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1、病毒制备与转染Virus Production and Transduction,实用生物医学实验技术,病毒的定义,Avirus(the word is from theLatinvirusreferring topoisonand noxious substances)is a smallinfectious agentthat replicates only inside the living cellsof other organisms.,Viruses can infect all types of life forms,from animalsandplantstobacteria
2、andarchea,SARS病毒,艾滋病病毒,致病之毒素，极其微小，结构简单、以寄生、复制进行繁殖的一类非细胞型微生物,Virus particles(known asvirions)consist of two or three parts:thegenetic materialmade from eitherDNAorRNA,longmoleculesthat carry genetic information;aproteincoat that protects these genesanenvelopeoflipidsthat surrounds the protein coat wh
3、en they are outside a cell.,病毒的组成,病毒的特点,病毒能增殖、遗传和演化，因而具有生命最基本的特征，其主要特点是：形体极其微小，一般都能通过细菌滤器，必须在电子显微镜下才能观察没有细胞构造，其主要成分仅为核酸（DNA或RNA）和蛋白质两种既无产能酶系，也无蛋白质和核酸合成酶系，只能利用宿主活细胞内现成代谢系统合成自身的核酸和蛋白质成分。以核酸和蛋白质等“元件”的装配实现其大量繁殖。在离体条件下，能以无生命的生物大分子状态存在，并长期保持其侵染活力。有些病毒的核酸还能整合到宿主的基因组中，并诱发潜伏性感染。,病毒的分类,有不同类别病毒共上百万个，目前有详细了解的只有
4、约5000个 从遗传物质分类：DNA病毒、RNA病毒、蛋白质病毒（如：朊病毒）The vast majority of viruses have RNA genomes 从病毒结构分类：真病毒（Euvirus，简称病毒）和亚病毒（Subvirus，比病毒更为简单，仅具有某种核酸不具有蛋白质，或仅具有蛋白质而不具有核酸，包括类病毒、拟病毒、朊病毒）从寄主类型分类：噬菌体（细菌病毒）、植物病毒（如烟草花叶病毒）、动物病毒（如禽流感病毒、天花病毒、HIV等）从性质来分：温和病毒（HIV）、烈性病毒（狂犬病毒）。,病毒的生命周期,Viruses provide simple systems that
5、can be used to manipulate and investigate the functions of cells.Viruses have been useful in the study ofgeneticsand helped our understanding of the basic mechanisms ofmolecular genetics,such asDNA replication,transcription,RNA processing,translation,proteintransport,andimmunology.Mostly in the form
6、 of viral vectors:introducing foreign DNAs into cells and for gene therapy and industrial production of vaccines Biological weapons,病毒在生物医学中的应用,逆转录病毒（Retrovirus）腺病毒（Adenovirus）腺相关病毒（Adeno-associated virus）仙台病毒（Sendai Virus）慢病毒（Lentivirus）,生物医学研究中常用的病毒表达系统,逆转录病毒（Retrovirus）,Aretrovirusis a single-str
7、andedRNA virusthat stores its nucleic acid in the form of an mRNAgenome,100nm in size Retro-reverse,RNA-DNA-RNA-protein Reverse transcribe-integrate-transcribe-translate-package,1.free virus,2.attach,3.endocytosis,4.RNA to DNA,5.integrate,6.dormant,7.transcribe to mRNA,8.translate,9.package into new
8、 virus and exit,三个核心基因：gag-编码病毒的核心蛋白，起保护作用；pol-编码逆转录酶；env-编码病毒的被膜糖蛋白,逆转录病毒,分类：（1）单嗜性逆转录病毒（ecotropic retrovirus），只感染小鼠和少数几个品种的大鼠；（2）兼嗜性逆转录病毒（amphotropic retrovius），能感染小鼠的细胞，也能感染其他种属动物的细胞；用得较多（3）异嗜性逆转录病毒（xenotropic retrovirus），能感染多种动物细胞，但不能感染小鼠细胞。（4）全嗜性逆转录病毒（pantropic retrovirus）,逆转录病毒表达系统,1.Packaging
9、 Cells,e.g.Phoenix cells(an adenovirus Ad5-transformed human embryonic kidney cell line 293T,transfected with two MMLV packaging gene constructs:CMV-Env-PolyA,and RSV-Gag/Pol-Tyt2-PolyA.)Use passage number 20,10 for reprogramming.2.Retroviral plasmid DNA(usually based on MMLV based with LTRs,the pac
10、kaging signal sequence and containing the gene of interest,a promotor and a selectable marker like antibiotic resistance,Inserts of up to 6.5 kb can be efficiently packaged)and control vector with GFP.Ecotropic vectors infect rodent cells,amphotropic or xenotropic are able to infect human cells3.Tra
11、nsfection reagent,e.g.lipofectamine2000,30004.Medium,DMEM with 10%FBS and 1x penicillin-streptomycin5.Polybrene 1000 x(4 mg/mL)in PBS,stock sterile filtered,逆转录病毒表达系统,Retroviral vectors are useful for integrating genetic material into the host cell genomemaking stable cell lines Transduction is effi
12、cient in dividing cells but low in non-dividing cells such as muscle and neurons Transduction efficiencies of 90%are achievable for most mitotic cell types The copy number per cell can be easily controlled by varying the multiplicity of infection(MOI),腺病毒（Adenovirus）,Medium-sized(90100nm),nonenvelop
