CN101605891A - The genetics of PrP gene melts and uses target to decide the cell of promoter trap strategy method production as the serum-free recombinant proteins of therapeutical agent - Google Patents

Abstract

The invention provides the human somatocyte of the immortalization that does not contain prion protein (PrP) system, wherein, two allelotrope of described PrP gene are rejected fully.The present invention also provides and has been used to produce the method for described clone and uses it for the purposes of producing the human recombination protein that is suitable as biologics.

Description

The genetics of PrP gene melts and uses target to decide the cell of promoter trap strategy method production as the serum-free recombinant proteins of therapeutical agent

The invention provides the human somatocyte of the immortalization that does not contain prion protein (PrP) system, wherein, two allelotrope of described PrP gene are rejected fully.The present invention also provides the method that is used to produce described clone, and it is used to produce the fixed proteic application of people of target that is suitable as biologics.

Background technology

Protein virus is an infectious agent, and it can cause the central nervous system spongiform encephalopathy of animal, and is different with viroid with virus, and Protein virus lacks nucleic acid fully, and can tolerate proteolytic enzyme.Infectious particles has been accredited as PrP ^Sc, it is PrP ^cThe isomeric form of (being the normal cell prion protein).Described Protein virus hypothesis (Prusiner, Proc.Natl.Acad.Sci.95,13363-13383, (1988)) is thought PrP ^ScMolecule itself can be with PrP ^cMolecule changes into unusual conformation, by heterodimerization or by nuclear polymerization process.

Modal prion disease is a sheep and goat itch disease in the animal, and bovine spongiform encephalopathy (BSE).Four kinds of prion diseases in the mankind, have been identified: (1) Kuru disease, (2) Ke Laoyishi disease (CJD), (3) Gerstmann-

-Scheinker syndromes (GSS) and (4) mortality family insomnia (FFI).Described human prion disease may be sporadic, and is that genetics originates from or the infectivity origin.

First infectious prion disease is 250 years itch diseases as the sheep and goat disease that disclosed in the past.The itch disease was proved in the past and can propagates by experiment in 50 years.There is not evidence to show that the itch disease once propagated into the mankind.BSE is the 1985 (Wells etc. that disclose on the ox health of Britain at first, Veterinary record 121,419-420, (1987)), and be considered to propagate (Wilesmith etc. from meat and the bone meal of ruminating animal by edible, VeterinaryRecord 123,638-644, (1988)).This disease is by wide-scale distribution, peaked in 1992, occurred surpassing 180,000 clinical case in Britain, although mathematical statistics shows, there are 1,000,000 oxen of 1-2 to be subjected to infection, but, they even as big as just slaughtered before showing the clinical disease symptom and enter the human foods chain (Anderson etc., Nature 382,779-788, (1996)).BSE has propagated into up to 20 kinds of other species, comprises domestic and external cat (Wyatt etc., Veterinary Record 129:233-236, (1991); Kirkwood andCunningham, Veterinary Record 135,296-303 (1994)) and the ungulates of the foreign country in the british animal garden.In July, 1988, meat and bone meal that the propagation of BSE causes United Kingdom Government to limit from ruminating animal use as animal-feed, November in 1989, have stipulated that restriction is used for the human consumption with the ox internal organ.

The human prion transmission of disease is (the Gajdusek ﹠amp of report in the Fore people (Fore people) of later stage the 1950's in Papua New Guinea at first; Zigas, New EnglandJournal of Medicine 257,974-978 (1957)), and be considered to during ceremony anthropophagy and ritual, propagate.The existing document record of the iatrogenic infection of CJD system by the medicine equipment that polluted and graft direct inoculation CNS.(Buchanan etc., British Medical Journal302,824-824, (1991) also take place by intramuscularly people corpse somatotropin and gonad-stimulating hormone in iatrogenic infection; Brown etc., Transfusion 38:810-816, (1992)).

Variant CJD (vCJD) is the human prion disease, obviously causes by contact mad cow disease (BSE) infectious agent.VCJD at first reported out (Will etc., Lancet 347:921-5, (1996)) in the past in 10 years, it is the CJD sickness rate of systematicness monitoring Britain and clinical manifestation and the result that obtains.

For vCJD, opposite with other human prion diseases, the disease-related form of described prion protein and infectivity can detect (Hill, A.F. etc., Lancet 353:183-9 (1999) easily in the lymphoid tissue of whole health; Head, M.W. etc., Am.J.Pathol.164:142 (2004)), or even before the clinical disease outbreak, detect (Hilton, D.A. etc., Lancet 352:703-4 (1998)).This can cause such worry, and blood and blood products also may contain infective granule, and it has constituted the possible source of the iatrogenic infection of variant CJD.

Strengthened above-mentioned worry (Hunter etc., J.Gen.Virol.83:2897-905 (2002)) by the BSE propagation experimentation that animal blood before giving the sheep model infusion from this disease clinical onset and leukocytic cream carry out.

That carries out on animal model studies show that, most of Protein virus infectivity in the blood is relevant with cell, lower (the Brown P etc. of level in blood plasma, Transfusion 38:810-816 (1998)), and evidence suggests, any communicable existence all may weaken (Stenland, C.J. etc., Transfusion 42:1497-500 (2002) during separating plasma; Gregori, L. etc., Biologicals 32:1-10 (2004)).The blood transfusion of reply vCJD is propagated, and makes the blood supply delay of the donor that itself is exactly the blood ingredient acceptor, and this method just came into effect from 1980, so that reduce by three grades or the risk of more senior propagation that causes oneself's outburst.Yet, and can't get rid of blood plasma or blood products is propagated the possibility of described disease, because still do not have suitable blood testing (Aguzzi, A.﹠amp; Glatzel M., Nat.Clin.Pract.Neurol.2:321-329 (2006)).

The danger of propagating the Protein virus relative disease by people's based article is a serious health problem.For blood products, the method for prevention is control vCJD donor on the one hand, simultaneously by filtration, purification blood.

On the other hand, recombinant human protein's safety in production can avoid Protein virus by importing the danger from the blood-transmitted of contaminated donor.The proteic production of people is produced in such as intestinal bacteria (Escherichia coli) and yeast saccharomyces cerevisiae organisms such as (Saccharomyces cerevisiae), make the proteic production of a lot of people to synthesize on a large scale, but the plasmid stability of desirable protein product and insoluble and so on factor may restrict the application of said system.The posttranslational modification that most people's protein requirement is extra so that realize the function of described intrinsic protein, and therefore require they mammalian cell or even synthetic in species specific clone so that obtain the proteic blood plasma sample function of producing.

For example, for Factor IX, use such as Chinese hamster ovary cell non-human clones such as (CHO), be faced with the critical defect that the treatment albumen of purifying is polluted by the cell micro composition, this pollution can cause occurring in patient's body antigen-reactive (Refacto, Wyeth, package insert).

In addition, may have non-human glycosylation form, can in patient's body, produce antigen-reactive equally by inhuman expression system expressed proteins.The biological stability of thrombin and effectiveness mainly are subjected to their O-and the influence of N-glycosylation form.Periphery and terminal monose are even more important, because they are detected by the specific receptor from the cell of being responsible for their degradeds.Thrombin carries sialic acid residues as terminal monose.The change that sialic acid in the glycoprotein feeler is formed may produce different glycosylation forms.Therefore, when occurring modifying, can and render a service biological stability and produce fundamental influence.Therefore, the important factor that will consider when producing recombinant blood coagulation factor is the influence of assessment glycosylation to non-human production clone.In general, human cell system is more suitable for producing recombinant blood coagulation factor than non-human clone.Its reason may be not have external oligose to be incorporated into described oligose part between synthesis phase at recombinant protein.

For above-mentioned reasons, Mammals, particularly human system are optimized for recombinant human protein's production.Particularly immortalized cell line HEK293 and derivative thereof, f.e.FreeStyleHEK293F can express recombinant people albumen (EP 05 105 965.7).Because a lot of therapeutical agents are produced in mammlian system, are necessary to guarantee the security of isolating the said products from described system, guarantee that it does not have prion protein fully.On the one hand, consider and do not have the available communicable suitable detection method of Protein virus that is used to detect, on the other hand, propagation of Protein virus and infectivity depend on the expression of Normocellular prion protein, this prion protein may change into infectious prion albumen, and eliminating Protein virus reliable and the most feasible communicable method in described system is thoroughly to avoid described prion protein genetic expression by rejecting prion gene fully.

The knockout cell that the present invention discloses is the expression that production system has stoped described prion protein fully, and can provide and not contain the proteic recombinant human protein of infectious prion, and make the patient of the recombinant products treatment of producing in the described prion-free cell of acceptance, avoided the risk that polluted by Protein virus.

For the expression of special inhibition specific gene in eukaryotic cell, adopted genetic engineering and Protocols in Molecular Biology to develop some kinds of methods.Genetics melts the dna sequence dna that encode specific protein is eliminated in (being called " genetics rejecting " in the industry usually by the expert of this area) expression.The result of this elimination is that resulting " knockout cell " can not express the gene of described rejecting fully.On the contrary, " RNA interfering " (RNAi) technology is not eliminate described dna sequence dna, but introduces antisense or complementary sequence, can prevent to translate the gene that these are suppressed like this.In most of the cases, resulting " rejecting-inhibition " gene can only partly suppress, and allows some residual expression level.

Because mouse is as the widespread use of human diseases model, and since the muroid embryo do ( ES) the early stage utilizability of cell, these cells can carry out genetics and embryo operation easily, and the rejecting technology is mainly carried out (UK 1993 for Joyner A.L., Oxford Univ.Press) on mouse ES cells.Now, the rejecting technology of carrying out on mouse has developed into to be enough to not only allow to reject fully (" not having allelotrope "), but also can obtain conditional mutant.The fixed sudden change of target can temporarily induce or suppress, for example by add tsiklomitsin (tet-on, tet-off system, Gossen, M.and Bujard, H.Proc.Natl.Acad.Sci.USA 89:5547-5551 (1992)) in mouse feed; It can start by the cell or tissue specificity promoter, perhaps by ubiquitous, but starts by the promotor of developmental regulation, and this promotor may be closed during fetal development, and starts fast after birth.

The generation of rejecting ES clone always requires identical initial step: (1) produces in order to reject specific gene custom-designed target and decides construct; (2) this construct is imported the cell that described ES cell and (3) select and screen its expection gene that carries to be deleted surely by target.

The fixed construct of described target must always comprise choice box and with the logo area of the disallowable dna homolog of desire.These two elements are realizations and only detect and integrate institute's absolute demand in needed site; And described native gene homologous zone can start the homologous recombination between these two homologous dna fragments, and described choice box allows screening to carry the cell of the construct of being integrated.The most widely used choice box is neomycin phosphotransferase frame (" neo ") in ES rejecting technology, it comprises the ORF (Soriano etc. of coding by this kind of enzyme of mouse phosphoglyceric kinase (pGK) promotor startup, Cell 64,693-702 (1991)), and its downstream be the polyA transcription termination signal.When construct that described target is fixed imports the ES cell and selects with G418, have only those the cell of the described construct of stable integration can tolerate described microbiotic.In these resistant cells, the fixed construct of some target is replaced by endogenous original series.Subsequently can be by genome PCR or by the fixed result of target among the clone of genome Southern engram analysis screening and separating.In mouse ES cells, the fixed construct of described target is incorporated into the frequency of the frequency at described genomic expection position well below random integration, and this makes usually must hundreds of antibiotics resistance clones of screening before identifying the fixed clone of correct target.

A kind of possibility that the target of raising particular build body is decided frequency is the length that increases the homology zone, because have linear dependence (Hasty etc., Mol Cell Biol.1991 Nov between these two parameters; 11 (11): 5586-91 (1991)).In addition, developed multiple " trapping method ", these methods depend on the use of element of the target locating point of the described choice box of effective expression.For example, in described promoter trap strategy method, the fixed carrier of described target is design like this, and the fixed gene transcription mechanism of endogenous target can drive the expression that is cloned in the choice box in the fixed carrier of described target.In this case, described carrier comprises the zone with the fixed dna homolog of target, and described zone is without any promoter activity, and therefore, the major part that random integration takes place is cloned in the antibiotic-screening and can not survives.Generally, clone's enrichment that the promoter trap strategy screening can be fixed with target is 100 times.

Use the RNAi method, the PrP expression of gene in the sick mice infected neuroblast of the itch of cultivating oncocyte can only be up to now instantaneous and part reticent (Daude etc., J.Cell.Sci..2003Jul 1; 116 (Pt 13): 2775-9 (2003)).

Successfully reported the genetics target of prion gene in the mouse genome that uses different methods to realize and decided result's (table 1), so that its effective consequence of afunction and the possible relation of it and TSEs are described.

Table 1:The PrP mouse that obtains from document is rejected data

Reject title	Method	Antibiotic-screening	Homology arm (kb)	Host cell	The frequency of the reorganization that target is fixed
						Zurich I	Conventional	??G4180.3mg/ml	??n/a	??ES?AB1	??1/5000
The Edinburg	Conventional	G4180.2mg/ml ganciclovir 2 μ M	A left side: 1.2 right sides: 2.4	??E14	??1/800
						Nagasaki	Conventional	??G4180.2mg/ml??FIAU?200nM	A left side: 3.0 right sides: 9.0	??J1?ES	??1/321
??Rcm0	Conventional	??G4181mg/ml	??n/a	Neuroblastoma N2a	??n/a
						Zurich II	Condition	??G4180.4mg/ml	A left side: 1.4 right sides: 3.7	??E14.1	??82/1046(PCR)，??5/82

As basis " prion protein-unique theory " (Prusiner Nobel Lecture, Dec.8,1997) desired, the mouse homozygote of inactivation gene (promptly lacking PrP fully expresses) is actually the anti-prion infection, but, be apparent that most that it is viable and the energy normal development that above-mentioned all that mention are rejected the PrP mouse.Only identify relative gentle neuroscience phenotype (Rossi etc., EMBO J.20,4,694-702, (2001)) in Nagasaki, Rcm0 and Zurich II elimination test, this shows that the function of PrP gene is not indispensable.In addition, as what shown, the functional gene compensation mechanism that exists certain to produce by Dpl, this gene shows significantly and the homology of PrP, and this gene of mapping expression in the CNS of the mouse of rejecting PrP only the 16Kb in downstream raised (Moore etc., J.Mol.Biol.292,797-817 (1999)).

Above-mentioned rejecting technology has run into two large problems when being applied to somatocyte.At first, reject the finite information that a gene in the above-mentioned cell can only provide relevant this destructive functional consequence.Because the phenotype of the knockout cell in cultivating has not necessarily embodied the mutant biology, as final effect from the mouse of rejecting the ES cell.Secondly, prior problem is that the target of homologous recombination is decided frequency ratio low about two orders of magnitude (Hanson and Sedivy, Mol.CellBiol.15 (1): 45-51 (1995)) in the ES cell in the somatocyte.Fixed in order in somatocyte, to carry out the efficient gene target, using the promoter trap strategy method of no promoter vector is indispensable (Sedivy and Dutriaux, Trends Genet.15 (3): 88-90 (1999)), because they can strengthen 100-500 doubly usually.In importing somatocyte and when carrying out antibiotic-screening, described choice box can only be expressed after the promotor of the gene fixed from target is carried out homologous recombination.By by Genetix, the ClonePix technology that UK provides also can be improved the fixed gene of target.

Find at present, can set up the human somatocyte system clone of HEK293F clone (for example, from), wherein, the gene of coding prion protein sequence is at least one, preferred inactivation on two allelotrope, or for the triploid cell on three allelotrope inactivation.This purpose is that the homologous recombination by the rejecting carrier that carries no promotor selective marker realizes, its target fixed be incorporated in the genome after, can described selective marker be expressed by endogenous PrP promotor.The cell that resulting Protein virus is melted is adapted at being used for the proteic recombinant production of the fixed people of target with after the fixed proteic suitable expression vector transfection of coding target.After in conjunction with suitable protein purification and virally inactivated technology, this method provide be used to produce be fit to that the human treatment uses do not contain effective system prion protein, safety and recombinant human protein that have high activity.

Summary of the invention

The present invention relates to

(1) do not contain the immortalization somatocyte system of prion protein (PrP), wherein, two allelotrope of described PrP gene are deleted fully;

(2) be used to produce the method for the immortalized cell line that does not contain PrP defined in above-mentioned (1), this method comprises by rejecting the construct homologous recombination with PrP, deletes two allelotrope of the PrP ORF in the corresponding initiating cell subsequently;

(3) reject construct as defined PrP in top (2);

(4) immortalized cell line of defined described no PrP in top (1) is used for the no PrP recombinant production of people's albumen or derivatives thereof or its mutant (following all be referred to as " albumen that target is fixed ");

(5) preparation is used for the method for the clone of the fixed proteic no PrP recombinant production of target, this method comprises with the transfection carrier that comprises the proteic gene that replication orgin and the described people's target of coding are fixed, the immortalization host cell system of no PrP defined in the transfection above-mentioned (1), 5 ' end of people's protein gene that described thus target is fixed is connected with promotor, and its 3 ' end is connected with the polyA signal;

(6) the immortalized cell line stable transfection of no PrP is preferably under serum-free condition, with the transfection carrier transfection defined in above-mentioned (5); With

(7) be used to produce the method that the target that is used as medicine is decided the proteic no PrP recombinant production of people, this method comprises the human clone of the immortalization of the no PrP defined in the cultivation above-mentioned (6).

The method of embodiment of the present invention (7) is particularly suitable for producing recombinant human protein and treatment antibody, comprise thrombin, as factor VII/a, Factor IX, factors IX, the von Willebrand factor (vWF) and Adamts13 and somatomedin as granulocyte colony-stimulating factor (G-CSF) or rHuGM-CSF (GM-CSF), do not have prion protein to pollute.For this method, can use the human clone of following immortalization, for example, have HEK 293 clones that the prion protein sequence is melted in the present invention.This clone can be by can selecting or selective marker with containing, and for example, neomycin phosphotransferase ORF lacks the carrier that it self promotor and translation start, and comes the human clone of transfection immortalization to obtain.

In a kind of preferred embodiment, described clone is that following human cell is, as the clone from HEK293F or Per.C6 cell (the human fetal retinal cell of immortalization).Other suitable cells are CHO (Chinese hamster ovary cell) and BHK (baby hamster kidney cell) cell.

Description of drawings

Fig. 1: the promoter trap strategy method that is used to melt human PrP gene

Fig. 2: 1.PrP K.O. construct pBS_Neo_P ^-_ R+L_Arm_2B

Fig. 3: preparation construct pBS_Neo_P ^-The cloning process of _ L+R_Arm_2B.Be cloned into the fragments sequence in the pBluescript carrier, i) do not have it self promotor and the neomycin gene of ATG, ii) be used for the left arm of homologous recombination, iii) be used for the right arm of homologous recombination

Fig. 4: the screening method of the PCR-based after G418 selects

Fig. 5: at stable integration be used to characterize cloned genes group Southern method after the fixed construct pBS_Neo-_P-_L+R_Arm_2B of target

Fig. 6: the genome PCR screening after integrating the fixed construct pBS_Neo_P-_R+L_Arm_2B of target.Dna marker: gene scale DNA ladder mixture; Positive control: the genomic dna of deciding the cytomixis colony of PrP from target.A1-8 and B1-8 are the cell clones of selecting by ClonePixFL.Clone with green circular ring marks is accredited as the PCR positive colony, because it is the 2.3kb band.

