCR design of primers of clone HA gene, according to the pGEMT-HA[Influenza A virus that has checked order (A/Chicken/Henan/12/2004 (H5N1))] sequence, according to the design of primers principle, use Primerpremier5.0 software design primer, primer is given birth to worker Bioisystech Co., Ltd by Shanghai and is synthesized.Sequence is respectively:
Sp 5’-ATAAGAAT
GCGGCCGCATGGAGAAAATAGT-3’(SEQ ID NO：3)
As 5’-CCG
CTCGAGAGAAATGCAAATTCTG-3’(SEQ ID NO：4)
X underscore in the primer sequence is represented restriction enzyme Not I and Xho I site respectively.
1.4 the PCR product is connected to the pBudCE4.1 carrier
Be inserted into the pBudCE4.1-NA carrier of cutting through same enzyme after cutting with restriction enzyme Not I and Xho I enzyme, be positioned at EF-1 α promotor after, obtain pBudCE4.1-NA-HA.
1.5, thereby carry out the amplification of plasmid and purifying and send the order-checking of order-checking company with plasmid with the carrier ordinary method transformed into escherichia coli DH5 α of aforementioned structure.
1.6DNA sequencing result DNA STAR software analysis, (the GenBank accession number of HA is AY950233.1 with Influenza A virus among the gene bank (A/Chicken/Henan/12/2004 (H5N1)) sequence, the GenBank accession number of NA is AY950248.1) comparison, judge that vector construction is correct.
Experimental result is seen Fig. 1 and 2, verifies figure as a result by PCR product figure and plasmid enzyme restriction, judges the success of pseudovirus plasmid construction, and the proteic gene of HA and NA is successfully cloned and is cloned on the carrier pBudCE4.1.
Liposome transfection method (available from Invitorgen lipofectamine2000) is adopted in the preparation of embodiment 2:H5N1 subtype avian influenza pseudovirus
2.1 will need the plasmid pNL4-3Luc of transfection
+Env
-Vpr
-(" Conserved amino acids W423and N424 in receptor-binding domain of SARS-CoV are potential targets fortherapeutic monoclonal antibody ", Chao Bian etc., Virology 383 (2009) 39-46; " Analysis of hemagglutinin-mediated entry tropism of H5N1 avianinfluenza ", Ying Guo etc., Virology Journal 2009,6:39; Vpr Is Required forEfficient Replication of Human Immunodeficiency Virus Type-1 inMononuclear Phagocytes, Connor etc., Vigology 206,935-944 (1995)), pBudCE4.1-NA-HA, egfp expression plasmid pGFP (" Hemagglutinin pseudotypedlentiviral particles:Characterization of a new method for avian H5N1influenza sero-diagnosis ", Isabelle Nefkens etc., Journal of ClinicalVirology 39 (2007) 27-33), vesicular stomatitis virus G protein expressing plasmid pVSVG (" InfectiousHepatitis C Virus Pseudo-particles Containing Functional E1-E2 EnvelopeProtein Complexes " Birke Bartosch etc., Journal of Experimental Medicine 197 (2003) 633-642) adopt a large amount of extractions of test kit and purifying (QIAGEN plasmid midipurification kit), preparation transfection plasmid.
2.2 get 1. pGFP of plasmid, 2. pBudCE4.1-NA-HA, pNL4-3,3. pVSVG, pNL4-3 and 4. empty pBudCE4.1, pNL4-3 adopt the method for liposome transfection to press the hereinafter difference of the amount shown in the 2.3rd part transfection 293T cell, with pGFP is the transfection positive control, with the empty carrier is the pseudovirus negative control, with the positive control (VSVG to any cell have very strong infectivity) of VSVG as pseudovirus.
2.3 transfection amount
1. change pGFP 10ug; Liposome 25ul
2. change pNL4-3Luc
+Env
-Vpr
-5ug, pBudCE4.1-NA-HA 5g; Liposome 25ul
3. change pNL4-3Luc
+Env
-Vpr
-5ug, pVSVG 5ug; Liposome 25ul
4. change pNL4-3Luc
+Env
-Vpr
-5ug, empty pBudCE4.1 5ug; Liposome 25ul
2.4 the results of pseudovirus and simple purification
Transfection HEK293T cell according to the above ratio, transfection after 48 hours 3000 change 5 minutes simply centrifugal, collect the pseudovirus supernatant, frozen in-70 ℃ or carry out that mdck cell is infectious to be measured at once.