13、ed(without an outer lipid bilayer)viruseswith double strandedDNAgenome,the largest nonenveloped viruses.In humans,there are 57 accepted human adenovirus serotypes(HAdV-1 to 57)in seven species(Human adenovirus A to G),Adenoviruses possess a linear dsDNAgenomeand are able toreplicatein thenucleusofve
14、rtebratecells using the hosts replication machinery.,Adenovirus life cycle,free virus,attach,by binding to CD46 or the Coxsackie/Adenovirus Receptor(CAR)Entry within an endosome Viral DNA is subsequently released and enter thenucleustranscribe to mRNA,translate,package into new virus and exit,腺病毒基因组
15、包含早期表达的与腺病毒复制相关的E1E4基因和晚期表达的与腺病毒颗粒组装相关的L1L5基因,腺病毒基因组,Two phases:an early and a late phase The early genes are responsible for expressing mainly non-structural,regulatoryproteins.Once the early genes have liberated adequate virus proteins,replication machinery,and replication substrates,replication o
16、f the adenovirus genome can occur.,The late phase of the adenovirus lifecycle is focused on producing sufficient quantities of structural protein to pack all the genetic material,1.A choice of adenoviral vectors that allow highly efficient generation of a recombinant adenovirus containing the gene o
17、f interest under the control of a promoter of choice.The vector also contains the elements required to allow packaging of the expression construct into virions(e.g.5 and 3 ITRs,encapsidation signal,adenoviral late genes)2.An optimized cell line,e.g.293A,the cell line contains a stably integrated cop
18、y of the E1 gene that supplies the E1 proteins(E1a and E1b)required to generate recombinant adenovirus.The flat morphology of the cells makes the titering procedure easier.3.Transfection reagent,e.g.lipofectamine2000,30004.Medium,DMEM with 10%FBS and 1x penicillin-streptomycin5.Re-infect cell line,腺
19、病毒表达系统,The rate of infection and the subsequent yield of recombinant protein are typically quite high,better than retroviral system The permissive host cell range is very wide.The virus has been used to infect many mammalian cell types(both replicative and non-replicative)for high expression of the
20、recombinant protein.Adenovirusesare especially useful in infecting cell lines that have lowtransfectionefficiency withliposome.Does not integrate into the host chromosome so does not activate or inactivate host genesExpression is transient in nature,as long as the viral genome is not degraded,1-2 we
21、eks depending on the cell type.Longer expression can be observed in slow dividing cells such as neurons.,腺病毒表达系统的特点,腺相关病毒(AAV),AAV was discovered in 1965 as a contaminant of adenovirus(Ad)preparations，11 AAVserotypeshave been described.Smallviruses infects humans and some other primate species,non-e
22、nveloped,approximately 22 nm A co-infecting helper virus is usually required for a productive infection to occur,The AAV genome is built of single-stranded DNA(ssDNA),which is about 4.7 KBThe genome comprises：inverted terminal repeats(ITRs)at both ends of the DNA strand,required for efficient multip
23、lication of the AAV genome and integration of the AAV DNA into the host cell genome(19th chromosome in humans)twoopen reading frames(ORFs):repandcap cap encodeds capsid protein VP1,VP2 and VP3,which interact together to form a capsid rep encodes four Rep proteins,Rep78,Rep68,Rep52,and Rep40,required
24、 for replication of DNA and integration into chromosome.Infect both dividing and non-dividing cells,stably integrate into the host cell genome at a specific site(designated AAVS1)in the humanchromosome19,腺相关病毒结构,腺相关病毒基因组,Schematic Map of AAV Genome,腺相关病毒,1.attach2.receptor-mediatedendocytosis（hepara
25、n sulfate proteoglycan）3.endosomal trafficking4.escape from the lateendosome5.translocation to thenucleus6.uncoating7.formation of double-stranded DNA replicative form of the AAV genome8.expression ofrepgenes9.genomereplication10.expression ofcapgenes,synthesis of progeny ssDNA particles11.assembly