Fig. 7: the Southern of the PrP K.O. cell after target is integrated a K.O. construct pBS_Neo_P-_R+L_Arm_2B surely analyzes: used following composition: dna marker: gene scale DNA ladder mixture; The WT:293F wild-type; K.O.: the K.O. clone who after target has been integrated a K.O. construct pBS_Neo_P-_R+L_Arm_2B surely, has identified.From the genomic dna and 5 ' of K.O. clone and 293F WT cell-, 3 '-with the Neo-probe hybridization.As desired, WT 293F cell and 5 '-show 10.8kb WT band with 3 '-probe, but there is not signal with the Neo-probe.For K.O. clone, can detect with the band of the 4.2kb of 3 '-probe and with the band of the 6.5kb of 3 '-probe, in addition, seen the band of 6.5kb with the Neo-probe.

Fig. 8: have the fish analysis that a target is decided the allelic PrP K.O. of PrP cell: 2 signals of collection of illustrative plates a:chr20 and Bac; 3 signals of collection of illustrative plates b:chr20 and Bac; 3 signals of collection of illustrative plates c:chr20, but Bac has only 2 signals.

Fig. 9: have the elisa assay that a target is decided PrP allelic PrP K.O. cell and 293F cell: analyzed target of carrying and decided the allelic two kinds of K.O. clones of PrP (K.O.1 and K.O.6), and compared with wild-type 293F cell.

Figure 10: 2.PrP K.O. construct pBS_Zeo_P ^-_ R+L_Arm.

Figure 11: have PCR screening cell clone or the blended cell colony that two PrP K.O. integrate: on gel, used following composition: dna marker; Gene scale DNA ladder mixture; Positive control: the genomic dna of deciding PrP cytomixis colony from target.T3-1 is the blended cell colony of selecting with zeocin.T3-2 is the blended cell colony of selecting with zeocin and G418.These two kinds of blended cell colonys all are accredited as the positive.

Figure 12: at stable integration be used to characterize cloned genes group Southern method after the fixed construct pBS_Neo-_P-_L+R_Arm_ of target.

Figure 13.: have the transfection efficiency that a target is decided the allelic PrP KO of PrP cell.Cell and comparison transfection efficiency with expression plasmid transfection equal amts.

Figure 14: FVIII (Figure 14 a), FIX (Figure 14 b) and G-CSFb (Figure 14 c) expression in having the fixed allelic PrP K.O. of the PrP cell of target.The FVIII of arbitrary unit, the expression and the 293F wild-type cell of FIX and G-CSFb/10E6 cell compare.

Embodiment

Used in this application to give a definition and shortenings:

" BAC " represents bacterial artificial chromosome." bp " represents base pair." G418 " is two kinds of different selection microbiotic with " zeocin "; To carry these microbiotic, make it can tolerate (" G418 as the construct of selective marker transfectional cell stably ^R" or " zeocin ^R") above-mentioned microbiotic." homologous recombination " expression has two mechanism that dna fragmentation reconfigures each other of homologous sequence." left arm " represents the subarea that includes near the PrP gene of exon 3 upstream." right arm " expression is near the zone in the exon 3 downstream of PrP gene." Neo " represents neomycin phosphotransferase gene." ORF " represents open reading frame." PCR " represents polymerase chain reaction, " PrP " expression prion gene or prion protein." HEK293F " represents a species specific human embryos kidney cell line.

Embodiment of the present invention (1) relate to the human somatocyte of the immortalization that does not contain prion protein (PrP) system, and wherein, two allelotrope of described PrP gene are rejected fully.

According to the present invention, described clone can be transfected and be cultivated under serum-free condition.

In addition preferably, described clone is by integrating adenoviral sequence by immortalization in its genome.Described clone can be selected from following one group of initiating cell: kidney, bladder, liver, lung, cardiac muscle, unstriated muscle, ovary and gastrointestinal cell.Preferably, described initiating cell is a human kidney cells system, as human foetus's nephrocyte.Particularly preferably be, described human foetus's nephrocyte is FreeStyle a 293 (293F cell; Invitrogen R79007), HEK 293 (293 cells; ATCC CRL-1573; Or 293T cell (DSM ACC 2494), preferably 293F cell (Invitrogen R79007) DSM ACC 305).

In another kind of preferred embodiment of the present invention, described PrP ORF be by can selecting or the rejecting trap of selectable marker gene carries out homologous recombination and deleted fully with carrying, described like this select or the expression of selective marker is started by endogenous PrP promotor.

In particularly preferred embodiments, the prion-free clone of embodiment (1) is prion-free 293F clone pf293F, and it comprises population mixtures in the middle of all, need to separate the separating clone of described final knockout cell system and by its deutero-modifier.

In embodiment of the present invention (2), described rejecting construct may be fit to delete the complete PrP ORF on two allelotrope.In addition, described rejecting construct can carry identical or different no promotor selectable marker gene, and its side chain is and two sequences of the intragenic insertion of the PrP of initiating cell site homologous.But, preferably, but described rejecting construct carries different selectable marker genes or selective marker.Described rejecting construct can also carry one of following function sequence: poly A sequence, recombinase recognition sequence and IRES etc.

The length of the homologous sequence of described rejecting construct can be 1-10kb, preferably approximately 6kb, and preferably be equivalent to the sequence of the PrP ORF upstream and downstream of described initiating cell system.Particularly preferably be, described homologous sequence is the sequence shown in SEQ ID NOs:2 and 3.The coding positive selectable marker suitable selective marker including, but not limited to, neomycin phosphotransferase, zeocin, Totomycin; And described selective marker comprises fluorescent mark, as GFP and Dsred and enzyme, as LacZ.

Particularly preferably be, described rejecting construct has the one or more sequences shown in SEQ ID NOs:1 and 16.

Below in conjunction with HEK 293 or HEK 293F cell the present invention is done further explanation.In above-mentioned clone of the present invention, the coding region gene of PrP is deleted fully by promoter trap strategy.For the coding region of deleting the PrP gene in the HEK 293F cell needs three successive steps:

1. reject the coding region of the PrP on (below be referred to as " K.O. "-) fixed allelotrope of construct target with the PrP that contains the Xin Meisu selective marker.

2. identify and separate and only carry a target and decide PrP allelotrope and the allelic clone of wild-type PrP.This step is essential, because the genetics ununiformity of parent HEK 293F cell, carries the PrP gene of 3 copies in 75% colony, carries 2 copies in 25% colony.

3. with the coding region of the PrP on fixed all the other allelotrope of second PrP K.O. construct target, this time comprise the zeocin selective marker in the construct.

With a PrP K.O. construct (carry Xin Meisu as selective marker, Fig. 2) be stably transfected in the HEK293F cell after, under some different antibiotic concentrations, separate G418 ^RClone, and the screening of the method by PCR-based, this screening can identify the fixed result of target (Fig. 4, Fig. 6).Identify by genome Southern engram analysis then to carry the clone that the PrP-target is integrated surely, so that assessment:

Whether (a) described target is decided 5 ' and its 3 ' end that are incorporated into it of construct correct

(b) target has been decided how many PrP allelotrope (, two or three) and how many still intact (wild-type: one or two) has been arranged

Take turns among the K.O. second, remaining the PrP allelotrope that second PrP K.O. construct (carrying zeocin rather than Xin Meisu as selective marker) is used for rejecting described clone, wherein, an allelotrope is decided by the Xin Meisu target, and second PrP allelotrope intact still.To isolating zeocin ^RThe clone carries out after PCR screening and the genome Southern analysis, analyzes by RT-PCR and has confirmed melting fully of PrP gene, so that confirm to lack PrP mRNA, and confirms to lack fully PrP albumen by the Western engram analysis.

Then gained that can this paper is disclosed completely PrP K.O. clone be used to guarantee that the prion-free of human recombination protein expresses.

In transfection, antibiotic-screening, clone and separate, in the whole process of screening and amplification, cell is cultivated (for example in the FreeStyle substratum or in the Octapharma indoor cultivation base) under serum-free condition.After the genetics of setting up and finish final PrP K.O. cell clone and phenotype sign, found research cell bank (referring at the material of 6.1. and the culture method of method 293F cell partly).Then, this PrP K.O. clone, below being referred to as " prion-free 293F clone " can be with any interested stable gene ground transfection (Factor IX for example, factors IX, G-CSF, vWF, GM-CSF, factor VII/VIIa or antibody), under serum-free condition, carry out (referring to EP to be examined 05105965, the full content of this application is done this paper reference by receipts) fully according to the present invention.

Usually in serum free medium, grow by carry out the cell that stable transfection obtains with prion-free 293F cell, for example infect with the pcDNA3.1 construct that carries gene of interest, described cell inoculation in semisolid methylcellulose gum type substratum, is contained the microbiotic that is fit to clonal selection and the antibody for crossing by the high yield clone's of fluoroscopic examination mark.Use ClonePixFL (Genetix) a large amount of (for example, tens thousand of) clone to be analyzed then, so that select hundreds of the best clones of production subsequently at cell quantity and secretion recombinant protein.Different with other currently known methodss, wherein, nonproductive clone and mixing clone are random chooses, and ClonePixFL can identify and select the fastest clone of growth simultaneously, and it also is the highest clone of output, and it is from unicellular.The cell of being selected increases on microtiter plate, and subsequently at the rotation test tube, in Tissue Culture Flask and the fermentor tank, finishes whole processes under serum-free condition.

Here, whole stable transfection process is carried out under serum-free condition.In addition, in whole amplification and cell cultivation process subsequently, any contact can not take place with serum or animal derived albumen in cell.

During increasing, measure by ELISA, at robustness, the high speed of growth, vigor, easily the throughput of separability and active recombinant protein is selected best clone.In this chosen process, quantity is reduced to once more only a small amount of best clone of production.Except throughput, correct cDNA sequence, mRNA content and the cell performance when fermentation are the standards of identifying the best clone who is used for succeeding transfer culture.For this reason, the clone's that selects cell be paved plate again, analyze and select, and then increase according to the method described above and select with ClonePixFL.Succeeding transfer culture is an important step, produces the clone so that only be chosen in the best that does not have heritable variation in the clone's who paves plate the subpopulation.After succeeding transfer culture, the selected clone of stock under serum-free condition again.The recombinant human protein who is expressed by the subclone of final selection is done more detailed biological chemistry to be characterized.

In addition, can use ClonePixFL to separate the K.O. clone, go out the clone that the PrP gene has been rejected fully with fluorescently-labeled antibody test from semisolid medium.

Embodiment

Material and method

1. The device that is used for Protocols in Molecular Biology

Device	Supplier	Type	Catalog number (Cat.No.)	Note
					The agarose gel electrophoresis groove	??BioRad	??SUB-CELL?GT
The electrophoresis power supply	??BioRad	??PAC?3000
					The UV-photophore	??Vilber?Lourmat
Whizzer	??Heraeus	??Biofuge?fresco		??Max.13,000rpm
					Hot mixing tank	??Eppendorf	??5436
Water-bath	??HAAKE	??Type?002-9917
					37 ℃ of incubators	??Menmert	??Modell?300
Refrigerator	??Liebherr			??+2-8℃
					Refrigerated tank	??Liebherr		??-	??-20℃
Pure water system	??Millipore	??Milli-Q	??-
					Transfer pipet	??Gilson	??P2，P20，P200，??P1000	??-	??-
??Yellow?Tips	??Josef?Peske?oHG	??～200μl
					??Blue?Tips	??Josef?Peske?oHG	??～1000μl
Filter tip, 5-250 μ l	??Peske(Mμlti)	Im Rack, aseptic	??1491-11
					Filter tip, 100-1000 μ l	??Peske(Mμlti)	Im Rack, aseptic	??1420-111
Spectrophotometer	??Beckman	??DU?530
					The gel register system	??BioSciTec.??Science?Group	??Gelscript??versionl.1
1 μ glass slide glass	??Ibidi	??ibiTreat	??81826

2. the reagent and the test kit that are used for Protocols in Molecular Biology

Reagent	Supplier	Sequence number	Holding conditions	Note
					The Pfx polysaccharase	??Invitrogen	??11708-013	??-20℃
NotI	??NEB	??R0189S	??-20℃

??EcoRI	??NEB	??R0142S	??-20℃
					??KpnI	??NEB	??R0142S	??-20℃
??NotI	??NEB	??R0146S	??-20℃
					??Concert TM?Rapid?Plasmid??Miniprep?System	??GIBCO	??11453-024	??RT
QIAquick PCR purification kit	??Qiagen	??28104	??RT
					The Qia PhastGel extracts test kit	??Qiagen	??28704	??RT
DNA ladder mixture	??MBI	??SM0331/3	??-20℃
					??SmartLadder	??Eurogentec	??MW-1700-07	??-20℃
Agarose	??Sigma	??A9539	??RT
					Tryptones	??Sigma	??T9410	??RT
Yeast extract	??GIBCO	??30393-029	??RT
					??NaCl	??Roth	??9265.1	??RT
Agar	??Sigma	??A5054	??RT
					The T4 ligase enzyme	??NEB	??#M0202S	??-20℃
SURE 2 surpasses competent cell	??Stratagene	??200152	??-80℃
					One shot Top10F ' competent cell	??Invitrogen	??44-0300	??-80℃
HiSpeed plasmid Midi medicine box	??Qiagen	??12643	??RT
					EndoFree plasmid Maxi medicine box	??Qiagen	??12362	??RT
DNAeasy blood and organize medicine box	??QIAGEN	??69504	??RT
					Gentra purified genes cell medicine box	??QIAGEN	??158767	??RT

3. bacterial growth media

The LB substratum: Tryptones 10g, yeast extract 5g, NaCl 10g is dissolved into 1 liter of H ₂O, autoclaving then.

The LB/amp agar plate: the agar with 1.5% adds in the LB substratum that contains 100 μ g/ml penbritins

4. the material that is used for the 293/293F cell cultures

4.1. clone

293 clones: 293 clones are to have the permanent cell system of tactophily under the condition of serum.This clone is (Graham etc., 1977 of setting up with the initial human embryo kidney that the human adenovirus 5 type DNA by chopping transformed; Harrison etc., 1977).Described E1A adenoviral gene is expressed in described cell, and has participated in the transfer activation of some viral promotors, makes these cells can produce the albumen of high level.

293F clone: FreeStyle 293F clone is the variant of 293 clones, and it is adapted at FreeStyle 293 and expresses suspension growth in the substratum.Initiating cell system obtains in the Robert Horlick from Pharmacopeia company in 1988 there.Described FreeStyle 293F clone is from described 293 clones, and expection and can using together from FreeStyle 293 (the Invitrogen Catalog no.K9000-01) expression system that Invitrogen company obtains, or use other serum free mediums, as the indoor cultivation base of Octapharma company.FreeStyle 293F cell is adapted at FreeStyle 293 and expresses suspension culture in the substratum.Described FreeStyle 293F clone has following feature:

-by the first Master Cell Bank culture preparation from generation to generation, this culture carried out again clone by limiting dilution to it from parent 293F cell.Described 293 clone the culture of deriving only keeps 30-35 generation altogether under serum-free condition.

-suitable high-density, serum-free, suspension growth; Can express in the substratum at FreeStyle 293 and keep.

-have high transfection efficiency with the 293fectin transfection

-suspension culture can be expressed transfection in the substratum at FreeStyle 293, does not need to change substratum.

-allow with the large volume transfectional cell.

4.2. be used for the device of 293/293F cell cultures

Device	Supplier	Type	Catalog number (Cat.No.)	Note
					Aseptic extraction hood	??Heraeus	??HeraSafe
Incubator	??Kendro	??BDD6220	??-	??8％CO 2，??37℃
					The track shaking table	??GFL	??3005		Put into CO 2Incubator
??CO 2The shaking table incubator	??Kühner?AG	??ISF-W-1	??SM1503
					Microscope	??Zeiss	??Axiovert?25	??-	??-
Whizzer	??Kendro	??Megafuge?1.0	??-
					Refrigerator	??Liebherr	??200381	??-	??+2-8℃
-20 ℃ of refrigerated tanks	??Liebherr
					-80 ℃ of refrigerated tanks	??Heraeus	??HFC?586basic	??-	??-
Water-bath	??Memmert?GmbH	??WB?14	??-	??-
					Hemocytometer (Neubauer)	??Peske	??Neubauer??improved	??-	0.100mm dark 0.0025mm 2
The hemocytometer glass cover-plate	??Peske		??03-0000	??20×26×0.4??mm

The artificial cell counter	??Rexel，UK	??ENM	?-	?-
					Isopropanol bath	??Nalgene	1 ℃ of freezing container	?5100-0001	250 ml Virahols to be filled
Biostore (liquid nitrogen, submergence)	??Air?liquide	??Arpege?110
					Erlenmeyer flask, disposable use	??Corning	125,250,500,1000ml has the strainer vent cap	?431143-431147	Polycarbonate
Disposable transfer pipet 1mL	??Schubert????&??Weiss		?9.380.431
					Disposable transfer pipet 2mL	??Falcon/Costar	??-	?356507	?-
Disposable transfer pipet 5mL	??Nunc	??-	?159625	?-
					Disposable transfer pipet 10mL	??Nunc	??-	?159633	?-
Disposable transfer pipet 25mL	??Nunc	??-	?159641	?-
					Disposable transfer pipet 50mL	??Nunc	??-	?159668	?-
Move the liquid supplementary unit	??Sigma-Aldrich	??Accujet	?356555	?-
					The 15ml centrifuge tube	??Peske		?17-1200	Sterile packed
The 50ml centrifuge tube	??Peske		?17-1020	Sterile packed
					The adhesive-stick type label that is used for the low temperature bottle	??CILS	Thin from the lamination label	?LSL7-W-5-TN
The low temperature bottle	??Simport?plastics??(CAN)		?T-311-2	?1.8ml
					The operation support of low temperature bottle	??TPP	??100×200×25??mm	?99016	Fig. 1 referring to SOP
Low temperature box	??Simport	??136×136×50??mm	?T314-2100
					Photometer	??Dynex	??MRX
Spectrophotometer	??Beckman	??DU530
					Fluorescent microscope	??Olympus	??BX-41

4.3. be used for the reagent of 293/293F cell cultures

Reagent	Supplier	Sequence number	Holding conditions	Note
					Serum free medium FreeStyle 293	??Invitrogen	??12338-026	+ 8 ℃, shading	??-
?Lipofectamine2000CD	??Invitrogen	??12566-014	??4℃

??OptiProSFM	??Invitrogen	??12309-019	??4℃
					??DMSO	??Sigma	??D-2650	Room temperature, shading	Not at+8 ℃ or more preserve under the low temperature, because material may crystallization
80% ethanol+1%MEK	??Zefa	??ADR§/36	Room temperature	Be used for sterilization
					Virahol
100%p.a.	??Sigma??Aldrich	??I0398	Room temperature is because of being that combustible solvent seals and ventilate to preserve	Be used for isopropanol bath
					??Opti-MEM?I	??Invitrogen	??51985-034	+ 8 ℃, shading	??-
??Zeocin	??Invitrogen	??R250-01	??At-20℃	??-
					??G418	??PAA	??P11-012	??At4℃	??-
??Lipofectamin?2000CD	??Invitrogen	??12566-014	??4℃
					??OptiPRO?SFM	??Invitrogen	??12309-019	??4℃
??Colcimid	??Biochrom??AG	??L6221	??-20℃
					??Tween?20	??Sigma	??P9416	??RT
??Albuminativ?40g/l	??Octapharma	Lot number: 4417136161

4.4. be used for FISH, the reagent of immunostaining and ELISA

Reagent	Supplier	Sequence number	Holding conditions	Note
					No. 20 karyomit(e) whole chromosome dyeing probes	??Aquarius	??LPP20G	??-20℃
The anti-stripping agent of DAPI	??Aquarius	??DES?150L	??-20℃
					Analysis of protein thing (Bradford)	??BioRad	??500-0006	??4℃
Prion protein EIA test kit	??SpiBio	??589751	??-20℃
					??3F4(first?Ab)	??Sigma	??054K1525	??-20℃
Goat anti-mouse-FITC (secondary Ab)	??Novus	??NB720-F	??4℃
					??SAF32FITC(1，3mg/ml)	??Spibio	??No?odernr.	??-20℃
??SAF32(200μg)	??Spibio	??No?ordernr.	??-20℃
					??POM?17(1mg/ml)	??Dr.Aguzzi?Lab，??Zurich，ETH		??-20℃
??POM?12(1mg/ml)	??Dr.Aguzzi?Lab，??Zurich，ETH		??-20℃

4.5. be used for the initial sum standard growth substratum of 293F cell

FreeStyle 293 substratum, additive-free

FreeStyle 293 expresses substratum and can be used for the growth of FreeStyle 293F cell, keeps and transfection.Can express substratum from the FreeStyle 293 that Invitrogen company obtains is the serum-free prescription of determining, it is the high-density for 293 cells, the special exploitation of suspension culture and transfection.This substratum does not contain human and animal's starting point composition, and with the Glutamax-I preparation, so that improve stability and prolong shelf-lives.