Transfection is observed after 48 hours and observe the hole of changeing pGEP under fluorescent microscope, and 80% cell expressing green fluorescent protein (GEP) shows that transfection efficiency is higher.
The infective mensuration of embodiment 3:H5N1 bird flu pseudovirus (is example with 24 orifice plates)
Experimental technique:
1, infect the day before yesterday of bed board in 24 orifice plates, making every hole mdck cell number is 5 * 10
4
2, behind the 24h, draw original training liquid, every hole adds 100 μ l pseudovirus supernatants (being three kinds of pseudovirus supernatants of embodiment 2 preparations) and the mixture of 900 μ lMDCK cell culture fluids infects mdck cell.
3, behind the infection 24h, add the fresh mdck cell nutrient solution of 1ml.
4, behind the infection 48h, abandon supernatant, add lysate (Promega product), cracking under the normal temperature by 100 μ l/ holes.
5, will go up the cracked cell conditioned medium is transferred in the 1.5mlEP pipe, 12000 left the heart 10 minutes, get 10 μ lysis supernatants then, 10 μ l luciferase Assay Reagent are (available from promega company, article No. E1500), mix the back rapidly and use the fluorescence detector fluorescence intensity, repeat the record experimental result twice.
Experimental result sees the following form 1:
Table 1: pseudovirus infects back luciferase expression value
VSVG 263577 234334 248955.5 20677.923602
H5N1 15490 15117 15303.5 263.75082938
Negative control 3,369 2,749 3,059 438.40620434
Blank 159 263 211 73.539105243
Interpretation of result: blank is a cell conditioned medium, negative control is the virus that empty pBudCE4.1 carrier package comes out, can analyze from above-mentioned experimental result, compare positive control VSVG, though infectivity not as good as VSVG the H5N1 pseudovirus of preparation has certain infection ability, in external process that can the simulated virus infected cell.Adopt the method for separating transfection HA and NA expression plasmid, under all the same situation of other conditions, the luciferase fluorescent value of twice experiment gained is respectively 1042 and 1192.
This H5N1 pseudovirus called after H5N1 subtype avian influenza pseudovirus ABRC (pseudo-type H5N1 avianinfluenza virus-ABRC), this pseudovirus has been preserved in Chinese typical culture collection center (CCTCC on December 27th, 2009, China Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University preservation center, 430072), deposit number is CCTCC NO:V200917.
Neutralizing antibody activity in the serum after the immunity of experimental example 4:H5N1 bird flu pseudovirus detection combined vaccine
With H5N1 DNA combined vaccine immune mouse.This vaccine constitutes p-HA for the viral HA gene DNA of coding A/Chick n/H nan/12/2004 (H5N1) vaccine, the gene of HA utilizes NotI and XholI double enzyme site to be connected to the EF-1a promotor back of pBudCE4.1; Adopt German QIAGEN company in take out-take out greatly test kit and obtain a large amount of P-HA as dna vaccination; Carry out intramuscular injection in mouse (female Balb/C mouse in 4-6 age in week) two back leg upper arm musculus quadriceps, PBS/ mouse of 50 μ g/100 μ l, after the injection, use the electrode of 5mm distance at once, the muscle that inserts injection areas in the pin hole both sides shocks by electricity, and the electric shock instrument is El ctro SquarPorator T 30M; BTX, San Di go, CA.Every three all immunity once, altogether immunity is three times, gets the detection that mice serum is used for present embodiment, this serum called after HA3# in immune for the third time back 7 days.
The detection step is as follows:
1. mdck cell bed board (24 orifice plate), 5 * 10
4Individual/hole
2. behind the 24h, pseudovirus+900ul of 100ul is diluted good vaccine immunity serum (with training liquid doubling dilution, extent of dilution 1: 100,500,1000,2000,4000,8000), put in the incubator 37 ℃ 1 hour, then original mdck cell training liquid is siphoned away, add the training liquid that contains pseudovirus and dilution antibody.