26、of completevirions12.release from the infected cell.,AAV causes a very mildimmune response,not currently known to causedisease-make AAV a very attractive candidate for creating viral vectors forgene therapy AAV can infect both dividing and quiescent cells Development of AAVs as gene therapy vectors,
27、however,has eliminated this integrative capacity by removal of therepandcapfrom the DNA of the vector.The cloning capacity of the vector is relatively limited and most therapeutic genes require the complete replacement of the viruss 4.8 kilobase genome,cloning capacity4.5KB,腺相关病毒表达系统,AAV Helper-Free
28、 System,eliminating the requirement for live helper virus the AAV Helper-Free System provides a safer and more convenient gene delivery system Most of the adenovirus gene products required for the production of infective AAV particles are supplied on an additional plasmid(e.g.Cell Biolabs pHelper pl
29、asmids contains E2A,E4,and VA RNA genes)that is co-transfected into cells with human AAV vector DNA The remaining adenoviral gene product is supplied by the 293 host cells,which stably express the adenovirus E1 gene.,腺相关病毒表达系统,AAV Helper-Free System,AAV-DJ,AAV serotypes(AAV1-AAV11)differ broadly in
30、transduction efficacies and tissue tropisms.DNA family shuffling technology was used to create a complex library of hybrid capsids from eight different wild-type viruses Stringent selection of AAV variants on human liver cells and with human anti-AAV antisera result in AAV-DJ(a clone named after the
31、 first two authors)-superior in vitro transduction efficacies in comparison with any other wild type serotypes.(up to 100,000-fold better than AAV-8 or AAV-9,also substantially better than AAV-2.,1.One day before transfection,plate sufficient 293 cells or 293AAV cells to achieve 70-80%confluence on
32、the day of transfection.2.Cotransfect cells with pAAV Expression vector,pAAV-DJ and pHelper,ratio of vectors at 1:1:1 Calcium Phosphate transfection method is preferred for AAV production.3.48-72 hours after transfection,harvest cells.4.Centrifuge the cell suspension.Remove the supernatant and resus
33、pend the cell pellet in DMEM or sterile PBS.5.Freeze and thaw the cell suspension four times by placing it alternately in a dry ice/ethanol bath and a water bath of 37C.Remove cell debris by centrifugation at 10,000 g for 10 min and collect the supernatant as AAV crude lysate.6.AAV crude lysate can
34、be used directly or purified/concentrated if needed.For long term storage,store supernatant at-80C in aliquots.,腺相关病毒制备,SeV(mouse parainfluenza virus type 1,hemagglutinating virus of Japan(HVJ)is a single-strand RNA virus,spherical shape and an average diameter of 260 nm A SeV virion consists of the
35、 nucleocapsid(genomic RNA complexed with NP,P and L proteins),an envelope(a lipid bilayer with F and HN proteins,responsible for insert into cell membrane)and a matrix(M protein)connecting the nucleocapsid and envelope First isolation in the 1950s in Japan,仙台病毒（Sendai Virus）,SeV is responsible for a
36、 highly transmissible respiratory tract infection in mice,hamsters,guinea pigs,and rats through both air and direct contact routesSeV is neither tumorigenic nor pathogenic to humans Low species and cell specificity-infect most cell types Do not integrate into host genome Induce transient but very st
37、rong gene expression,仙台病毒的特点,APPLICATION OF SeV VECTORS IN GENE THERAPY,Recombinant SeV vector can induce strongex-gene expression in the cardiovascular system,retinal epithelium,hepatocytes,colonic epithelium,neurons,dendritic cells and in human hematopoietic stem cells.This remarkably wide host ra