4.6. be used for the freezing substratum of 293F cell

FreeStyle 293 substratum+10%DMSO

5. molecular biology method

5.1 use QIAGEN DNAeasy Blood ﹠amp; Tissue test kit (cat No.69504) or Gentra cell reagent box (QIAGEN Cat No.158767) isolation of genomic DNA from cell precipitation is used for PCR and Southern analyzes.

5.2 isolation of genomic DNA is used for pcr analysis from the flat board of 96-hole:

● use twice in the described cell of PBS rinsing.

● in each hole, add molten born of the same parents' damping fluid (0, the 6ml Proteinase K should add [10mM Tris, 10mg EDTA, 10mMNacl, 0.5%sarcosyl] in the molten born of the same parents' damping fluid of 12ml to) of 50 μ l according to following table

● cultivate described flat board down at 50 ℃ and spend the night, should seal film with Parafilm and cover described flat board.

● with the speed of 2500rpm centrifugal 1 minute.In each hole, add the NaCl/ETOH (the 5M NaCl of 150 μ l and the cooling dehydrated alcohol of 10ml) of 100 μ l and at room temperature rocked described dull and stereotyped 30 minutes.Nucleic acid precipitates with thread latticed form.

● with the speed of 2500rpm centrifugal 1 minute, the described flat board that carefully overturns, so that outwell solution, nucleic acid remains adhered on the described flat board, inhales the seal excess liquid on paper handkerchief.

● use the hyperchannel pipette in each hole, to drip the described nucleic acid of 80% ethanol rinsing 3 times of 100 μ l.In each washing step, rocked described dull and stereotyped 30 minutes.Carefully outwell ethanol with centrifugal 1 minute of the speed of 2500rpm and by each upset.

● genomic dna is dissolved among the TE of 50 μ l, and seals the film covering with Parafilm.Described flat board is put into that 37 ℃ incubator spends the night or cultivated 1-2 hour down, so that dissolving fully at 50 ℃.

● flat board preservation under 4 ℃ that will have genomic dna is stand-by, or preserves down at-20 ℃.

6. with Lipofectamin 2000CD transfectional cell:

Before the beginning, confirm that cell is healthy, and survival rate＞90%.

1. in transfection the day before yesterday, prepare suspension culture with not containing antibiotic growth medium, its cell density is 0.8-1.1 * 10 ⁶Cell/ml.

2. on transfection same day: preparation density is 1.1 * 10 ⁶The suspension culture of viable cell/ml.

3. in order to carry out transfection each time, use following reagent dosage and volume to prepare mixture, be used for being with or without the cell transfecting that Albuminative carries out each milliliter under as the condition of additive at growth medium:

● at the OptiMEM of 34 μ l ^TMSFM or OptiPRO ^TMDilution 0.5-1.5 μ g DNA among the SFM

● at the OptiMEM of 34 μ l ^TMSFM or OptiPro ^TMThe Lipofectamin of dilution 1-10 μ l among the SFM ^TM2000CD

4. described mixture is added in the flask/flat board that contains cell and substratum.

5. under 37 ℃, on flat board, cultivate described cell, or at CO ₂In the incubator, on the track shaking table, rocked suspension culture 24-96 hour with the speed of 125rpm.

6.3. the selection of transfection, the amplification of culture, with cell inoculation on the flat board of 96-hole, and Preparing to use ClonePixFL to clone selects:

1. after transfection, microbiotic zeocin or G418 are added in the culture of transfection to select.

2. after selecting, collect the antibiotics resistance cell, so that isolation of genomic DNA and screen by pcr analysis.

3. the culture that will have positive PCR The selection result increases 5 * 10 ⁵Cell/ml.

4. seed cells on the 96 hole flat boards that growth medium is housed, density is 1000 cells/well.

5. if cell density reaches 50%, the preparation replica plate is used for PCR and cell freezing.

6. when the cell density that reaches 80%, described flat board can be used for the PCR screening.

7. collect the PCR positive cell and with the density of 200-250 cells/well it is inoculated on the 96 hole flat boards once more; Or the described PCR positive cell and be inoculated on the flat board of 96-hole of increasing respectively with the density of 200-250 cells/well.

8. the preparation replica plate is used for PCR, and carries out cell freezing when cell density reaches 50%.

9. when the cell density that reaches 80%, described flat board can be used for the PCR screening.

10. described PCR positive cell is increased 5 * 10 ⁵Cell/ml is so that carry out inoculation test.

11. with different density with described cell inoculation in semisolid medium, so that seek optimum cell density, so that can under the situation that does not contact other colonies, select single colony easily by ClonePixFL.

12. select the single cell colony with ClonePixFL.

13. synchronously the colony of selecting is placed on the flat board of 96-hole and amplification, the replica of preparation cell is used for freezing and pcr analysis.

7.FISH analyze: determine No. 20 chromosomal quantity in 293F cell and the pf293F cell:

Before beginning, confirm that cell is healthy and survival rate＞85%.

1. prepare suspension culture with the 5-10ml growth medium, making cell density is 1-3 * 10 ⁶Cell.

2. 0.2 μ g/ml Colchiceinamidum is added in the substratum, and on the track shaking table with the speed rotating and culturing of 125rpm 30 minutes-1 hour.

3. pass through with centrifugal 10 minutes harvested cells of the speed of 1100rpm.

4. taking-up supernatant liquor stays 500 μ l and suspended sediment again.

5. at room temperature in 5-10ml 75mM KCl, cultivated 8-10 minute.First milliliter KCl drips of solution is added in the described cell.

6. add the 2-3ml stationary liquid and centrifugal 10 minutes with the speed of 1100rpm.

7. cell precipitation is transferred in the microcentrifuge test tube, and in this test tube, carried out subsequently all fixing washing operations.Importantly, cell will suspend fully again.

8. with stationary liquid washed cell 3-6 time, after each washing step, with the speed of 6000rpm in microcentrifuge with cell centrifugation 1 minute.

9. slide glass is necessary for room temperature, makes slide glass pass through hot steam second with 2-3, so that make its surface wettability (bath temperature is 75-80 ℃)

10. 10-30 μ l cell suspending liquid is placed on the slide glass, does not allow liquid dried.

11. when the surface becomes particulate state, allow described slide glass pass through hot steam once more with 1-2 time second.

12. on its surface has the metal plate of thermograde, be dried at once.

13. cultivating slide glass in incubated at room temperature slide glass 2-3 days or under 65 ℃ spends the night.

14. slide glass is immersed in 2 * SSC, among the pH 7.0 2 minutes.

15. slide glass is put into ethanol series liquid each 2 minutes dewater (70%, 85% and 100%)

(chromosome dyeing probe, Cytocell/Aquarius LPP20G), and allow it be warmed up to room temperature 16. from-20 ℃ of following taking-up probes.

17. guarantee that by mixing with transfer pipet repeatedly probe solution is even.

18. take out 5-10 μ l probe solution, and be placed on the slide glass, cover with 24 * 24mm cover glass, and seal with rubbery collosol or transparent nail polish

19. sex change is 2 minutes under 75 ℃ (+/-1 ℃), and hybridization is spent the night under 37 ℃ (+/-1 ℃).

20. carefully remove the vestige of cover glass and all glue

21. with 0.4 * SSC (pH 7.0) washed of 72 ℃ (+/-1 ℃) 2 minutes.

22. drain the washings on the slide glass and at room temperature use 2 * SSC, 0.005%Tween20 (pH 7.0) washing 30 seconds.

23. drain slide glass and add the anti-stripping agent of DAPI of 10 μ l

24. cover and made in 10 minutes in the dark colour developing with cover glass.

25. use fluorescence microscope.

8. immunostaining: analyze the PrP egg on the cell surface of 293F cell and pf293F cell White amount:

Before beginning, guarantee that cell is healthy and survival rate＞85%.

1. with 1-3 * 10 ⁵Cells/well (1 μ slide glass, density Ibidi) are inoculated in the 30-100 μ l growth medium, and 37 ℃ of following overnight incubation.

2. if cell well-grown and become and stick together shape just begins dyeing

3. careful sucking-off substratum, and with the careful rinsing cell of PBS, do not rock.

4. at room temperature added ice-cold fixing agent (50% ethanol, 50% methyl alcohol) 1 minute.

5. use PBS washed cell twice 5 minutes at once

6. cover cell with 8%BSA/PBS and at room temperature cultivated 1 hour.In the moist incubator of sealing, cultivate, air-dry to prevent the fixed cell.

7. with 1 * PBS washed cell twice, 5 minute

8. remove unnecessary PBS gently, and be used in former antibody diluting among the 1%BSA/PBS (3F4, POM12, POM17, SAF32, SAF32FITC 6H4) covers cell, and at room temperature cultivated 1-2 hour.In the moist incubator of sealing, cultivate, air-dry to prevent tissue slice.

9. use PBS washed cell twice, 5 minute

10. remove unnecessary PBS gently, and (goat anti-mouse-FITC) covers cell, and at room temperature cultivates 1-2 hour to be used in the secondary antibody of diluting among the 1%BSA/PBS.In the moist incubator of sealing, cultivate, air-dry to prevent tissue slice.

11. use the PBS washed cell in the dark three times, 5 minutes

12. on slide glass, add the anti-stripping agent of Dapi-, lay cover glass and under fluorescent microscope, check sample.

9.ELISA analyze: to contain the allelic 293F cell of one or two PrP and The pf293F cell carries out the quantitative analysis of PrP protein content:

1. collect 2 * 10 ⁷Cell and centrifugal 5 minutes with the speed of 800rpm

2. remove substratum

3. with the cold PBS washed cell precipitation of 5ml

4. the speed with 800rpm makes described cell centrifugation 5 minutes.

5. remove PBS

6. with 1ml cold PBS washed cell throw out, and with the speed of 6000rpm centrifugal 5 minutes, remove PBS

7. the cell precipitation thing can direct molten born of the same parents or freezing, so that preserve down at-80 ℃

8. molten born of the same parents' damping fluid that 1000 μ l are cold adds in the described cell precipitation

9. allow molten cytosol by 20,23 and No. 26 syringe needles

10. pass through the protein concentration of the molten cytosol of Bradford analysis to measure cell

Embodiment 1:With the PrP K.O. construct pBS_Neo_P that contains Xin Meisu ^-_ R+L_Arm_2B (SEQ ID NO:1) carries out the detailed description of first round rejecting.

A. target is decided method:The PrP that thoroughly eliminates human HEK 293 or HEK 293F cell by promoter trap strategy expresses.Design this method (referring to Fig. 1) and be cell (translation initiation codon is got rid of, and it belongs to the PrP gene) in order to select PrP gene ORF wherein to be replaced specially by neomycin phosphotransferase ORF.

Plasmid construction body 2B (referring to Fig. 2) is made of 4 major portions: 1. carrier main chain (not illustrating in the accompanying drawings): pBluescript, 2. the frame of " neo " (=neomycin phosphotransferase) brachymemma, complete ORF by this gene is formed, do not have it self translation initiation codon, follow by transcription termination signal (referring to SEQ ID NO.1).Key issue: the frame of this neo brachymemma does not carry it self any promotor.Described neo ORF is designed to begin translation from the initial ATG codon of PrP gene." left arm " 3.neo of upstream district, its sequence and PrP intron identical (referring to SEQ ID NO.2) between E2 and E3." right arm " 4.neo in downstream district, its sequence identical with the PrP district in PrP ORF downstream (referring to SEQ ID NO.3).

(a) arrives host 293F cell with this construct transfection when carrying out following operation, (b) the genome neutralization (c) that is incorporated into them is subsequently selected with G418, the purpose of this method is that enrichment has the homologous recombination cell of (representing with two black " X " among Fig. 1), neo ORF downstream and translational control sequence (P that this reorganization can cause PrP to transcribe, E1, integration E2).

The cell of only having integrated the transfection of described construct in the sub-downstream of function on could survival in G418 selects.Therefore, the expection of the cell of all survivals all carries the integration (therefore being referred to as promoter trap strategy) from the sub-downstream of function on.Because construct arm and PrP gene have very long homology (having about 6kb homologous sequence altogether), estimate that homologous recombination is special integration at described PrP locus, it has caused the integration shown in the 3rd row of Fig. 1.

B: target is decided PrP K.O. construct (pBS_Neo_P ^- _ R+L_Arm_2B) generation:The generation that target is decided construct comprises three successive clone steps (Fig. 3), each step comprises: the pcr amplification step that is used for producing " inserting fragment ", described insertion fragment is inserted the Connection Step of carrier, and will connect mixture and be transformed in the intestinal bacteria and by the correct clone of restriction enzyme digestion selection.

1. prepare the pBS_Neo_P-_2B construct:, pcDNA3.1+ carrier (Invitrogen) as template, is used following program synthetic oligonucleotide: 2B-Neo-F:5 '-GGCAA by pcr amplification neo ORF GAATTCGCAGA GCAGTCATTATGATTGAACAAGATGGATTGCAC-GCAG-3 ' (SEQ ID NO.4)

2B-Neo-R：5’-GGACCG CTCGAG-ATGCTTCCGGCTCGTATGTTGT-3’(SEQ?ID?NO.5)，

It has EcoRI and XhoI restriction site (underlining the place).With the product (1244bp) of EcoRI and XhoI digest amplification and be connected on the pBluescript II KS+ (Stratagene) that EcoRI/XhoI-digested.To connect mixture then is transformed in the One shot Top10F ' competent cell (Invitrogen).By screening with the plasmid DNA of single bacterium colony preparation with the EcoRI/XhoI restrictive diges-tion.

2. prepare the pBS_Neo_P-_L_Arm_2B construct: by the left arm of the fixed construct of the described target of pcr amplification, with BAC dna clone 186 (BACPAC Resources Center, BPCR, http://bacpac.chori.org/) as template, and use following program synthetic oligonucleotide:

2B-L-F：5’-GGCAA GCGGCCGC-CTCTGTCTAGGAACACTGCTGTG-3’(SEQ?ID?NO.6)

2B-L-R：5’-GGCAA GAATTC-AAAATGAAGAGGAGAACGTCAGAGTC-3’(SEQ?ID?NO.7)，

They have NotI and EcoRI restriction site (underlining the place) respectively.With EcoRI and XhoI digest amplification product (1929bp) and be connected on the pBS_Neo_P-_2B that EcoRI/NotI-digested.To connect mixture then is transformed in the One shot Top10F ' competent cell (Invitrogen).By screening with the plasmid DNA of single bacterium colony preparation with the EcoRI/NotI restrictive diges-tion.

3. prepare the pBS_Neo_P-_R+L_Arm_2B construct: by the right arm of the fixed construct of the described target of pcr amplification, BAC dna clone 186 that will be identical with top B.2 part is used as template, and uses following program synthetic oligonucleotide:

2B-R-F：5’-GGACCG CTCGAG-TGTGTACCGAGAACTGGGGTGATG-3’(SEQ?ID?NO.8)

2B-R-R：5’-GGCGG GGTACC-GCAGAATCTCTGAGCTCACCTCAG-3’(SEQ?ID?NO.9)，

They have XhoI and KpnI restriction site (underlining the place) respectively.

With XhoI and KpnI digest amplification product (4.6Kb) and be connected on the pBS_Neo_P-_L_Arm_2B that EcoRI/NotI digested.To connect mixture then is transformed into SURE and 2 surpasses competent cell (Stratagene).By screening by the plasmid DNA of single bacterium colony preparation with the XhoI/KpnI restrictive diges-tion.On resulting construct pBS_Neo_P-_R+L_Arm_2B (sequence in the clone district shown in SEQ IDNO.1), the side of neo ORF (, and not having the choice box of it self translation initiation codon) without any promoter sequence:

-upstream is to include the subarea, and neck is preceding in the exon 3 of PrP gene in human genome; With

-downstream is the PrP district, in human genome, follow PrP ORF closely after.

4.pBS_Neo_P-_R+L_Arm_2B the linearizing of construct: by digesting the construct linearizing that described target is fixed with the KpnI restriction, use the QIA PhastGel to extract test kit (referring to Molecular Biology Methods, 5.2) purifying, SmartLadder mark (Eurogentec) electrophoresis with 5 μ l carries out quantitative analysis on 0.8% sepharose by 1 μ l aliquots containig is placed on then.

C. the pBS_Neo_P-_R+L_Arm_2B construct is imported the human cell:Use Lipofectamin 2000CD reagent (Invitrogen, referring to transfection method, 6.2) the construct pBS_Neo_P-_R+L_Arm_2B that target is fixed import host cell (293F, Invitrogen).After transfection 48 hours, cell is taped against on the culture dish of 10cm, density is 1.25-1.5 * 10 ⁶Cell.Antibiotic-screening is when inoculation, and antibiotic concentration is 30-120 μ gG418/ml.Carried out substratum every 2/3 day and change, cultivated 14-21 days.

D. the fixed colony screening of target:When cell reached fusion, the preparation genomic dna used QIA DNAeasy to organize test kit (referring to Molecular BiologyMethods, 5.3), with 1 * 10 when antibiotic-screening ⁶-1 * 10 ⁷G418 ^RCell preparation genomic dna, the method that discloses before perhaps using are layered on the ES cell that (Ramirez-Solis etc., 1992) prepare genomic dna on the 96 hole flat boards.From each genomic dna preparation, preparation PCR mixture, it contains the 80-300ng genomic dna, 1xPCR damping fluid, each Oligonucleolide primers of 200nM, 5mM MgCl ₂, the Expand Fidelity polysaccharase (Roche) of 200nM dNTP and 1.33 units.The sequence of the synthetic oligonucleotide that is used to screen is as follows:

K.O.-F1：5′CGACTCAGTGTCATTCCCTGCAGTCTC?3′(SEQ?ID?NO.10)

K.O.-R1：5′CATAGCCGAATAGCCTCTCCACCCAAG?3′(SEQ?ID?NO.11)

Loop parameter is as follows: 94 ℃ 2 minutes; [94 ℃, 15s; 71,6 ℃, 30s; 72 ℃ of 100s] * 16 circulations; [94 ℃, 15s; 71,6 ℃, 30s; 72 ℃ of 100s+ prolong 3 seconds in each successive circulation] * 26 circulations; 72 ℃ of 7min.Genome DNA sample obtains the PCR product of 2.3kb, shows to have the fixed allelotrope of one or several targets in this cell colony.Use the Southern-engram analysis further to analyze the fixed clone of target.