System's contrast: VSVG+900ul trains liquid
Positive control: H5N1 pseudovirus+900ul trains liquid
Negative control: H5N1 pseudovirus+900ul training liquid+irrelevant serum
The pseudovirus of transfection empty carrier+900ul trains liquid
Blank: directly add training liquid
3. behind the 48h, add the fresh training liquid of 1ml, add antibody (extent of dilution 1: 100,500,1000,2000,4000,8000) in proportion in the training liquid
4. behind the 72h, survey the luciferase fluorescent value, detection method is with embodiment 3.
Experimental result is as shown in table 2 below.
Table 2: pseudovirus detects neutralizing antibody activity in the combined vaccine mice immunized serum
VSVG 263577 234334 248955.5 20677.923602
H5N1 15490 15117 15303.5 263.75082938
Negative control 3,369 2,749 3,059 438.40620434
Blank 159 263 211 73.539105243
Irrelevant serum 11,978 11,878 11,928 70.710678119
Interpretation: from above-mentioned interpretation, serum behind the vaccine immunity has certain neutralizing antibody activity, along with the continuous decline of antibody dilution ratio, the infectivity of virus also constantly descends, and illustrates that this serum has the ability of certain blocking virus infected cell.Comparatively speaking, irrelevant serum can not be blocked infecting of pseudovirus.
Experimental example 5:H5N1 bird flu pseudovirus detects the neutralization activity of monoclonal antibody
Detect HA1 protein immunization with prokaryotic expression, and the neutralization activity of three strain monoclonal antibodies of the HA1 protein screening by prokaryotic expression, whether can filter out in order to the albumen of judging prokaryotic expression and to have the active antibody of neutralization, three strain monoclonal antibodies are own prepared in laboratory, difference called after 98#, 157#, 145# (perhaps use the antibody of article No., see www.antibodies-online.com) as ABIN235650 or ABIN235652.
Experimental technique
1. mdck cell bed board (24 orifice plate), 5 * 10
4Individual/hole
2. behind the 24h, pseudovirus+900ul of 100ul is diluted good vaccine immunity serum (with training liquid doubling dilution, extent of dilution 1: 500,1000,2000), put in the incubator 37 ℃ 1 hour, then original mdck cell training liquid is siphoned away, add the training liquid that contains pseudovirus and dilution antibody.
System's contrast: VSVG+900ul trains liquid
Positive control: H5N1 pseudovirus+900ul trains liquid
Negative control: H5N1 pseudovirus+900ul training liquid+irrelevant serum
The pseudovirus of transfection empty carrier+900ul trains liquid
Blank: directly add training liquid
3. behind the 48h, add the fresh training liquid of 1ml, add antibody (extent of dilution 1: 500,1000,2000) in proportion in the training liquid
4. behind the 72h, survey the luciferase fluorescent value, detection method is with embodiment 3.
The result is presented in the following table 3.
Table 3: pseudovirus detects the neutralizing antibody activity of associating three strain monoclonal antibodies
Blank 220 192 206 19.798989873
Negative control 830 3,700 2,265 2029.396462
H5N1 944001 1210325 1077163 188319.50639
Positive serum 1: 2,000 228,686 196,804 212,745 22543.978398
Positive serum 1: 1,000 33,241 28,792 31016.5 3145.9180695
98#1∶500 896501 1016522 956511.5 84867.662985
98#1∶1000 685896 868423 777159.5 129066.07945
98#1∶2000 850840 890260 870550 27874.149314
157#1∶500 702574 672574 687574 21213.203436
157#1∶1000 1013693 1490089 1251891 336862.84213
157#1∶2000 1018750 932475 975612.5 61005.637547
145#1∶500 1131068 1000068 1065568 92630.988335
145#1∶1000 891936 995591 943763.5 73295.153404
145#1∶2000 1049224 1253406 1151315 144378.4768
Irrelevant serum 1: 500 1,363,406 1,361,106 1,362,256 1626.3455967
Irrelevant serum 1: 1,000 900,510 951,655 926082.5 36164.976324
Irrelevant serum 1: 2,000 1,264,273 1,563,686 1413979.5 211716.96268
Interpretation: compare (this serum contains neutralizing antibody) with positive serum, three strain monoclonal antibodies all can not fine blocking-up pseudovirus infect, the albumen of prokaryotic expression illustrates owing to, can not well be induced the generation of neutralizing antibody because the difference of protein translation effect.