38、nge partly depends on the fact that the primary SeV receptor,sialic acid,is distributed universally among animal cellsSeV vectors rely for their gene expression only on virus-encoded RNA polymerase and tubulin,a ubiquitous conserved cytoskeletal proteinReplication-defective SeV vectors expressing FG
39、F2 have been developed for treatment of critical limb ischemiaApplications of SeV vectors to cancer gene therapy have been investigated at the preclinical stageSeV induces syncytia,used to merge a monoclonalB cell,exposed to a chosen antigen,and amyelomatumor cell to producehybridomas,The SeV vector
40、 stands unique among other vector systems in genomic reprogramming because it can express the reprogramming genes without chromosomal integration.SeV vectors can reprogram the nuclei of various human cells,including CD34+cord blood cells,activated T lymphocytes and monocytes High efficiency,Applicat
41、ion of SeV in Cellular Reprogramming,Lentivirus(lente-,Latin for slow)is amemberof Retrovirusfamily,characterized by a longincubation period.Thevirionsare spherical and 80100nm in diameter,enveloped,with tinyspikes(about 8nm)Lentiviruses can deliver a significant amount ofviralRNA into theDNAof theh
42、ost celland being able to infect non-dividing cells.Lentivirus expression system is a useful research tool to introduce a gene product or shRNA intocells or animal models for prolonged expression,慢病毒(Lentivirus),慢病毒结构,Genomess-RNA,three main genes coding for the viral proteins in the order:5-gag-pol
43、-env-3 and tworegulatory genes,tat and rev,Structural ProteinThe lentiviral proteome consists of five major structural proteins:Gp120glycosylatedsurface envelope protein SU,encoded by the viral geneenv.Largest 120 KDa.Gp41 glycosylated transmembrane envelope protein TM,also encoded by the viral gene
44、env.2nd largest 41 KDa.P24 non-glycosylatedcapsidprotein CA,encoded by the viral genegag.3rd largest 24 KDa.P17 non-glycosylatedmatrixprotein MA,also encoded bygag.4th largest 17 KDa.Non-glycosylated capsid protein NC,also encoded bygag.5th largest 7-11 KDa.,慢病毒表达系统,Lentivirus vector based on the hu
45、man immunodeficiency virus-1(HIV-1)has become a promising vector for gene transfer studies.The advantageous feature of lentivirus vector is the ability of gene transfer and integration into dividing and non-dividing cells.The pseudotyped envelope with vesicular stomatitis virus envelope G(VSV-G)prot
46、ein broadens the target cell range.Lentiviral vectors have been shown to deliver genes to neurons,lymphocytes and macrophages,cell types that previous retrovirus vectors could not be used.Lentiviral vectors have also proven to be effective in transducing brain,liver,muscle,and retina in vivo without
47、 toxicity or immune responses.Lentivirus system is widely used to integrate siRNA efficiently in a wide variety of cell lines and primary cells both in vitro and in vivo.,慢病毒表达系统,第二代慢病毒表达系统,a transfer plasmid,asinglepackaging plasmid,and an envelope plasmid,第三代，4质粒系统，更安全，课后阅读,慢病毒的制备,For producing le
48、ntiviral particles,you typically need 4 components:A lentiviral vector or“transfer vector”containing the shRNA or transgene and the flanking LTRs A packaging vectororset of packaging plasmids An envelope vector,Packaging cells:293T or 293FT 来源于SV40大T抗原转化的人胚肾细胞 stably express the neomycin resistance
49、gene,成功制备慢病毒的要点,1.Packaging Cells,细胞质量要高，尽量用早代的细胞，冻存一批早代细胞以备后用，最佳转染密度70-80%2.Lenti plasmid DNA,最大DNA载荷12KB，质粒抽提，纯度，浓度，比例：Lenti:psPAX2:pMD2.G=12:8:43.Transfection reagent,e.g.CaCl2,lipofectamine 2000,3000，争取转染效率达到最佳，最好每次用GFP质粒做参照4.Medium,不能对细胞生长有负作用，FBS，双抗等5.Polybrene 1000 x(4 mg/mL)in PBS,stock ster
50、ile filtered6.Test virus by transduction of 293cells or other cells,慢病毒的浓缩,1.超速离心法2.利用蛋白质浓缩离心管3.PEG(polyethylene glycol)沉淀浓缩法，亲水性很高，在溶液里会吸收大量水分，减少病毒颗粒之间的距离，使病毒聚集在一起，提高病毒的相对浓度，达到沉淀浓缩的目的,滴度测定,DETERMINATION OF TOTAL VECTOR CONCENTRATION USING ANTI-p24 IMMUNOASSAYBIOLOGICAL TITRATION OF LENTIVECTORS USI