E. carry a target and decide the sign that the allelic target of PrP is cloned surely:

1. genome PCR screening (Fig. 4): as the disclosed method of superincumbent D part isolation of genomic DNA from the blended cell colony or in the isolated cells colony.All PrP K.O. screening PCR reactions are all used over against shining and bearing contrast and carry out.Being used for over against the template of photograph is from determine to comprise the 200ng genomic dna that target is decided the allelic cell colony mixture of PrP with PrP K.O.PCR sieve method; Negative contrast water replaces genomic dna as template.The appearance of 2.2kb PCR product on sepharose after electrophoresis shown that corresponding cell colony has the fixed PrP cell allelotrope of at least one target.By this method the clone who is selected by ClonePixFL is analyzed, the result as shown in Figure 6.

2. genome Southern analyzes (Fig. 7): the 293F cell clone of deciding PrP by the target of said gene group PCR evaluation is done further genetics sign by the Southern engram analysis.Separate and after capillary transfers on the Hybond+ film at the genome dna electrophoresis of EcoRI-digestion, resulting trace carries out radioactivity hybridization (referring to the red arrow among Fig. 5) with custom-designed dna probe, so that the correct and complete integration of checking neo ORF.5 '-and 3 '-probe be 5 '-and 3 '-external region homologous with the integration site of expection.The Neo probe is used to verify whether the K.O. construct is incorporated on the fixed locus of target or only is random integration.5 '-and 3 '-probe be by the pcr amplification method, use in B.2 disclosed BAC dna clone 186 preparations of present embodiment.The Neo probe is with plasmid pcDNA3.1 (+) amplification, and it comprises the Xin Meisu frame, referring to the general introduction of following form:

The probe title	The DNA probe length	The sequence that is used for the PCR primer of dna probe	Target is decided the Southern collection of illustrative plates of an allelic cell expection of PrP
				5 '-probe 1	??251??bp	??5’-K.O.-F1(SEQ?ID?No.12)：??5’-AGCTTTACCGTCCAGTCTTC-3’??5’-K.O.-R1(SEQ?ID?No.13)：??5’-GGTCTTGATGGCGATA?ACTC-3’	4.2kb and 10.8kb
5 '-probe 2	??252??bp	??5’-K.O.-F2(SEQ?ID?No.14)：??5’-GAGTTATCGCCATCAAGACC-3’??5’-K.O.-R2(SEQ?ID?No.15)：??5’-CATGAGAACCAACGCTAGAG-3’	4.2kb and 10.8kb
				New 3 '-probe	??288??bp	New-3 '-probe-forward (SEQ ID No.28): 5 '-CTAGAGGTCCAGGTCATCTTG-3 ' New-3 '-probe-reverse (SEQ ID No.29): 5 '-TCAGGGAAATTGGGGATCCTG-3 '	6.5kb and 10.8kb
Neo-visits	??304??bp	Neo-probe-F (SEQ ID No.21): 5 '-AGCGAGCACGTACTCGGATG-3 '	??6.5kb

Pin

Neo-probe-R (SEQ ID No.22): 5 '-AAGCACGAGGAAGCGGTCAG-3 '

Figure 5 illustrates the genome Southern method of carrying out with construct pBS_Neo-_P-_L+R_Arm_2B.After genome being carried out EcoRI digestion and radioactivity hybridization, target shows the bands of a spectrum of 4.2kb when deciding the clone and 5 ' of PrP-dna probe hybridization, show the bands of a spectrum of 6.5kb when hybridizing with 3 '-dna probe, and with any probe in detecting to the bands of a spectrum from the fixed allelic wild-type of target not all be 10.8kb.Adopt the Neo-probe, 6.5kb DNA band should be decided to detect on the allelotrope at the PrP target, and for wild-type 293F cell, should not have bands of a spectrum to be detected.

The clone who shows correct collection of illustrative plates in the time of will be with all 3 probe in detecting on PCR and Southern trace is accredited as the cell that on allelotrope target is decided PrP K.O..By FISH, ELISA and immunostaining are further analyzed these positive colonies.

3.FISH analyze (Fig. 8):

Human PrP gene is positioned on No. 20 karyomit(e).293F cell and WCP (whole chromosome dyeing) probe hybridization is so that dye to No. 20 karyomit(e)s.This fish analysis shows that 25% 293F wild-type cell has No. 20 karyomit(e)s of 2 copies, and 75% 293F cell has No. 20 karyomit(e)s of 3 copies.Say technically, use existing method to be difficult to and may reject the 3PrP gene hardly.Therefore, the fish analysis that carries out with No. 20 karyomit(e) WCP dyeing probe helps to differentiate that having carried a target decides the allelic cell clone of PrP, whether has two or three No. 20 karyomit(e)s.In addition, BACDNA clones 186 probes and crosses over 100kb, comprises described PrP gene, and is No. 20 chromosomal parts, equally it is used for FISH hybridization, so that detect possible chromosome deletion and transposition.

After a K.O. construct pBS_Neo_P-_R+L_arm_2B target is integrated surely, can observe three kinds of different FISH figure of PrP K.O. clone:

A. dye two karyomit(e)/mid-terms with No. 20 chromosome probes, (Fig. 8 a) all to show the BAC signal.

B. dye three karyomit(e)/mid-terms, the three shows BAC signal (Fig. 8 b).

C. dye (because No. 20 chromosomal arms translocate to another karyomit(e), this karyomit(e) can only partly dye) three karyomit(e)/mid-terms, have only two No. 20 complete karyomit(e)s to show BAC signal (Fig. 8 c).

Statistical analysis to some PrP K.O. clones is summarized in the following form:

Clone	(collection of illustrative plates a) for 2x chr.20 2xBAC	No. 20 karyomit(e) 3x of 3x BAC (collection of illustrative plates b)	3x chr.20 2x Bac (collection of illustrative plates c)	Other	The total cell Nr. that analyzes
						??K.O.1	??4(12％)	??1(3％)	??29(85％)	??--	??34
??K.O.2	??--	??22(69％)	??10(31％)	??--	??32
						??K.O.3	??2(5％)	??35(87％)	??--	??3(8％)	??40
??K.O.4	??11(27，5％)	??28(70％)	??--	??1(2，??5％)	??40
						??K.O.5	??--	??4(15％)	??21(78％)	??2(7％)	??27
??K.O.6	??--	??1(4％)	??23(96％)	??--	??24

K.O. cell with two BAC signals is used to reject second PrP gene, and no matter each cell exists two or three No. 20 chromosome copies.Therefore, select two following K.O. clones:

K.O.1: there is the colony with two painted No. 20 chromosome copies in this clone, and the two all has the FISH signal (account for detected cell 12%) of BAC.Bigger colony (85%) has shown three painted No. 20 chromosome copies.One of above-mentioned karyomit(e) can only partly dye, and is translocated to another karyomit(e).Have only two complete painted No. 20 karyomit(e)s to show the FISH signal of BAC.

The cell of K.O.6:96% has chromosomal 3 signals No. 20, and 2 signals of BAC.

K.O.1 and K.O.6 are further analyzed by ELISA, so that determine whether their PrP protein level is about 1/2nd of wild-type 293F cell.

4.ELISA test (Fig. 9)

Carry out elisa assay,, and compare with wild-type 293F cell so that the PrP protein concentration in the PrP K.O. cell is carried out quantitatively.Clone K.O.1 and K.O.6 have a PrPK.O. allelotrope, compare with WT 293F cell, can express 1/2nd PrP albumen (Fig. 9) consistently.The conclusion that these data are also additionally supported and additionally confirmed to obtain after the fish analysis.

Embodiment 2:Carry out second detailed description of taking turns rejecting with the PrP K.O. construct that contains zeocin.

It is identical with example 1 described method that this target is decided method, and different is the microbiotic difference.Promptly zeocin has replaced Xin Meisu on the 2nd PrP K.O. construct, so antibiotics resistance clone's selection is carried out with zeocin rather than G418.

A. the clone contains the 2nd PrP K.O. construct of zeocin frame: the Xin Meisu ORF on a PrP K.O. construct pBS_Neo_P-_L+R_Arm_2B is not had the zeocin ORF of himself promotor and ATG translation initiation codon to replace.By the described zeocin frame of pcr amplification,, do not use following PCR primer from himself ATG of plasmid pcDNA3.1-Zeo (+).

Zeo-F：

5’-GGCAAGAATTCGCAGAGCAGTCATTATGGCCAAGTTGACCAGTGCCGTTCC-3’(SEQ?ID?No.27)

Zeo-R：5’-GGACCGCTCGAGTCAGTCCTGCTCCTCGGCCAC-3’(SEQ?ID?No.18)

The length of resulting PCR product is 412bp.

With EcoRI and XhoI digestion pBS_Neo_P-_R+L_Arm_2B plasmid, and be connected with the Zeocin frame of pcr amplification, this frame comprises the restriction site of EcoRI and XhoI at its end.The plasmid that obtains by this connection is called as pBS_Zeo_P-_R+L_Arm (Figure 10) (SEQ IDNO.16), and is used to fixed the 2nd PrP allelotrope of target.With BamHI with the 2nd PrP K.O. construct pBS_Zeo_P-_R+L_Arm linearizing, so that carry out transfection according to the disclosed method of part B.4.

B. With the 2nd PrP K.O. construct, pBS_Zeo_P_R+L_Arm imports has a PrP The allelic PrP K.O. cell of gene: use Lipofectamin 2000CD reagent with the 2nd PrP K.O. construct, the pBS_Zeo_P_R+L-Arm importing is identified Have a target and decide PrP The cell that allelic target is fixed(referring to transfection method, the 19th page).After the transfection 48 hours, cell is taped against on the plate of 10cm or in the shaking table flask of 125ml further suspension culture, cell density is 1.25-1.5 * 10 ⁶Cell/ml.In order to carry out antibiotic-screening, G418 and zeocin are added in the culture by following concentration: G4180-30 μ g/ml, zeocin 0-30 μ g/ml.Changed substratum every 2/3 day, cultivated 14-30 days.

C. screen the fixed clone of zeocin-target: when cell obtains merging under antibiotic-screening, fully according to the method for screening the one K.O. prepare genomic dna (the 25th page, D).

PCR screening method and routine 1D method are similar, and different is, the reverse primer of PCR is designed to the homology with zeocin ORF.

Preparation PCR mixture contains the 20-300ng genomic dna, 1xPCR damping fluid, each Oligonucleolide primers 200nM, 1.25mM MgCl ₂, the Expand Fidelity polysaccharase (Roche) of 200nM dNTP and 1.33 units.The sequence of the synthetic oligonucleotide that is used to screen is as follows:

Zeo-K.O.-F2：5′CTCCTCTTCCTCCCATCTTACC?3′(SEQ?ID?NO.19)

Zeo-K.O.-R2：5′CGAAGTCGTCCTCCACGAAGTC?3′(SEQ?ID?NO.20)

Loop parameter is as follows: 94 ℃ of 4min; [94 ℃, 15s; 63 ℃, 30s; 72 ℃ of 100s] * 16 circulations; [94 ℃, 15s; 63 ℃, 30s; 72 ℃ of each successive circulations of 100s+ prolong 3s] * 24 circulations; 72 ℃ of 7min.Genome DNA sample has obtained 2.3kb PCR product, shows to have the fixed allelotrope of one or several target in this cell colony.

D. have two targets and decide the sign that the allelic target of PrP is cloned surely:

1. genome PCR screening (Figure 11): for whether identification of cell or cytomixis colony have the allelotrope that two targets decide PrP, carry out two independently PCR screen.The PCR primer is used to confirm that to Zeo-K.O.-F2 and Zeo-K.O.-R2 the target of the 2nd PrP Ko.O. construct (pBS_Zeo_P-_R+L_Arm) integrates surely, and primer is used to confirm that to K.O.-F1 and K.O.-F2 the target of a PrP K.O. construct (pBS_Neo_P-_R+L_Arm_2B) integrates surely simultaneously.

From blended cell colony or isolated cells colony, extract genomic dna according to the method that above-mentioned C part is disclosed.All PrP K.O. screening PCR reactions are all used over against shining and bearing contrast and carry out.Being used for over against the template of photograph is 200ng genomic dna from the cell colony mixture, detect by PrP K.O.PCR screening side in advance at this colony allelotrope of deciding PrP that hits, what it at first used is that primer is to Zeo-K.O.-F2 and Zeo-K.O.-R2; Described negative contrast water has replaced genomic dna and has done template.The appearance of 2.3kb PCR product after agarose gel electrophoresis has shown that corresponding cell colony has the cell that carries zeocin frame rather than PrP gene, and it is second to take turns the result of K.O..This result as shown in figure 11.Be accredited as PCR male clone with primer Zeo-K.O.-F2 and Zeo-K.O.-R2, use K.O.-F1 and K.O.-R1 to be further analyzed by PCR, surely integrate existence so that determine the target of a K.O. construct whether still, perhaps it is replaced (Figure 11) by the 2nd K.O. construct.

When twice PCR was all positive, this clone was further analyzed by the Southern-engram analysis.

2.Southern trace sieve method (Figure 12) is similar with example 1 described method.

The genome Southern method of carrying out with construct pBS_Zeo-_P-_L+R_Arm as shown in figure 11.With EcoRI genome is digested with radioactivity hybridization after, target shows the bands of a spectrum of 4.2kb when deciding the clone and 5 ' of PrP-dna probe hybridization, show the bands of a spectrum of 5.7kb when hybridizing, and with two kinds of probe in detecting the time, all show the bands of a spectrum of 10.8kb from the fixed allelic wild-type of target not with 3 '-dna probe.For the fixed allelotrope of PrP target, use the Zeo probe in detecting to 5.7kb DNA band, and for wild-type 293F cell, detect less than bands of a spectrum.

The difference of the Southern collection of illustrative plates that the target of two kinds of K.O. constructs is integrated surely only detects when using 3 '-probe and Zeo-or Neo probe.

● use 3 '-probe:, should detect the band of 6.5kb, and, the band of 5.7kb also should occur for the 2nd K.O. construct for a K.O. construct.In both cases, the WT band all is 10.8kb.

● use the Neo-probe: the band that after integrating a K.O. construct, should detect 6.5kb.

● use the Zeo-probe: the target for a K.O. construct is integrated surely, does not have detected signal, and generally detects the band of 5.7kb for the 2nd K.O. construct.

● be used for the dna probe of Southern analysis and the collection of illustrative plates of expection thereof and be summarized in following form:

The probe title	The length of DNA probe	The sequence that is used for the PCR primer of dna probe	Target is decided the Southern collection of illustrative plates of an allelic cell expection of PrP	Target is decided the Southern collection of illustrative plates of two allelic cell expections of PrP
					5 '-probe 1	??251??bp	??5’K.O.-F1(SEQ?ID?No.12)：??5’-AGCTTTACCGTCCAGTCTTC-3’??5’-K.O.-R1(SEQ?ID?No.13)：??5’-GGTCTTGATGGCGATAACTC-3’	4.2kb and 10.8kb	4.2kb and 10.8kb
??5’-	??252	??5’-K.O.-F2(SEQ?ID?No.14)：	4.2kb and	4.2kb and

Probe 2	?bp	??5’-GAGTTATCGCCATCAAGACC-3’??5’-K.O.-R2(SEQ?ID?No.15)：??5’-CATGAGAACCAACGCTAGAG-3’	??10.8kb	??10.8kb
					New 3 '-probe	?288?bp	New-3 '-probe-forward (SEQ ID No.28): 5 '-CTAGAGGTCCAGGTCATCTTG-3 ' New-3 '-probe-reverse (SEQ ID No.29): 5 '-TCAGGGAAATTGGGGATCCTG-3 '	6.5kb and 10.8kb	5.7kb, 6.5 kb and 10.8kb
Neo-probe	?304?bp	Neo-probe-F (SEQ ID No.21): 5 '-AGCGAGCACGTACTCGGATG-3 ' Neo-probe-R (SEQ ID No.22): 5 '-AAGCACGAGGAAGCGGTCAG-3 '	??6.5kb	??6.5kb
					Zeo-probe	?376?bp	Zeo-probe-F (SEQ ID No.23) 5 '-ATGGCCAAGTTGACCAGTGCCG-3 ' Zeo-probe-R (SEQ ID No.24) 5 '-GTCAGTCCTGCTCCTCGGCCAC:-3 '	No bands of a spectrum	??5.7kb

3.FISH analyze: find that for indoor serum-free adaptability wild-type 293F cell, No. 20 chromosomal copy increases along with time lengthening.Importantly, should get rid of and have two targets and decide the allelic K.O. cell of PrP, it still also contains the 3rd No. 20 chromosomal situations of not touching with wild type gene seat PrP.Therefore, two targets of being identified that have are decided the allelic K.O. cell of PrP and should further be hybridized with BAC 186 probes and No. 20 karyomit(e) WCP dyeing probes.For PrP K.O. clone completely, should not observe above 2BAC signal.

4.ELISA analyze: carry out this analysis according to method 9 is described.In the pf293F cell, should not detect PrP albumen.

5. immunostaining:, on cell surface, do not have the signal of antibody staining for the pf293F cell.

Embodiment 3:For preparing the prion-free 293F clone pf293F serum-free transfection that the prion-free recombinant protein carries out.

3.1 have the transfection that a target is decided the allelic pf293F cell of PrP.

With pcDNA3.1-FVIII (SEQ ID NO.26), pcDNA3.1-FIX (SEQ ID NO.17) and pcDNA3.1-hyg (+)-G-CSFb (SEQ ID NO.25) carrier transfection have a target and decide the allelic PrP KO clone of PrP, so that express human FVIII, FIX and G-CSFb albumen.

Figure 13 illustrates deletion after the one PrP allelotrope transfection efficiency and suitable (Figure 10) of wt293F cell.The FVIII of each 10E6 cell, the expression of the activity unit of FIX and G-CSFb and 293F wild-type cell similar (Figure 14).This has shown that the rejecting of a PrP gene neither can influence the transfection ability of this cell, can not reduce the quantity of the recombinant protein of being produced again.

3.2 the serum-free transfection and the production of human recombination protein in the prion-free 293F clone fully

Successful ablation Protein virus ORF, having caused novel cell is the generation of pf293F, it can produce the therapeutical agent of complete prion-free.Pollute this new clone for fear of Protein virus, for example the Protein virus from culture medium raw material pollutes, all programs (comprising stable transfection, cell cultures and fermentation) are all carried out (referring to PCT/EP2006063705) under serum-free condition, and use the ClonePixFL method.

Specifically, carry out double digested,, excise the fragment of the 1.4kb of the open reading frame that comprises human plasma thromboplastin component from carrier pTG36 and the pcDNA3.1-FIX disclosed in the WO 01/70968 by using HindIII and NotI.This fragment is connected on the fragment of the 5.6kb that obtains by HindIII and NotI double digested carrier pcDNA3.1Hygro (+)-zz (from V870-20, available from Invitrogen), has obtained shown carrier pcDNA3.1-FIX as SEQ ID NO.17.The suspension culture of preparation 28ml, the pf293F cell density of living is 10 ⁶Prepare fat-DNA mixture by following steps, use

The plasmid DNA of I (Invitrogen) dilution 30 μ g, making cumulative volume is 1ml, and uses

I dilutes 40 μ l's

Making cumulative volume is 1ml.After at room temperature cultivating 5 minutes, the DNA that diluted is added to

In, making cumulative volume is 2ml.The sample of transfection was at room temperature cultivated 20 minutes in the dark.The transfection mixture of 2ml added to (final cell density is 1 * 10 in the 28ml pf293F suspension culture ⁶Cell/ml).The pf293F cell of transfection contains 8%CO in 37 ℃/air ₂Wet gas in, on the track shaking table, cultivated 72 hours, rotating speed is 125rpm.