Experiment conclusion: by experiment 3,4,5, illustrate that H5N1 subtype avian influenza pseudovirus can be used for the research of bird flu neutralizing antibody as effective detection means.
The foregoing description only is illustrative, and nonrestrictive.Protection scope of the present invention will be limited by claims of the application.
Sequence table
＜110〉Shanghai Inst. of Life Science, CAS
＜120〉preparation method and its usage of H5N1 subtype avian influenza pseudovirus
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Claims (10)
1. bird flu pseudovirus, this virus contains the HA gene and the NA gene of packaging plasmid and highly pathogenic avian influenza virus.
2. bird flu pseudovirus as claimed in claim 1 is characterized in that, described packaging plasmid expressing luciferase is as reporter gene.
3. bird flu pseudovirus as claimed in claim 1 or 2 is characterized in that described packaging plasmid is selected from pNL4-3Luc
+Env
-Vpr
-PHR ' CMV-Luc and pCMVR Δ 8.2.
4. as each described bird flu pseudovirus among the claim 1-3, it is characterized in that the HA gene of described highly pathogenic avian influenza virus and NA gene are selected from the HA gene and the NA gene of avian influenza virus H1 hypotype, H5 hypotype and H7 hypotype.
5. as each described bird flu pseudovirus among the claim 1-4, it is characterized in that described bird flu pseudovirus is that deposit number is the bird flu pseudovirus of CCTCC NO:V200917.
6. a method for preparing the bird flu pseudovirus is characterized in that, this method comprises:
Use contains the HA gene of highly pathogenic avian influenza virus and the plasmid and the packaging plasmid cotransfection host cell of NA gene,
Cultivate this host cell and
From the substratum supernatant, collect the bird flu pseudovirus.
7. method as claimed in claim 6 is characterized in that described packaging plasmid is selected from pNL4-3Luc
+Env
-Vpr
-PHR ' CMV-Luc and pCMVR Δ 8.2.
8. the purposes of bird flu pseudovirus is characterized in that, is used to measure tiring or seeking avian influenza virus surface neutralizing antibody bonded epi-position of the intravital neutralizing antibody of the infected, perhaps is used for external drug screening or vaccine effect evaluation.
9. purposes as claimed in claim 8 is characterized in that, described bird flu pseudovirus is that deposit number is the bird flu pseudovirus of CCTCC NO:V200917.
10. a bird flu pseudovirus preparation system is characterized in that, this system comprises packaging plasmid and contains the HA gene of highly pathogenic avian influenza virus and the plasmid of NA gene.
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| CN104830908A (en) * | 2015-06-02 | 2015-08-12 | 中国食品药品检定研究院 | Pseudovirus packaging system and application thereof |
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| CN103376317A (en) * | 2012-04-12 | 2013-10-30 | 徐超 | On-site determination method for applicability and economical efficiency of different vaccines |
| CN103376317B (en) * | 2012-04-12 | 2015-11-18 | 徐超 | The on-the-spot decision method of different vaccine applicability and economy |
| CN104513820A (en) * | 2013-09-30 | 2015-04-15 | 中国人民解放军军事医学科学院微生物流行病研究所 | DNA fragment and application thereof in preparation of H5N1-subtype flu Guassia luciferase reporter virus |
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| CN105177043A (en) * | 2015-09-17 | 2015-12-23 | 杭州庆正鸿科技有限公司 | Preparation method of H7N9 pseudoviruses and applications of H7N9 pseudoviruses |
| CN110184284A (en) * | 2019-05-22 | 2019-08-30 | 华南农业大学 | The recombinant fowl influenza virus for carrying NanoLuc gene and its application in living imaging mouse model |
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Application publication date: 20110921 |