In the substratum based on semi-solid methylcellulose gum, this substratum contains suitable microbiotic and is used for colony screening with the pf293F cell inoculation of transfection as stated above, and detects the highest production clone of output with the antibody that mark is crossed by fluorescence.Use ClonePixFL (Genetix) to analyze a large amount of (thousands of) clone, analyzed the proteic secretion of the fixed FIX of cell quantity and target, produce the clone so that only select hundreds of best FIX subsequently.Different with other currently known methodss, nonproductive clone and blended clone also are random chooses, and the use of ClonePixFL makes it possible to select the clone of quick growth, and they only are from the single celled high clone of production.The cell of selecting increases on the microtitration flat board, subsequently at centrifuge tube, increases under serum-free condition in cell cultures flask and the fermentor tank, so that finish whole procedure.

FIX production of identifying by aforesaid method is cloned in serum-free FreeStyle 293 and expresses in the substratum and cultivate.According to standard method separation and the fixed albumen of purifying target.In addition,, can use the purification process that is disclosed in the optimization among the PCT/EP 2006/061148, comprise that for example SD-handles for the therapeutical agent of production safety.

The Pf293F cell also can be used for the serum-free production of recombinant human FVIII and G-CSF, uses expression vector pcDNA3.1-hyg (+)-G-CSFb (SEQ ID No.25) and pcDNA3.1-FVIII (SEQ IDNo.26) to carry out.

Sequence table:

SEQ ID NO.1:K.O. carrier pBS_Neo_P-_R+L_Arm_2B

Molecule: 10623bps DNA, ring-type

Type begins to stop title/description

Gene 673 2580 PrP left arms

Gene 2,608 3807 Neo

Gene 3,817 8411 PrP right arms

Gene 9,637 10494 AmpR

SEQ ID Nos.2 and 3:A PrP left side and right arm

SEQ ID Nos.4-15:Primer

SEQ ID NO.16:K.O. carrier pBS_Zeo_P-_R+L_Arm

Molecule: 9790bps DNA, ring-type

Type begins to stop title/description

Gene 673 2581 PrP left arms

Gene 2,605 2976 Zeo

Gene 2,983 7583 PrP right arms

Gene 8,805 9662 AmpR

SEQ?ID?NO.17：pcDNA3.1-FIX，

Molecule: 6960bps DNA, ring-type

Type begins to stop title/description

Zone 209 863 CMV promotors

Zone 895 911 MCS "

Gene 939 2324 hFIX

Gene 2,328 2339 SV40 '/SV40polya+ intron

Zone 2,340 2370 ' MCS

Zone 2,381 2595 BGH pA

Zone 2,658 3071 f1 starting points

Zone 3,136 3460 SV40 promotors

Gene 3,478 4501 HygR

Zone 4,514 4886 SV40pA

Zone 5,819 5146 C PUC starting points

Gene 6,824 5964 C AmpR (complementary strand)

SEQ ID Nos 18-24: primer

SEQ?ID?NO.25：pcDNA3.1-hyg(+)-G-CSFb

Molecule: 6237bps dna circle shape

209 863 CMV promoter regions

970 1584 GCSFb genes

1,658 1872 BGH pA zones

1,935 2348 f1 starting areas

2,413 2737 SV40 promoter regions

2,755 3778 HygR genes

3,791 4163 SV40pA zones

5096 4423C PUC starting areas

6101 5241C AmpR genes

SEQ?ID?NO.26：pcDNA3.1-FVIII

Molecule: 9975bps dna circle shape

1 655 CMV promoter regions

783 3082 hFVIII genes

Human FVIII structural domain A1 and A2

783 839 signal peptide zones

Signal peptide for hFVIII

840 1826 A1 zones

HFVIII A1 structural domain

1,977 2972 A2 zones

HFVIII A2 structural domain

3,084 3107 hinge area

3,105 5162 hFVIII genes

Human factor FVIII structural domain A3, C1, C2

3,243 4226 A3 zones

HFVIII A3 structural domain

4,227 4670 C1 zones

HFVIII C3 structural domain

4,683 5141 C2 zones

HFVIII C2 structural domain

5,188 5402 BGH pA zones

5,465 5878 f1 starting areas

5,943 6267 SV40 promoter regions

6,285 7308 HygR genes

7,321 7693 SV40pA zones

8626 7953C pUC starting areas

9631 8771C AmpR genes

SEQ ID Nos 27-29: primer

Sequence table

<110>Octapharma?AG

＜120〉genetics of PrP gene melts

Use target to decide the cell of promoter trap strategy method production as the serum-free recombinant proteins of therapeutical agent

<130>072819WO

<160>29

<170>PatentIn?version?3.3

<210>1

<211>10622

<212>DNA

＜213〉artificial

<220>

＜223〉KO carrier pBS_NEO_P-_R+L_Arm_2B

<400>1

ctaaattgta?agcgttaata?ttttgttaaa?attcgcgtta?aatttttgtt?aaatcagctc????60

attttttaac?caataggccg?aaatcggcaa?aatcccttat?aaatcaaaag?aatagaccga???120

gatagggttg?agtgttgttc?cagtttggaa?caagagtcca?ctattaaaga?acgtggactc???180

caacgtcaaa?gggcgaaaaa?ccgtctatca?gggcgatggc?ccactacgtg?aaccatcacc???240

ctaatcaagt?tttttggggt?cgaggtgccg?taaagcacta?aatcggaacc?ctaaagggag???300

cccccgattt?agagcttgac?ggggaaagcc?ggcgaacgtg?gcgagaaagg?aagggaagaa???360

agcgaaagga?gcgggcgcta?gggcgctggc?aagtgtagcg?gtcacgctgc?gcgtaaccac???420

cacacccgcc?gcgcttaatg?cgccgctaca?gggcgcgtcc?cattcgccat?tcaggctgcg???480

caactgttgg?gaagggcgat?cggtgcgggc?ctcttcgcta?ttacgccagc?tggcgaaagg???540

gggatgtgct?gcaaggcgat?taagttgggt?aacgccaggg?ttttcccagt?cacgacgttg???600

taaaacgacg?gccagtgagc?gcgcgtaata?cgactcacta?tagggcgaat?tggagctcca???660

ccgcggtggc?ggccgcctct?gtctaggaac?actgctgtga?acaaaaccaa?actcccgccc???720

taaaggagcc?tgcactcccg?tggagaacat?gaataataag?cacagaggaa?ataacataat???780

atctcaagta?gctgtaactg?ctccagagaa?taatgaagcc?aggaaagggg?gtgggctagg???840

gggtgctgtt?ttaggtagag?tgatgggaac?agccccactg?agcaaacttt?agccacatga???900

gtagctggaa?gaaaagcctt?ctaggaccag?ggaacagcaa?gtgcaacagc?cctgagacag???960

gatgggcttg?tcagtttgag?gagcagtggg?aggcctgaac?caggttacat?ggggcccagc??1020

cagtatggcc?acgactttgt?gttttatcca?gagtacaaag?gagcctcact?gagggacaag??1080

ggaagtggca?tgatgtgacc?cgcatattaa?gaggagagcg?ctcaatggca?gccaggggag??1140

gagcagggag?gctggttggg?aggctgttga?agaaatcagg?tgagaagtga?tggaagcacc??1200

gaataagatg?gtcatgttgg?aaaaattgag?aagctgaggt?gcttagcatt?gattttcaag??1260

gtagagctac?tgagatttgc?tgatagatcc?aatgtatgct?gggagagaaa?attcagtcac??1320

tctagagcat?tggctggatt?tgtcacccat?tgcagcgaat?ggagaaggtg?ctgacataaa??1380

agccctttag?actgaaagct?actgactgag?gggatggtgc?cctagtttga?tttcctgggg??1440

tgtatgagta?gcagggaggc?caagagctgg?gcctcacgag?attgtgggga?ctacatcagg??1500

gaagcaggcc?tgcaaagaac?ctgcaccaca?gacccccacc?taaaagagcc?tccaaaaacc??1560

ctaactggca?tgacctgagt?actttaactc?tgcttgtaac?tttcaaatat?attttgattt??1620

gcttatgacc?accaaggcaa?aacttcccat?ttcaataatg?ttagtagaaa?cataaacagg??1680

atgttaattt?atttatttgg?taattctttt?tatgaatatt?atccagttta?atcattagct??1740

ctgaaggaga?tgaaaaataa?ttttctaatt?tttagaaaaa?tttgcagcta?attgggtgat??1800

aaaggtaagg?ggtttctgag?ttcacaaaaa?tgttctaaaa?ttggcaacag?tttcggttgc??1860

acgtttcatt?aaatgtacta?aaaaccattg?aactgtacat?ggtatatggt?gaattatatg??1920

gtatgtgaat?tatatctcaa?caagctgggt?attgtttttt?taaaaaataa?aaataaaaaa??1980

ggagaaagag?agagagaaaa?acaattgcag?atcatcccag?cactgaggac?aagactaact??2040

tcagtgttcc?agtatatgcc?attataggtt?ttacggcaca?cagtgatgat?ttggagccta??2100

tgatttgacc?tagggtacag?caggtactgt?ttagcaatca?ttttactatt?gtcataggtc??2160

tctgctcttg?gagctaagtg?cccagggtaa?atgagatctt?taatttgaaa?agagattttt??2220

gatttgatga?atgtacattc?tccaaagggt?cataaattgt?cattctggat?gtttgatctg??2280

tttgttgttt?tggtacaaaa?attagaagaa?aataattcac?tcataaaatg?ttaaataatg??2340

aactaaaagt?cattcatcaa?gtccataact?tagggtcaca?tttgtccttg?gagcaggaga??2400

aagagttgtg?ttcacccttt?tcttactttt?gcttttgtcc?taagtgcttc?agagaagtac??2460

agggtggcaa?cagtgtttct?actgagcagc?tgataccatt?gctatgcact?cattcattat??2520

gcaggaaaca?tttagtaatt?tcaacataaa?tatgggactc?tgacgttctc?ctcttcattt??2580

tgaattcgca?gagcagtcat?tatgattgaa?caagatggat?tgcacgcagg?ttctccggcc??2640

gcttgggtgg?agaggctatt?cggctatgac?tgggcacaac?agacaatcgg?ctgctctgat??2700

gccgccgtgt?tccggctgtc?agcgcagggg?cgcccggttc?tttttgtcaa?gaccgacctg??2760

tccggtgccc?tgaatgaact?gcaggacgag?gcagcgcggc?tatcgtggct?ggccacgacg??2820

ggcgttcctt?gcgcagctgt?gctcgacgtt?gtcactgaag?cgggaaggga?ctggctgcta??2880

ttgggcgaag?tgccggggca?ggatctcctg?tcatctcacc?ttgctcctgc?cgagaaagta??2940

tccatcatgg?ctgatgcaat?gcggcggctg?catacgcttg?atccggctac?ctgcccattc??3000

gaccaccaag?cgaaacatcg?catcgagcga?gcacgtactc?ggatggaagc?cggtcttgtc??3060

gatcaggatg?atctggacga?agagcatcag?gggctcgcgc?cagccgaact?gttcgccagg??3120

ctcaaggcgc?gcatgcccga?cggcgaggat?ctcgtcgtga?cccatggcga?tgcctgcttg??3180

ccgaatatca?tggtggaaaa?tggccgcttt?tctggattca?tcgactgtgg?ccggctgggt??3240

gtggcggacc?gctatcagga?catagcgttg?gctacccgtg?atattgctga?agagcttggc??3300

ggcgaatggg?ctgaccgctt?cctcgtgctt?tacggtatcg?ccgctcccga?ttcgcagcgc????3360

atcgccttct?atcgccttct?tgacgagttc?ttctgagcgg?gactctgggg?ttcgaaatga????3420

ccgaccaagc?gacgcccaac?ctgccatcac?gagatttcga?ttccaccgcc?gccttctatg????3480

aaaggttggg?cttcggaatc?gttttccggg?acgccggctg?gatgatcctc?cagcgcgggg????3540

atctcatgct?ggagttcttc?gcccacccca?acttgtttat?tgcagcttat?aatggttaca????3600

aataaagcaa?tagcatcaca?aatttcacaa?ataaagcatt?tttttcactg?cattctagtt????3660

gtggtttgtc?caaactcatc?aatgtatctt?atcatgtctg?tataccgtcg?acctctagct????3720

agagcttggc?gtaatcatgg?tcatagctgt?ttcctgtgtg?aaattgttat?ccgctcacaa????3780

ttccacacaa?catacgagcc?ggaagcatct?cgagtgtgta?ccgagaactg?gggtgatgtt????3840

ttacttttca?cagtatgggc?tacacagcag?ctgttcaaca?agagtaaata?ttgtcacaac????3900

actgaacctc?tggctagagg?acatattcac?agtgaacata?actgtaacat?atatgaaagg????3960

cttctgggac?ttgaaatcaa?atgtttggga?atggtgccct?tggaggcaac?ctcccatttt????4020

agatgtttaa?aggaccctat?atgtggcatt?cctttcttta?aactataggt?aattaaggca????4080

gctgaaaagt?aaattgcctt?ctagacactg?aaggcaaatc?tcctttgtcc?atttacctgg????4140

aaaccagaat?gattttgaca?tacaggagag?ctgcagttgt?gaaagcacca?tcatcataga????4200

ggatgatgta?attaaaaaat?ggtcagtgtg?caaagaaaag?aactgcttgc?atttctttat????4260

ttctgtctca?taattgtcaa?aaaccagaat?taggtcaagt?tcatagtttc?tgtaattggc????4320

ttttgaatca?aagaataggg?agacaatcta?aaaaatatct?taggttggag?atgacagaaa????4380

tatgattgat?ttgaagtgga?aaaagaaatt?ctgttaatgt?taattaaagt?aaaattattc????4440

cctgaattgt?ttgatattgt?cacctagcag?atatgtatta?cttttctgca?atgttattat????4500

tggcttgcac?tttgtgagta?ttctatgtaa?aaatatatat?gtatataaaa?tatatattgc????4560

ataggacaga?cttaggagtt?ttgtttagag?cagttaacat?ctgaagtgtc?taatgcatta????4620

acttttgtaa?ggtactgaat?acttaatatg?tgggaaaccc?ttttgcgtgg?tccttaggct????4680

tacaatgtgc?actgaatcgt?ttcatgtaag?aatccaaagt?ggacaccatt?aacaggtctt????4740

tgaaatatgc?atgtacttta?tattttctat?atttgtaact?ttgcatgttc?ttgttttgtt????4800

atataaaaaa?attgtaaatg?tttaatatct?gactgaaatt?aaacgagcga?agatgagcac????4860

cacctcccgt?gtctgcagtt?gtatttcctg?gtgcttgccc?tgtgttgggg?actgttttgg????4920

gggttaatct?gagccaagtg?gcgctttctg?tcctcccttc?tcaagtgatg?gccgatggtt????4980

cacgcacttc?cccctgttcc?tgcccttgtc?ctcacttccc?agtcacccac?tagttcatct????5040

ctgcggcttt?tgcattttct?ccacaagcat?ctaagtgggc?ttagcactgg?taaactgcaa????5100

aggcactatt?gcagcaggag?gaacagtctg?ggagcttttt?tcagtcctgg?atttagaaat????5160

agattttctt?gattaaaatg?aaaattaaca?agctctaaag?aactgttgac?ccttgaacta????5220

cacagggatt?agaggcactg?acctgccgca?cagtcgaaaa?tctgcagaga?agtttttttt????5280

gttttgtttt?gttttttttg?agacggagtc?tcgctctgtc?gcccaggctg?gagtgcagtg????5340

gcgggatctc?ggctcactgc?aacctccgcc?tcccgggttc?aggcgattct?cctgcctcag????5400

cctcctgagt?agctgggact?acaggcatat?gccaccatgc?ccggctaatt?tttgtatttt????5460

tagtagagat?ggagtttcac?catattggcc?aggctgttct?caaactcggc?ctcaagtgat????5520

ctgctcgcct?cagccaccca?aagtgctagg?attacaagca?tgagccaccg?cgcccggcct????5580

gcatagaact?tttaactccc?ccaaaactta?attgctaata?gattaattgc?ctgctgttgg????5640

ctggaagcct?taccaataac?gtaaacagtt?ggttagcaca?tatttcacat?gtcatatata????5700

ctatatactg?tattcttacc?ataaagtaag?ttagagaaaa?tgttgttaaa?acaatgagga????5760

aggaaaagta?tatttactat?tcactgactg?gaagtggatc?atcataaaga?tcttcatctc????5820

atcttcctca?cattgaggaa?ggctgaggag?gaaggggagg?ggttggtctt?gctgtctcct????5880

gggtggcaga?ggcagaagaa?aatctgtgtc?tcgtggactc?agttccaacc?cgtggtgttc????5940

aagggtcacc?tgtgcctgct?ttatgcaccc?caaatcaggc?tctaaaatag?tctcaggtcc????6000

tgtgagcctt?agtgacacac?tcttctcaga?ttcaatacct?ttgatctgaa?aggtgacttt????6060

gatttgtcat?aatcttccaa?ctcattctct?acatgtttcg?acgcaccagc?actgtgatgt????6120

tcttggttat?ccttggcaga?agttttctcc?tgcaactaat?ttgctaaagg?cagggggtaa????6180

tcagaagtga?cagtggttga?aatcacaggc?caatatctta?gcaaacgtct?gtgttgtaaa????6240

tggggaagtg?cctctggtgg?ggtcttcaag?gatcactcca?gcctggctag?ggaaactcta????6300

ggggagaggc?acttgaaatt?attgtactca?taggtcctag?agagggaggc?atgtcatgcc????6360

aagcaggggg?ctggattgga?ggcgtgccca?gggattgggt?gcaactcagc?gtgtatgaga????6420

cagaaagaga?gagaactcct?gggcaagtgc?ctttactggg?agccaggatg?gaggacacaa????6480

gcacaaggtg?taaggggatc?tcactggtgt?gtttgaatgt?cactaagtca?cagtcagggg????6540

aagacaagaa?gtggaacttg?tggcagggac?cagccttatt?acactggcac?ctggttacct????6600

ggacagggtg?cccactgcct?atttgtagga?tgtcaaggta?tcaggaaaat?atgaagtttt????6660

taaaaattta?caatataatg?ccaacctcct?acggtgatca?gaggatcagc?tttgtattaa????6720

agatggaaag?ataccaatca?tcacccacaa?aactactaaa?acagaaagaa?tgacaaatac????6780

aattctcggc?caggcacagt?tgctcacgcc?tgtaatccct?gcactttggg?aggccgaggc????6840

aggcagatca?cttgaggtca?ggagttcaag?accagcctgg?ccaacatggt?gaaaccccca????6900

ttccctacta?aaattacaaa?aaattagctg?ggtgtggtcg?cacgtgcctg?taatcccagc????6960

tactcaggag?gctgaggcag?gagaatcgct?tataaccggg?aggcagaggc?tgcagtgagc????7020

cgagatcccg?ccattgcact?ccagcctggc?ggtaagagcg?aaactccgtc?tcaaaaaaat????7080

aaaataaaat?aaacttttca?ttacctgcct?ataataattt?ctcaaaaaga?gtcaactgag????7140

gagacttaaa?gtgttcgaag?catagctact?tgtaggacca?gagtgaataa?tatccacctg????7200

tatctttccc?aggcctcatg?tttcctttgt?tatttcaaac?gtgtgagtca?tgcaaaagga????7260

atttttacta?aaaaggaaaa?gaaaaaaatt?taaaatgacc?agcataaaca?taattgagtt????7320

caatccccca?gaaagggtta?ttcggggaac?agccaggcgc?tgcccttgtt?tttccccctc????7380

cccatctggg?gctaagacca?aaaggggtct?gtctttggag?ggaaatctgg?gtctgtacag????7440

cttcccactc?taaggggagc?cttaatccca?gccggaacct?gagcttctcc?aggcaaggcc????7500

tgaccttgcc?gactgatcta?agcattctgc?gcttcatttt?caccccagaa?aggtgacttc????7560

ctcctggctg?gggggctcct?cccctaggac?tcagctcatc?ctgaactaat?aaccgcgtta????7620

tgtccatcct?ttgttccgtt?ctttgagtcc?cacccatgtt?cttcaggaaa?gctgcagctg????7680

gtccacgtgc?cttgcctgtg?cttctgtaga?actacccctc?ccttctctgc?ctgcagcagg????7740

gctcctggaa?cttgaatgtg?cacttgaatc?tgcaggtcct?gactgtaggt?ctgtgtttct????7800

aacatgctgc?tgggtgacgc?tgtgcccgtc?ctgtggtcgg?cattgtaaca?aggctccgcg????7860

ggttcccacg?ccggagtctg?cgtcagaaac?ctctggaggg?cttgttatac?acagatggct????7920

gaaccccatc?cccagagttt?ctgattccct?agtaccaggg?tggggtctga?gaatttgcat???7980

ttctaacaaa?ttcccaggtg?atactgatgc?tgctggtgcg?gagaggtcac?atggaaacca???8040

ctgctcggca?gatgcaaaag?aagcagaagc?cgccccaacc?ctgaaatact?cacaaagctg???8100

ctgtggagac?cagacacctg?ccgggggaat?gagagcctgg?ggatcctgcc?tgggaggaag???8160

agagagctgg?gagctctatg?ggtgaatgcc?aatggaggtg?caaggaggtg?actaactcct???8220

gccgtctgaa?aatagtgaag?cacagccgag?gcctgaagcc?cacccttagt?gagagatata???8280

gttaaacttg?gaaatggtgc?ccagtcactt?gaaaacagca?cttctcagat?tcagatgtac???8340

acatgaacca?cctgaggatc?ttattaaaat?gtgggttctg?aggctgtagc?tctgaggtga???8400

gctcagagat?tctgcggtac?ccagcttttg?ttccctttag?tgagggttaa?ttgcgcgctt???8460

ggcgtaatca?tggtcatagc?tgtttcctgt?gtgaaattgt?tatccgctca?caattccaca???8520

caacatacga?gccggaagca?taaagtgtaa?agcctggggt?gcctaatgag?tgagctaact???8580

cacattaatt?gcgttgcgct?cactgcccgc?tttccagtcg?ggaaacctgt?cgtgccagct???8640

gcattaatga?atcggccaac?gcgcggggag?aggcggtttg?cgtattgggc?gctcttccgc???8700

ttcctcgctc?actgactcgc?tgcgctcggt?cgttcggctg?cggcgagcgg?tatcagctca???8760

ctcaaaggcg?gtaatacggt?tatccacaga?atcaggggat?aacgcaggaa?agaacatgtg???8820

agcaaaaggc?cagcaaaagg?ccaggaaccg?taaaaaggcc?gcgttgctgg?cgtttttcca???8880

taggctccgc?ccccctgacg?agcatcacaa?aaatcgacgc?tcaagtcaga?ggtggcgaaa???8940

cccgacagga?ctataaagat?accaggcgtt?tccccctgga?agctccctcg?tgcgctctcc???9000

tgttccgacc?ctgccgctta?ccggatacct?gtccgccttt?ctcccttcgg?gaagcgtggc???9060

gctttctcat?agctcacgct?gtaggtatct?cagttcggtg?taggtcgttc?gctccaagct???9120

gggctgtgtg?cacgaacccc?ccgttcagcc?cgaccgctgc?gccttatccg?gtaactatcg???9180

tcttgagtcc?aacccggtaa?gacacgactt?atcgccactg?gcagcagcca?ctggtaacag???9240

gattagcaga?gcgaggtatg?taggcggtgc?tacagagttc?ttgaagtggt?ggcctaacta???9300

cggctacact?agaaggacag?tatttggtat?ctgcgctctg?ctgaagccag?ttaccttcgg???9360

aaaaagagtt?ggtagctctt?gatccggcaa?acaaaccacc?gctggtagcg?gtggtttttt???9420

tgtttgcaag?cagcagatta?cgcgcagaaa?aaaaggatct?caagaagatc?ctttgatctt???9480

ttctacgggg?tctgacgctc?agtggaacga?aaactcacgt?taagggattt?tggtcatgag???9540

attatcaaaa?aggatcttca?cctagatcct?tttaaattaa?aaatgaagtt?ttaaatcaat???9600

ctaaagtata?tatgagtaaa?cttggtctga?cagttaccaa?tgcttaatca?gtgaggcacc???9660

tatctcagcg?atctgtctat?ttcgttcatc?catagttgcc?tgactccccg?tcgtgtagat???9720

aactacgata?cgggagggct?taccatctgg?ccccagtgct?gcaatgatac?cgcgagaccc???9780

acgctcaccg?gctccagatt?tatcagcaat?aaaccagcca?gccggaaggg?ccgagcgcag???9840

aagtggtcct?gcaactttat?ccgcctccat?ccagtctatt?aattgttgcc?gggaagctag???9900

agtaagtagt?tcgccagtta?atagtttgcg?caacgttgtt?gccattgcta?caggcatcgt???9960

ggtgtcacgc?tcgtcgtttg?gtatggcttc?attcagctcc?ggttcccaac?gatcaaggcg??10020

agttacatga?tcccccatgt?tgtgcaaaaa?agcggttagc?tccttcggtc?ctccgatcgt??10080

tgtcagaagt?aagttggccg?cagtgttatc?actcatggtt?atggcagcac?tgcataattc??10140

tcttactgtc?atgccatccg?taagatgctt?ttctgtgact?ggtgagtact?caaccaagtc??10200

attctgagaa?tagtgtatgc?ggcgaccgag?ttgctcttgc?ccggcgtcaa?tacgggataa??10260

taccgcgcca?catagcagaa?ctttaaaagt?gctcatcatt?ggaaaacgtt?cttcggggcg??10320

aaaactctca?aggatcttac?cgctgttgag?atccagttcg?atgtaaccca?ctcgtgcacc??10380

caactgatct?tcagcatctt?ttactttcac?cagcgtttct?gggtgagcaa?aaacaggaag??10440

gcaaaatgcc?gcaaaaaagg?gaataagggc?gacacggaaa?tgttgaatac?tcatactctt??10500

cctttttcaa?tattattgaa?gcatttatca?gggttattgt?ctcatgagcg?gatacatatt??10560

tgaatgtatt?tagaaaaata?aacaaatagg?ggttccgcgc?acatttcccc?gaaaagtgcc??10620

ac?????????????????????????????????????????????????????????????????10622

<210>2

<211>1909

<212>DNA

＜213〉artificial

<220>

＜223〉left arm PrP homology zone

<400>2

ccgcctctgt?ctaggaacac?tgctgtgaac?aaaaccaaac?tcccgcccta?aaggagcctg??????60

cactcccgtg?gagaacatga?ataataagca?cagaggaaat?aacataatat?ctcaagtagc?????120

tgtaactgct?ccagagaata?atgaagccag?gaaagggggt?gggctagggg?gtgctgtttt?????180

aggtagagtg?atgggaacag?ccccactgag?caaactttag?ccacatgagt?agctggaaga?????240

aaagccttct?aggaccaggg?aacagcaagt?gcaacagccc?tgagacagga?tgggcttgtc?????300

agtttgagga?gcagtgggag?gcctgaacca?ggttacatgg?ggcccagcca?gtatggccac?????360

gactttgtgt?tttatccaga?gtacaaagga?gcctcactga?gggacaaggg?aagtggcatg?????420

atgtgacccg?catattaaga?ggagagcgct?caatggcagc?caggggagga?gcagggaggc?????480

tggttgggag?gctgttgaag?aaatcaggtg?agaagtgatg?gaagcaccga?ataagatggt?????540

catgttggaa?aaattgagaa?gctgaggtgc?ttagcattga?ttttcaaggt?agagctactg?????600

agatttgctg?atagatccaa?tgtatgctgg?gagagaaaat?tcagtcactc?tagagcattg?????660

gctggatttg?tcacccattg?cagcgaatgg?agaaggtgct?gacataaaag?ccctttagac?????720

tgaaagctac?tgactgaggg?gatggtgccc?tagtttgatt?tcctggggtg?tatgagtagc?????780

agggaggcca?agagctgggc?ctcacgagat?tgtggggact?acatcaggga?agcaggcctg?????840

caaagaacct?gcaccacaga?cccccaccta?aaagagcctc?caaaaaccct?aactggcatg?????900

acctgagtac?tttaactctg?cttgtaactt?tcaaatatat?tttgatttgc?ttatgaccac?????960

caaggcaaaa?cttcccattt?caataatgtt?agtagaaaca?taaacaggat?gttaatttat????1020

ttatttggta?attcttttta?tgaatattat?ccagtttaat?cattagctct?gaaggagatg????1080

aaaaataatt?ttctaatttt?tagaaaaatt?tgcagctaat?tgggtgataa?aggtaagggg????1140

tttctgagtt?cacaaaaatg?ttctaaaatt?ggcaacagtt?tcggttgcac?gtttcattaa????1200

atgtactaaa?aaccattgaa?ctgtacatgg?tatatggtga?attatatggt?atgtgaatta??1260

tatctcaaca?agctgggtat?tgttttttta?aaaaataaaa?ataaaaaagg?agaaagagag??1320

agagaaaaac?aattgcagat?catcccagca?ctgaggacaa?gactaacttc?agtgttccag??1380

tatatgccat?tataggtttt?acggcacaca?gtgatgattt?ggagcctatg?atttgaccta??1440

gggtacagca?ggtactgttt?agcaatcatt?ttactattgt?cataggtctc?tgctcttgga??1500

gctaagtgcc?cagggtaaat?gagatcttta?atttgaaaag?agatttttga?tttgatgaat??1560

gtacattctc?caaagggtca?taaattgtca?ttctggatgt?ttgatctgtt?tgttgttttg??1620

gtacaaaaat?tagaagaaaa?taattcactc?ataaaatgtt?aaataatgaa?ctaaaagtca??1680

ttcatcaagt?ccataactta?gggtcacatt?tgtccttgga?gcaggagaaa?gagttgtgtt??1740

cacccttttc?ttacttttgc?ttttgtccta?agtgcttcag?agaagtacag?ggtggcaaca??1800

gtgtttctac?tgagcagctg?ataccattgc?tatgcactca?ttcattatgc?aggaaacatt??1860

tagtaatttc?aacataaata?tgggactctg?acgttctcct?cttcatttt??????????????1909

<210>3

<211>4601

<212>DNA

＜213〉artificial

<220>

＜223〉right arm PrP homology zone

<400>3

tgtgtaccga?gaactggggt?gatgttttac?ttttcacagt?atgggctaca?cagcagctgt????60

tcaacaagag?taaatattgt?cacaacactg?aacctctggc?tagaggacat?attcacagtg???120

aacataactg?taacatatat?gaaaggcttc?tgggacttga?aatcaaatgt?ttgggaatgg???180

tgcccttgga?ggcaacctcc?cattttagat?gtttaaagga?ccctatatgt?ggcattcctt???240

tctttaaact?ataggtaatt?aaggcagctg?aaaagtaaat?tgccttctag?acactgaagg???300

caaatctcct?ttgtccattt?acctggaaac?cagaatgatt?ttgacataca?ggagagctgc???360

agttgtgaaa?gcaccatcat?catagaggat?gatgtaatta?aaaaatggtc?agtgtgcaaa???420

gaaaagaact?gcttgcattt?ctttatttct?gtctcataat?tgtcaaaaac?cagaattagg???480

tcaagttcat?agtttctgta?attggctttt?gaatcaaaga?atagggagac?aatctaaaaa???540

atatcttagg?ttggagatga?cagaaatatg?attgatttga?agtggaaaaa?gaaattctgt???600

taatgttaat?taaagtaaaa?ttattccctg?aattgtttga?tattgtcacc?tagcagatat???660

gtattacttt?tctgcaatgt?tattattggc?ttgcactttg?tgagtattct?atgtaaaaat???720

atatatgtat?ataaaatata?tattgcatag?gacagactta?ggagttttgt?ttagagcagt???780

taacatctga?agtgtctaat?gcattaactt?ttgtaaggta?ctgaatactt?aatatgtggg???840

aaaccctttt?gcgtggtcct?taggcttaca?atgtgcactg?aatcgtttca?tgtaagaatc???900

caaagtggac?accattaaca?ggtctttgaa?atatgcatgt?actttatatt?ttctatattt???960

gtaactttgc?atgttcttgt?tttgttatat?aaaaaaattg?taaatgttta?atatctgact??1020

gaaattaaac?gagcgaagat?gagcaccacc?tcccgtgtct?gcagttgtat?ttcctggtgc??1080

ttgccctgtg?ttggggactg?ttttgggggt?taatctgagc?caagtggcgc?tttctgtcct??1140

cccttctcaa?gtgatggccg?atggttcacg?cacttccccc?tgttcctgcc?cttgtcctca??1200

cttcccagtc?acccactagt?tcatctctgc?ggcttttgca?ttttctccac?aagcatctaa??1260

gtgggcttag?cactggtaaa?ctgcaaaggc?actattgcag?caggaggaac?agtctgggag??1320

cttttttcag?tcctggattt?agaaatagat?tttcttgatt?aaaatgaaaa?ttaacaagct??1380

ctaaagaact?gttgaccctt?gaactacaca?gggattagag?gcactgacct?gccgcacagt??1440

cgaaaatctg?cagagaagtt?ttttttgttt?tgttttgttt?tttttgagac?ggagtctcgc??1500

tctgtcgccc?aggctggagt?gcagtggcgg?gatctcggct?cactgcaacc?tccgcctccc??1560

gggttcaggc?gattctcctg?cctcagcctc?ctgagtagct?gggactacag?gcatatgcca??1620

ccatgcccgg?ctaatttttg?tatttttagt?agagatggag?tttcaccata?ttggccaggc??1680

tgttctcaaa?ctcggcctca?agtgatctgc?tcgcctcagc?cacccaaagt?gctaggatta??1740

caagcatgag?ccaccgcgcc?cggcctgcat?agaactttta?actcccccaa?aacttaattg??1800

ctaatagatt?aattgcctgc?tgttggctgg?aagccttacc?aataacgtaa?acagttggtt??1860

agcacatatt?tcacatgtca?tatatactat?atactgtatt?cttaccataa?agtaagttag??1920

agaaaatgtt?gttaaaacaa?tgaggaagga?aaagtatatt?tactattcac?tgactggaag??1980

tggatcatca?taaagatctt?catctcatct?tcctcacatt?gaggaaggct?gaggaggaag??2040

gggaggggtt?ggtcttgctg?tctcctgggt?ggcagaggca?gaagaaaatc?tgtgtctcgt??2100

ggactcagtt?ccaacccgtg?gtgttcaagg?gtcacctgtg?cctgctttat?gcaccccaaa??2160

tcaggctcta?aaatagtctc?aggtcctgtg?agccttagtg?acacactctt?ctcagattca??2220

atacctttga?tctgaaaggt?gactttgatt?tgtcataatc?ttccaactca?ttctctacat??2280

gtttcgacgc?accagcactg?tgatgttctt?ggttatcctt?ggcagaagtt?ttctcctgca??2340

actaatttgc?taaaggcagg?gggtaatcag?aagtgacagt?ggttgaaatc?acaggccaat??2400

atcttagcaa?acgtctgtgt?tgtaaatggg?gaagtgcctc?tggtggggtc?ttcaaggatc??2460

actccagcct?ggctagggaa?actctagggg?agaggcactt?gaaattattg?tactcatagg??2520

tcctagagag?ggaggcatgt?catgccaagc?agggggctgg?attggaggcg?tgcccaggga??2580

ttgggtgcaa?ctcagcgtgt?atgagacaga?aagagagaga?actcctgggc?aagtgccttt??2640

actgggagcc?aggatggagg?acacaagcac?aaggtgtaag?gggatctcac?tggtgtgttt??2700

gaatgtcact?aagtcacagt?caggggaaga?caagaagtgg?aacttgtggc?agggaccagc??2760

cttattacac?tggcacctgg?ttacctggac?agggtgccca?ctgcctattt?gtaggatgtc??2820

aaggtatcag?gaaaatatga?agtttttaaa?aatttacaat?ataatgccaa?cctcctacgg??2880

tgatcagagg?atcagctttg?tattaaagat?ggaaagatac?caatcatcac?ccacaaaact??2940

actaaaacag?aaagaatgac?aaatacaatt?ctcggccagg?cacagttgct?cacgcctgta??3000

atccctgcac?tttgggaggc?cgaggcaggc?agatcacttg?aggtcaggag?ttcaagacca??3060

gcctggccaa?catggtgaaa?cccccattcc?ctactaaaat?tacaaaaaat?tagctgggtg??3120

tggtcgcacg?tgcctgtaat?cccagctact?caggaggctg?aggcaggaga?atcgcttata??3180

accgggaggc?agaggctgca?gtgagccgag?atcccgccat?tgcactccag?cctggcggta??3240

agagcgaaac?tccgtctcaa?aaaaataaaa?taaaataaac?ttttcattac?ctgcctataa??3300

taatttctca?aaaagagtca?actgaggaga?cttaaagtgt?tcgaagcata?gctacttgta??3360

ggaccagagt?gaataatatc?cacctgtatc?tttcccaggc?ctcatgtttc?ctttgttatt??3420

tcaaacgtgt?gagtcatgca?aaaggaattt?ttactaaaaa?ggaaaagaaa?aaaatttaaa??3480

atgaccagca?taaacataat?tgagttcaat?cccccagaaa?gggttattcg?gggaacagcc??3540

aggcgctgcc?cttgtttttc?cccctcccca?tctggggcta?agaccaaaag?gggtctgtct??3600

ttggagggaa?atctgggtct?gtacagcttc?ccactctaag?gggagcctta?atcccagccg??3660

gaacctgagc?ttctccaggc?aaggcctgac?cttgccgact?gatctaagca?ttctgcgctt??3720

cattttcacc?ccagaaaggt?gacttcctcc?tggctggggg?gctcctcccc?taggactcag??3780

ctcatcctga?actaataacc?gcgttatgtc?catcctttgt?tccgttcttt?gagtcccacc??3840

catgttcttc?aggaaagctg?cagctggtcc?acgtgccttg?cctgtgcttc?tgtagaacta??3900

cccctccctt?ctctgcctgc?agcagggctc?ctggaacttg?aatgtgcact?tgaatctgca??3960

ggtcctgact?gtaggtctgt?gtttctaaca?tgctgctggg?tgacgctgtg?cccgtcctgt??4020

ggtcggcatt?gtaacaaggc?tccgcgggtt?cccacgccgg?agtctgcgtc?agaaacctct??4080

ggagggcttg?ttatacacag?atggctgaac?cccatcccca?gagtttctga?ttccctagta??4140

ccagggtggg?gtctgagaat?ttgcatttct?aacaaattcc?caggtgatac?tgatgctgct??4200

ggtgcggaga?ggtcacatgg?aaaccactgc?tcggcagatg?caaaagaagc?agaagccgcc??4260

ccaaccctga?aatactcaca?aagctgctgt?ggagaccaga?cacctgccgg?gggaatgaga??4320

gcctggggat?cctgcctggg?aggaagagag?agctgggagc?tctatgggtg?aatgccaatg??4380

gaggtgcaag?gaggtgacta?actcctgccg?tctgaaaata?gtgaagcaca?gccgaggcct??4440

gaagcccacc?cttagtgaga?gatatagtta?aacttggaaa?tggtgcccag?tcacttgaaa??4500

acagcacttc?tcagattcag?atgtacacat?gaaccacctg?aggatcttat?taaaatgtgg??4560

gttctgaggc?tgtagctctg?aggtgagctc?agagattctg?c??????????????????????4601

<210>4

<211>53

<212>DNA

＜213〉artificial

<220>

＜223〉primer

<400>4

ggcaagaatt?cgcagagcag?tcattatgat?tgaacaagat?ggattgcacg?cag????53

<210>5

<211>34

<212>DNA

＜213〉artificial

<220>

＜223〉primer

<400>5

ggaccgctcg?agatgcttcc?ggctcgtatg?ttgt?????????????????????????34

<210>6

<211>36

<212>DNA

＜213〉artificial

<220>

＜223〉primer

<400>6

ggcaagcggc?cgcctctgtc?taggaacact?gctgtg???????????????????????36

<210>7

<211>37

<212>DNA

＜213〉artificial

<220>

＜223〉primer

<400>7

ggcaagaatt?caaaatgaag?aggagaacgt?cagagtc??????????????????????37

<210>8

<211>36

<212>DNA

＜213〉artificial

<220>

＜223〉primer

<400>8

ggaccgctcg?agtgtgtacc?gagaactggg?gtgatg????????????????????36

<210>9

<211>35

<212>DNA

＜213〉artificial

<220>

＜223〉primer

<400>9

ggcggggtac?cgcagaatct?ctgagctcac?ctcag?????????????????????35

<210>10

<211>27

<212>DNA

＜213〉artificial

<220>

＜223〉primer

<400>10

cgactcagtg?tcattccctg?cagtctc??????????????????????????????27

<210>11

<211>27

<212>DNA

＜213〉artificial

<220>

＜223〉primer

<400>11

catagccgaa?tagcctctcc?acccaag??????????????????????????????27

<210>12

<211>20

<212>DNA

＜213〉artificial

<220>

＜223〉primer

<400>12

agctttaccg?tccagtcttc??????????????????????????????????????20

<210>13

<211>20

<212>DNA

＜213〉artificial

<220>

＜223〉primer

<400>13

ggtcttgatg?gcgataactc??????????????????????????????????????20

<210>14

<211>20

<212>DNA

＜213〉artificial

<220>

＜223〉primer

<400>14

gagttatcgc?catcaagacc????????????????????????????????????20

<210>15

<211>20

<212>DNA

＜213〉artificial

<220>

＜223〉primer

<400>15

catgagaacc?aacgctagag????????????????????????????????????20

<210>16

<211>10158

<212>DNA

＜213〉artificial

<220>

＜223〉KO carrier PBS_Zeo_P-_R+L_Arm

<400>16

ctaaattgta?agcgttaata?ttttgttaaa?attcgcgtta?aatttttgtt?aaatcagctc????60

attttttaac?caataggccg?aaatcggcaa?aatcccttat?aaatcaaaag?aatagaccga???120

gatagggttg?agtgttgttc?cagtttggaa?caagagtcca?ctattaaaga?acgtggactc???180

caacgtcaaa?gggcgaaaaa?ccgtctatca?gggcgatggc?ccactacgtg?aaccatcacc???240

ctaatcaagt?tttttggggt?cgaggtgccg?taaagcacta?aatcggaacc?ctaaagggag???300

cccccgattt?agagcttgac?ggggaaagcc?ggcgaacgtg?gcgagaaagg?aagggaagaa???360

agcgaaagga?gcgggcgcta?gggcgctggc?aagtgtagcg?gtcacgctgc?gcgtaaccac???420

cacacccgcc?gcgcttaatg?cgccgctaca?gggcgcgtcc?cattcgccat?tcaggctgcg???480

caactgttgg?gaagggcgat?cggtgcgggc?ctcttcgcta?ttacgccagc?tggcgaaagg???540

gggatgtgct?gcaaggcgat?taagttgggt?aacgccaggg?ttttcccagt?cacgacgttg???600

taaaacgacg?gccagtgagc?gcgcgtaata?cgactcacta?tagggcgaat?tggagctcca???660

ccgcggtggc?ggccgcctct?gtctaggaac?actgctgtga?acaaaaccaa?actcccgccc???720

taaaggagcc?tgcactcccg?tggagaacat?gaataataag?cacagaggaa?ataacataat???780

atctcaagta?gctgtaactg?ctccagagaa?taatgaagcc?aggaaagggg?gtgggctagg???840

gggtgctgtt?ttaggtagag?tgatgggaac?agccccactg?agcaaacttt?agccacatga???900

gtagctggaa?gaaaagcctt?ctaggaccag?ggaacagcaa?gtgcaacagc?cctgagacag???960

gatgggcttg?tcagtttgag?gagcagtggg?aggcctgaac?caggttacat?ggggcccagc??1020

cagtatggcc?acgactttgt?gttttatcca?gagtacaaag?gagcctcact?gagggacaag??1080

ggaagtggca?tgatgtgacc?cgcatattaa?gaggagagcg?ctcaatggca?gccaggggag??1140

gagcagggag?gctggttggg?aggctgttga?agaaatcagg?tgagaagtga?tggaagcacc??1200

gaataagatg?gtcatgttgg?aaaaattgag?aagctgaggt?gcttagcatt?gattttcaag??1260

gtagagctac?tgagatttgc?tgatagatcc?aatgtatgct?gggagagaaa?attcagtcac??1320

tctagagcat?tggctggatt?tgtcacccat?tgcagcgaat?ggagaaggtg?ctgacataaa??1380

agccctttag?actgaaagct?actgactgag?gggatggtgc?cctagtttga?tttcctgggg??1440

tgtatgagta?gcagggaggc?caagagctgg?gcctcacgag?attgtgggga?ctacatcagg??1500

gaagcaggcc?tgcaaagaac?ctgcaccaca?gacccccacc?taaaagagcc?tccaaaaacc??1560

ctaactggca?tgacctgagt?actttaactc?tgcttgtaac?tttcaaatat?attttgattt??1620

gcttatgacc?accaaggcaa?aacttcccat?ttcaataatg?ttagtagaaa?cataaacagg??1680

atgttaattt?atttatttgg?taattctttt?tatgaatatt?atccagttta?atcattagct??1740

ctgaaggaga?tgaaaaataa?ttttctaatt?tttagaaaaa?tttgcagcta?attgggtgat??1800

aaaggtaagg?ggtttctgag?ttcacaaaaa?tgttctaaaa?ttggcaacag?tttcggttgc??1860

acgtttcatt?aaatgtacta?aaaaccattg?aactgtacat?ggtatatggt?gaattatatg??1920

gtatgtgaat?tatatctcaa?caagctgggt?attgtttttt?taaaaaataa?aaataaaaaa??1980

ggagaaagag?agagagaaaa?acaattgcag?atcatcccag?cactgaggac?aagactaact??2040

tcagtgttcc?agtatatgcc?attataggtt?ttacggcaca?cagtgatgat?ttggagccta??2100

tgatttgacc?tagggtacag?caggtactgt?ttagcaatca?ttttactatt?gtcataggtc??2160

tctgctcttg?gagctaagtg?cccagggtaa?atgagatctt?taatttgaaa?agagattttt??2220

gatttgatga?atgtacattc?tccaaagggt?cataaattgt?cattctggat?gtttgatctg??2280

tttgttgttt?tggtacaaaa?attagaagaa?aataattcac?tcataaaatg?ttaaataatg??2340

aactaaaagt?cattcatcaa?gtccataact?tagggtcaca?tttgtccttg?gagcaggaga??2400

aagagttgtg?ttcacccttt?tcttactttt?gcttttgtcc?taagtgcttc?agagaagtac??2460

agggtggcaa?cagtgtttct?actgagcagc?tgataccatt?gctatgcact?cattcattat??2520

gcaggaaaca?tttagtaatt?tcaacataaa?tatgggactc?tgacgttctc?ctcttcattt??2580

tgaattcgca?gagcagtcat?tatggccaag?ttgaccagtg?ccgttccggt?gctcaccgcg??2640

cgcgacgtcg?ccggagcggt?cgagttctgg?accgaccggc?tcgggttctc?ccgggacttc??2700

gtggaggacg?acttcgccgg?tgtggtccgg?gacgacgtga?ccctgttcat?cagcgcggtc??2760

caggaccagg?tggtgccgga?caacaccctg?gcctgggtgt?gggtgcgcgg?cctggacgag??2820

ctgtacgccg?agtggtcgga?ggtcgtgtcc?acgaacttcc?gggacgcctc?cgggccggcc??2880

atgaccgaga?tcggcgagca?gccgtggggg?cgggagttcg?ccctgcgcga?cccggccggc??2940

aactgcgtgc?acttcgtggc?cgaggagcag?gactgacacg?tgctacgaga?tttcgattcc??3000

accgccgcct?tctatgaaag?gttgggcttc?ggaatcgttt?tccgggacgc?cggctggatg??3060

atcctccagc?gcggggatct?catgctggag?ttcttcgccc?accccaactt?gtttattgca??3120

gcttataatg?gttacaaata?aagcaatagc?atcacaaatt?tcacaaataa?agcatttttt????3180

tcactgcatt?ctagttgtgg?tttgtccaaa?ctcatcaatg?tatcttatca?tgtctgtata????3240

ccgtcgacct?ctagctagag?cttggcgtaa?tcatggtcat?agctgtttcc?tgtgtgaaat????3300

tgttatccgc?tcacaattcc?acacaacata?cgagccggaa?gcatctcgag?tgtgtaccga????3360

gaactggggt?gatgttttac?ttttcacagt?atgggctaca?cagcagctgt?tcaacaagag????3420

taaatattgt?cacaacactg?aacctctggc?tagaggacat?attcacagtg?aacataactg????3480

taacatatat?gaaaggcttc?tgggacttga?aatcaaatgt?ttgggaatgg?tgcccttgga????3540

ggcaacctcc?cattttagat?gtttaaagga?ccctatatgt?ggcattcctt?tctttaaact????3600

ataggtaatt?aaggcagctg?aaaagtaaat?tgccttctag?acactgaagg?caaatctcct????3660

ttgtccattt?acctggaaac?cagaatgatt?ttgacataca?ggagagctgc?agttgtgaaa????3720

gcaccatcat?catagaggat?gatgtaatta?aaaaatggtc?agtgtgcaaa?gaaaagaact????3780

gcttgcattt?ctttatttct?gtctcataat?tgtcaaaaac?cagaattagg?tcaagttcat????3840

agtttctgta?attggctttt?gaatcaaaga?atagggagac?aatctaaaaa?atatcttagg????3900

ttggagatga?cagaaatatg?attgatttga?agtggaaaaa?gaaattctgt?taatgttaat????3960

taaagtaaaa?ttattccctg?aattgtttga?tattgtcacc?tagcagatat?gtattacttt????4020

tctgcaatgt?tattattggc?ttgcactttg?tgagtattct?atgtaaaaat?atatatgtat????4080

ataaaatata?tattgcatag?gacagactta?ggagttttgt?ttagagcagt?taacatctga????4140

agtgtctaat?gcattaactt?ttgtaaggta?ctgaatactt?aatatgtggg?aaaccctttt????4200

gcgtggtcct?taggcttaca?atgtgcactg?aatcgtttca?tgtaagaatc?caaagtggac????4260

accattaaca?ggtctttgaa?atatgcatgt?actttatatt?ttctatattt?gtaactttgc????4320

atgttcttgt?tttgttatat?aaaaaaattg?taaatgttta?atatctgact?gaaattaaac????4380

gagcgaagat?gagcaccacc?tcccgtgtct?gcagttgtat?ttcctggtgc?ttgccctgtg????4440

ttggggactg?ttttgggggt?taatctgagc?caagtggcgc?tttctgtcct?cccttctcaa????4500

gtgatggccg?atggttcacg?cacttccccc?tgttcctgcc?cttgtcctca?cttcccagtc????4560

acccactagt?tcatctctgc?ggcttttgca?ttttctccac?aagcatctaa?gtgggcttag????4620

cactggtaaa?ctgcaaaggc?actattgcag?caggaggaac?agtctgggag?cttttttcag????4680

tcctggattt?agaaatagat?tttcttgatt?aaaatgaaaa?ttaacaagct?ctaaagaact????4740

gttgaccctt?gaactacaca?gggattagag?gcactgacct?gccgcacagt?cgaaaatctg????4800

cagagaagtt?ttttttgttt?tgttttgttt?tttttgagac?ggagtctcgc?tctgtcgccc????4860

aggctggagt?gcagtggcgg?gatctcggct?cactgcaacc?tccgcctccc?gggttcaggc????4920

gattctcctg?cctcagcctc?ctgagtagct?gggactacag?gcatatgcca?ccatgcccgg????4980

ctaatttttg?tatttttagt?agagatggag?tttcaccata?ttggccaggc?tgttctcaaa????5040

ctcggcctca?agtgatctgc?tcgcctcagc?cacccaaagt?gctaggatta?caagcatgag????5100

ccaccgcgcc?cggcctgcat?agaactttta?actcccccaa?aacttaattg?ctaatagatt????5160

aattgcctgc?tgttggctgg?aagccttacc?aataacgtaa?acagttggtt?agcacatatt????5220

tcacatgtca?tatatactat?atactgtatt?cttaccataa?agtaagttag?agaaaatgtt????5280

gttaaaacaa?tgaggaagga?aaagtatatt?tactattcac?tgactggaag?tggatcatca????5340

taaagatctt?catctcatct?tcctcacatt?gaggaaggct?gaggaggaag?gggaggggtt????5400

ggtcttgctg?tctcctgggt?ggcagaggca?gaagaaaatc?tgtgtctcgt?ggactcagtt????5460

ccaacccgtg?gtgttcaagg?gtcacctgtg?cctgctttat?gcaccccaaa?tcaggctcta????5520

aaatagtctc?aggtcctgtg?agccttagtg?acacactctt?ctcagattca?atacctttga????5580

tctgaaaggt?gactttgatt?tgtcataatc?ttccaactca?ttctctacat?gtttcgacgc????5640

accagcactg?tgatgttctt?ggttatcctt?ggcagaagtt?ttctcctgca?actaatttgc????5700

taaaggcagg?gggtaatcag?aagtgacagt?ggttgaaatc?acaggccaat?atcttagcaa????5760

acgtctgtgt?tgtaaatggg?gaagtgcctc?tggtggggtc?ttcaaggatc?actccagcct????5820

ggctagggaa?actctagggg?agaggcactt?gaaattattg?tactcatagg?tcctagagag????5880

ggaggcatgt?catgccaagc?agggggctgg?attggaggcg?tgcccaggga?ttgggtgcaa????5940

ctcagcgtgt?atgagacaga?aagagagaga?actcctgggc?aagtgccttt?actgggagcc????6000

aggatggagg?acacaagcac?aaggtgtaag?gggatctcac?tggtgtgttt?gaatgtcact????6060

aagtcacagt?caggggaaga?caagaagtgg?aacttgtggc?agggaccagc?cttattacac????6120

tggcacctgg?ttacctggac?agggtgccca?ctgcctattt?gtaggatgtc?aaggtatcag????6180

gaaaatatga?agtttttaaa?aatttacaat?ataatgccaa?cctcctacgg?tgatcagagg????6240

atcagctttg?tattaaagat?ggaaagatac?caatcatcac?ccacaaaact?actaaaacag????6300

aaagaatgac?aaatacaatt?ctcggccagg?cacagttgct?cacgcctgta?atccctgcac????6360

tttgggaggc?cgaggcaggc?agatcacttg?aggtcaggag?ttcaagacca?gcctggccaa????6420

catggtgaaa?cccccattcc?ctactaaaat?tacaaaaaat?tagctgggtg?tggtcgcacg????6480

tgcctgtaat?cccagctact?caggaggctg?aggcaggaga?atcgcttata?accgggaggc????6540

agaggctgca?gtgagccgag?atcccgccat?tgcactccag?cctggcggta?agagcgaaac????6600

tccgtctcaa?aaaaataaaa?taaaataaac?ttttcattac?ctgcctataa?taatttctca????6660

aaaagagtca?actgaggaga?cttaaagtgt?tcgaagcata?gctacttgta?ggaccagagt????6720

gaataatatc?cacctgtatc?tttcccaggc?ctcatgtttc?ctttgttatt?tcaaacgtgt????6780

gagtcatgca?aaaggaattt?ttactaaaaa?ggaaaagaaa?aaaatttaaa?atgaccagca????6840

taaacataat?tgagttcaat?cccccagaaa?gggttattcg?gggaacagcc?aggcgctgcc????6900

cttgtttttc?cccctcccca?tctggggcta?agaccaaaag?gggtctgtct?ttggagggaa????6960

atctgggtct?gtacagcttc?ccactctaag?gggagcctta?atcccagccg?gaacctgagc????7020

ttctccaggc?aaggcctgac?cttgccgact?gatctaagca?ttctgcgctt?cattttcacc????7080

ccagaaaggt?gacttcctcc?tggctggggg?gctcctcccc?taggactcag?ctcatcctga????7140

actaataacc?gcgttatgtc?catcctttgt?tccgttcttt?gagtcccacc?catgttcttc????7200

aggaaagctg?cagctggtcc?acgtgccttg?cctgtgcttc?tgtagaacta?cccctccctt????7260

ctctgcctgc?agcagggctc?ctggaacttg?aatgtgcact?tgaatctgca?ggtcctgact????7320

gtaggtctgt?gtttctaaca?tgctgctggg?tgacgctgtg?cccgtcctgt?ggtcggcatt????7380

gtaacaaggc?tccgcgggtt?cccacgccgg?agtctgcgtc?agaaacctct?ggagggcttg????7440

ttatacacag?atggctgaac?cccatcccca?gagtttctga?ttccctagta?ccagggtggg????7500

gtctgagaatt?tgcatttct?aacaaattcc?caggtgatac?tgatgctgct?ggtgcggaga????7560

ggtcacatgg?aaaccactgc?tcggcagatg?caaaagaagc?agaagccgcc?ccaaccctga????7620

aatactcaca?aagctgctgt?ggagaccaga?cacctgccgg?gggaatgaga?gcctggggat????7680

cctgcctggg?aggaagagag?agctgggagc?tctatgggtg?aatgccaatg?gaggtgcaag????7740

gaggtgacta?actcctgccg?tctgaaaata?gtgaagcaca?gccgaggcct?gaagcccacc???7800

cttagtgaga?gatatagtta?aacttggaaa?tggtgcccag?tcacttgaaa?acagcacttc???7860

tcagattcag?atgtacacat?gaaccacctg?aggatcttat?taaaatgtgg?gttctgaggc???7920

tgtagctctg?aggtgagctc?agagattctg?cggtacccag?cttttgttcc?ctttagtgag???7980

ggttaattgc?gcgcttggcg?taatcatggt?catagctgtt?tcctgtgtga?aattgttatc???8040

cgctcacaat?tccacacaac?atacgagccg?gaagcataaa?gtgtaaagcc?tggggtgcct???8100

aatgagtgag?ctaactcaca?ttaattgcgt?tgcgctcact?gcccgctttc?cagtcgggaa???8160

acctgtcgtg?ccagctgcat?taatgaatcg?gccaacgcgc?ggggagaggc?ggtttgcgta???8220

ttgggcgctc?ttccgcttcc?tcgctcactg?actcgctgcg?ctcggtcgtt?cggctgcggc???8280

gagcggtatc?agctcactca?aaggcggtaa?tacggttatc?cacagaatca?ggggataacg???8340

caggaaagaa?catgtgagca?aaaggccagc?aaaaggccag?gaaccgtaaa?aaggccgcgt???8400

tgctggcgtt?tttccatagg?ctccgccccc?ctgacgagca?tcacaaaaat?cgacgctcaa???8460

gtcagaggtg?gcgaaacccg?acaggactat?aaagatacca?ggcgtttccc?cctggaagct???8520

ccctcgtgcg?ctctcctgtt?ccgaccctgc?cgcttaccgg?atacctgtcc?gcctttctcc???8580

cttcgggaag?cgtggcgctt?tctcatagct?cacgctgtag?gtatctcagt?tcggtgtagg???8640

tcgttcgctc?caagctgggc?tgtgtgcacg?aaccccccgt?tcagcccgac?cgctgcgcct???8700

tatccggtaa?ctatcgtctt?gagtccaacc?cggtaagaca?cgacttatcg?ccactggcag???8760

cagccactgg?taacaggatt?agcagagcga?ggtatgtagg?cggtgctaca?gagttcttga???8820

agtggtggcc?taactacggc?tacactagaa?ggacagtatt?tggtatctgc?gctctgctga???8880

agccagttac?cttcggaaaa?agagttggta?gctcttgatc?cggcaaacaa?accaccgctg???8940

gtagcggtgg?tttttttgtt?tgcaagcagc?agattacgcg?cagaaaaaaa?ggatctcaag???9000

aagatccttt?gatcttttct?acggggtctg?acgctcagtg?gaacgaaaac?tcacgttaag???9060

ggattttggt?catgagatta?tcaaaaagga?tcttcaccta?gatcctttta?aattaaaaat???9120

gaagttttaa?atcaatctaa?agtatatatg?agtaaacttg?gtctgacagt?taccaatgct???9180

taatcagtga?ggcacctatc?tcagcgatct?gtctatttcg?ttcatccata?gttgcctgac???9240

tccccgtcgt?gtagataact?acgatacggg?agggcttacc?atctggcccc?agtgctgcaa???9300

tgataccgcg?agacccacgc?tcaccggctc?cagatttatc?agcaataaac?cagccagccg???9360

gaagggccga?gcgcagaagt?ggtcctgcaa?ctttatccgc?ctccatccag?tctattaatt???9420

gttgccggga?agctagagta?agtagttcgc?cagttaatag?tttgcgcaac?gttgttgcca???9480

ttgctacagg?catcgtggtg?tcacgctcgt?cgtttggtat?ggcttcattc?agctccggtt???9540

cccaacgatc?aaggcgagtt?acatgatccc?ccatgttgtg?caaaaaagcg?gttagctcct???9600

tcggtcctcc?gatcgttgtc?agaagtaagt?tggccgcagt?gttatcactc?atggttatgg???9660

cagcactgca?taattctctt?actgtcatgc?catccgtaag?atgcttttct?gtgactggtg???9720

agtactcaac?caagtcattc?tgagaatagt?gtatgcggcg?accgagttgc?tcttgcccgg???9780

cgtcaatacg?ggataatacc?gcgccacata?gcagaacttt?aaaagtgctc?atcattggaa???9840

aacgttcttc?ggggcgaaaa?ctctcaagga?tcttaccgct?gttgagatcc?agttcgatgt???9900

aacccactcg?tgcacccaac?tgatcttcag?catcttttac?tttcaccagc?gtttctgggt???9960

gagcaaaaac?aggaaggcaa?aatgccgcaa?aaaagggaat?aagggcgaca?cggaaatgtt??10020

gaatactcat?actcttcctt?tttcaatatt?attgaagcat?ttatcagggt?tattgtctca??10080

tgagcggata?catatttgaa?tgtatttaga?aaaataaaca?aataggggtt?ccgcgcacat??10140

ttccccgaaa?agtgccac????????????????????????????????????????????????10158

<210>17

<211>6960

<212>DNA

＜213〉artificial

<220>

＜223〉carrier pcDNA3.1-FIX

<400>17

gacggatcgg?gagatctccc?gatcccctat?ggtgcactct?cagtacaatc?tgctctgatg????60

ccgcatagtt?aagccagtat?ctgctccctg?cttgtgtgtt?ggaggtcgct?gagtagtgcg???120

cgagcaaaat?ttaagctaca?acaaggcaag?gcttgaccga?caattgcatg?aagaatctgc???180

ttagggttag?gcgttttgcg?ctgcttcgcg?atgtacgggc?cagatatacg?cgttgacatt???240

gattattgac?tagttattaa?tagtaatcaa?ttacggggtc?attagttcat?agcccatata???300

tggagttccg?cgttacataa?cttacggtaa?atggcccgcc?tggctgaccg?cccaacgacc???360

cccgcccatt?gacgtcaata?atgacgtatg?ttcccatagt?aacgccaata?gggactttcc???420

attgacgtca?atgggtggag?tatttacggt?aaactgccca?cttggcagta?catcaagtgt???480

atcatatgcc?aagtacgccc?cctattgacg?tcaatgacgg?taaatggccc?gcctggcatt???540

atgcccagta?catgacctta?tgggactttc?ctacttggca?gtacatctac?gtattagtca???600

tcgctattac?catggtgatg?cggttttggc?agtacatcaa?tgggcgtgga?tagcggtttg???660

actcacgggg?atttccaagt?ctccacccca?ttgacgtcaa?tgggagtttg?ttttggcacc???720

aaaatcaacg?ggactttcca?aaatgtcgta?acaactccgc?cccattgacg?caaatgggcg???780

gtaggcgtgt?acggtgggag?gtctatataa?gcagagctct?ctggctaact?agagaaccca???840

ctgcttactg?gcttatcgaa?attaatacga?ctcactatag?ggagacccaa?gctggctagc???900

gtttaaactt?aagcttgcat?gccaattccg?caaaggttat?gcagcgcgtg?aacatgatca???960

tggcagaatc?accaggcctc?atcaccatct?gccttttagg?atatctactc?agtgctgaat??1020

gtacagtttt?tcttgatcat?gaaaacgcca?acaaaattct?gaatcggcca?aagaggtata??1080

attcaggtaa?attggaagag?tttgttcaag?ggaaccttga?gagagaatgt?atggaagaaa??1140

agtgtagttt?tgaagaagca?cgagaagttt?ttgaaaacac?tgaaagaaca?actgaatttt??1200

ggaagcagta?tgttgatgga?gatcagtgtg?agtccaatcc?atgtttaaat?ggcggcagtt??1260

gcaaggatga?cattaattcc?tatgaatgtt?ggtgtccctt?tggatttgaa?ggaaagaact??1320

gtgaattaga?tgtaacatgt?aacattaaga?atggcagatg?cgagcagttt?tgtaaaaata??1380

gtgctgataa?caaggtggtt?tgctcctgta?ctgagggata?tcgacttgca?gaaaaccaga??1440

agtcctgtga?accagcagtg?ccatttccat?gtggaagagt?ttctgtttca?caaacttcta??1500

agctcacccg?tgctgagact?gtttttcctg?atgtggacta?tgtaaattct?actgaagctg????1560

aaaccatttt?ggataacatc?actcaaagca?cccaatcatt?taatgacttc?actcgggttg????1620

ttggtggaga?agatgccaaa?ccaggtcaat?tcccttggca?ggttgttttg?aatggtaaag????1680

ttgatgcatt?ctgtggaggc?tctatcgtta?atgaaaaatg?gattgtaact?gctgcccact????1740

gtgttgaaac?tggtgttaaa?attacagttg?tcgcaggtga?acataatatt?gaggagacag????1800

aacatacaga?gcaaaagcga?aatgtgattc?gaattattcc?tcaccacaac?tacaatgcag????1860

ctattaataa?gtacaaccat?gacattgccc?ttctggaact?ggacgaaccc?ttagtgctaa????1920

acagctacgt?tacacctatt?tgcattgctg?acaaggaata?cacgaacatc?ttcctcaaat????1980

ttggatctgg?ctatgtaagt?ggctggggaa?gagtcttcca?caaagggaga?tcagctttag????2040

ttcttcagta?ccttagagtt?ccacttgttg?accgagccac?atgtcttcga?tctacaaagt????2100

tcaccatcta?taacaacatg?ttctgtgctg?gcttccatga?aggaggtaga?gattcatgtc????2160

aaggagatag?tgggggaccc?catgttactg?aagtggaagg?gaccagtttc?ttaactggaa????2220

ttattagctg?gggtgaagag?tgtgcaatga?aaggcaaata?tggaatatat?accaaggtat????2280

cccggtatgt?caactggatt?aaggaaaaaa?caaagctcac?ttaatgggat?cggtcgagcg????2340

gccgctcgag?tctagagggc?ccgtttaaac?ccgctgatca?gcctcgactg?tgccttctag????2400

ttgccagcca?tctgttgttt?gcccctcccc?cgtgccttcc?ttgaccctgg?aaggtgccac????2460

tcccactgtc?ctttcctaat?aaaatgagga?aattgcatcg?cattgtctga?gtaggtgtca????2520

ttctattctg?gggggtgggg?tggggcagga?cagcaagggg?gaggattggg?aagacaatag????2580

caggcatgct?ggggatgcgg?tgggctctat?ggcttctgag?gcggaaagaa?ccagctgggg????2640

ctctaggggg?tatccccacg?cgccctgtag?cggcgcatta?agcgcggcgg?gtgtggtggt????2700

tacgcgcagc?gtgaccgcta?cacttgccag?cgccctagcg?cccgctcctt?tcgctttctt????2760

cccttccttt?ctcgccacgt?tcgccggctt?tccccgtcaa?gctctaaatc?gggggctccc????2820

tttagggttc?cgatttagtg?ctttacggca?cctcgacccc?aaaaaacttg?attagggtga????2880

tggttcacgt?agtgggccat?cgccctgata?gacggttttt?cgccctttga?cgttggagtc????2940

cacgttcttt?aatagtggac?tcttgttcca?aactggaaca?acactcaacc?ctatctcggt????3000

ctattctttt?gatttataag?ggattttgcc?gatttcggcc?tattggttaa?aaaatgagct????3060

gatttaacaa?aaatttaacg?cgaattaatt?ctgtggaatg?tgtgtcagtt?agggtgtgga????3120

aagtccccag?gctccccagc?aggcagaagt?atgcaaagca?tgcatctcaa?ttagtcagca????3180

accaggtgtg?gaaagtcccc?aggctcccca?gcaggcagaa?gtatgcaaag?catgcatctc????3240

aattagtcag?caaccatagt?cccgccccta?actccgccca?tcccgcccct?aactccgccc????3300

agttccgccc?attctccgcc?ccatggctga?ctaatttttt?ttatttatgc?agaggccgag????3360

gccgcctctg?cctctgagct?attccagaag?tagtgaggag?gcttttttgg?aggcctaggc????3420

ttttgcaaaa?agctcccggg?agcttgtata?tccattttcg?gatctgatca?gcacgtgatg????3480

aaaaagcctg?aactcaccgc?gacgtctgtc?gagaagtttc?tgatcgaaaa?gttcgacagc????3540

gtctccgacc?tgatgcagct?ctcggagggc?gaagaatctc?gtgctttcag?cttcgatgta????3600

ggagggcgtg?gatatgtcct?gcgggtaaat?agctgcgccg?atggtttcta?caaagatcgt????3660

tatgtttatc?ggcactttgc?atcggccgcg?ctcccgattc?cggaagtgct?tgacattggg????3720

gaattcagcg?agagcctgac?ctattgcatc?tcccgccgtg?cacagggtgt?cacgttgcaa????3780

gacctgcctg?aaaccgaact?gcccgctgtt?ctgcagccgg?tcgcggaggc?catggatgcg????3840

atcgctgcgg?ccgatcttag?ccagacgagc?gggttcggcc?cattcggacc?gcaaggaatc????3900

ggtcaataca?ctacatggcg?tgatttcata?tgcgcgattg?ctgatcccca?tgtgtatcac????3960

tggcaaactg?tgatggacga?caccgtcagt?gcgtccgtcg?cgcaggctct?cgatgagctg????4020

atgctttggg?ccgaggactg?ccccgaagtc?cggcacctcg?tgcacgcgga?tttcggctcc????4080

aacaatgtcc?tgacggacaa?tggccgcata?acagcggtca?ttgactggag?cgaggcgatg????4140

ttcggggatt?cccaatacga?ggtcgccaac?atcttcttct?ggaggccgtg?gttggcttgt????4200

atggagcagc?agacgcgcta?cttcgagcgg?aggcatccgg?agcttgcagg?atcgccgcgg????4260

ctccgggcgt?atatgctccg?cattggtctt?gaccaactct?atcagagctt?ggttgacggc????4320

aatttcgatg?atgcagcttg?ggcgcagggt?cgatgcgacg?caatcgtccg?atccggagcc????4380

gggactgtcg?ggcgtacaca?aatcgcccgc?agaagcgcgg?ccgtctggac?cgatggctgt????4440

gtagaagtac?tcgccgatag?tggaaaccga?cgccccagca?ctcgtccgag?ggcaaaggaa????4500

tagcacgtgc?tacgagattt?cgattccacc?gccgccttct?atgaaaggtt?gggcttcgga????4560

atcgttttcc?gggacgccgg?ctggatgatc?ctccagcgcg?gggatctcat?gctggagttc????4620

ttcgcccacc?ccaacttgtt?tattgcagct?tataatggtt?acaaataaag?caatagcatc????4680

acaaatttca?caaataaagc?atttttttca?ctgcattcta?gttgtggttt?gtccaaactc????4740

atcaatgtat?cttatcatgt?ctgtataccg?tcgacctcta?gctagagctt?ggcgtaatca????4800

tggtcatagc?tgtttcctgt?gtgaaattgt?tatccgctca?caattccaca?caacatacga????4860

gccggaagca?taaagtgtaa?agcctggggt?gcctaatgag?tgagctaact?cacattaatt????4920

gcgttgcgct?cactgcccgct?ttccagtcg?ggaaacctgt?cgtgccagct?gcattaatga????4980

atcggccaac?gcgcggggag?aggcggtttg?cgtattgggc?gctcttccgc?ttcctcgctc????5040

actgactcgc?tgcgctcggt?cgttcggctg?cggcgagcgg?tatcagctca?ctcaaaggcg????5100

gtaatacggt?tatccacaga?atcaggggat?aacgcaggaa?agaacatgtg?agcaaaaggc????5160

cagcaaaagg?ccaggaaccg?taaaaaggcc?gcgttgctgg?cgtttttcca?taggctccgc????5220

ccccctgacg?agcatcacaa?aaatcgacgc?tcaagtcaga?ggtggcgaaa?cccgacagga????5280

ctataaagat?accaggcgtt?tccccctgga?agctccctcg?tgcgctctcc?tgttccgacc????5340

ctgccgctta?ccggatacct?gtccgccttt?ctcccttcgg?gaagcgtggc?gctttctcat????5400

agctcacgct?gtaggtatct?cagttcggtg?taggtcgttc?gctccaagct?gggctgtgtg????5460

cacgaacccc?ccgttcagcc?cgaccgctgc?gccttatccg?gtaactatcg?tcttgagtcc????5520

aacccggtaa?gacacgactt?atcgccactg?gcagcagcca?ctggtaacag?gattagcaga????5580

gcgaggtatg?taggcggtgc?tacagagttc?ttgaagtggt?ggcctaacta?cggctacact????5640

agaagaacag?tatttggtat?ctgcgctctg?ctgaagccag?ttaccttcgg?aaaaagagtt????5700

ggtagctctt?gatccggcaa?acaaaccacc?gctggtagcg?gtggtttttt?tgtttgcaag????5760

cagcagatta?cgcgcagaaa?aaaaggatct?caagaagatc?ctttgatctt?ttctacgggg????5820

tctgacgctc?agtggaacga?aaactcacgt?taagggattt?tggtcatgag?attatcaaaa????5880

aggatcttca?cctagatcct?tttaaattaa?aaatgaagtt?ttaaatcaat?ctaaagtata????5940

tatgagtaaa?cttggtctga?cagttaccaa?tgcttaatca?gtgaggcacc?tatctcagcg????6000

atctgtctat?ttcgttcatc?catagttgcc?tgactccccg?tcgtgtagat?aactacgata??6060

cgggagggct?taccatctgg?ccccagtgct?gcaatgatac?cgcgagaccc?acgctcaccg??6120

gctccagatt?tatcagcaat?aaaccagcca?gccggaaggg?ccgagcgcag?aagtggtcct??6180

gcaactttat?ccgcctccat?ccagtctatt?aattgttgcc?gggaagctag?agtaagtagt??6240

tcgccagtta?atagtttgcg?caacgttgtt?gccattgcta?caggcatcgt?ggtgtcacgc??6300

tcgtcgtttg?gtatggcttc?attcagctcc?ggttcccaac?gatcaaggcg?agttacatga??6360

tcccccatgt?tgtgcaaaaa?agcggttagc?tccttcggtc?ctccgatcgt?tgtcagaagt??6420

aagttggccg?cagtgttatc?actcatggtt?atggcagcac?tgcataattc?tcttactgtc??6480

atgccatccg?taagatgctt?ttctgtgact?ggtgagtact?caaccaagtc?attctgagaa??6540

tagtgtatgc?ggcgaccgag?ttgctcttgc?ccggcgtcaa?tacgggataa?taccgcgcca??6600

catagcagaa?ctttaaaagt?gctcatcatt?ggaaaacgtt?cttcggggcg?aaaactctca??6660

aggatcttac?cgctgttgag?atccagttcg?atgtaaccca?ctcgtgcacc?caactgatct??6720

tcagcatctt?ttactttcac?cagcgtttct?gggtgagcaa?aaacaggaag?gcaaaatgcc??6780

gcaaaaaagg?gaataagggc?gacacggaaa?tgttgaatac?tcatactctt?cctttttcaa??6840

tattattgaa?gcatttatca?gggttattgt?ctcatgagcg?gatacatatt?tgaatgtatt??6900

tagaaaaata?aacaaatagg?ggttccgcgc?acatttcccc?gaaaagtgcc?acctgacgtc??6960

<210>18

<211>33

<212>DNA

＜213〉artificial

<220>

＜223〉primer

<400>18

ggaccgctcg?agtcagtcct?gctcctcggc?cac?????????????????????33

<210>19

<211>22

<212>DNA

＜213〉artificial

<220>

＜223〉primer

<400>19

ctcctcttcc?tcccatctta?cc?????????????????????????????????22

<210>20

<211>22

<212>DNA

＜213〉artificial

<220>

＜223〉primer

<400>20

cgaagtcgtc?ctccacgaag?tc?????????????????????????????????22

<210>21

<211>20

<212>DNA

＜213〉artificial

<220>

＜223〉primer

<400>21

agcgagcacg?tactcggatg????????????????????????????????????20

<210>22

<211>20

<212>DNA

＜213〉artificial

<220>

＜223〉primer

<400>22

aagcacgagg?aagcggtcag????????????????????????????????????20

<210>23

<211>22

<212>DNA

＜213〉artificial

<220>

＜223〉primer

<400>23

atggccaagt?tgaccagtgc?cg?????????????????????????????????22

<210>24

<211>22

<212>DNA

＜213〉artificial

<220>

＜223〉primer

<400>24

gtcagtcctg?ctcctcggcc?ac?????????????????????????????????22

<210>25

<211>6237

<212>DNA

＜213〉artificial

<220>

＜223〉expression vector

<400>25

gacggatcgg?gagatctccc?gatcccctat?ggtgcactct?cagtacaatc?tgctctgatg????60

ccgcatagtt?aagccagtat?ctgctccctg?cttgtgtgtt?ggaggtcgct?gagtagtgcg???120

cgagcaaaat?ttaagctaca?acaaggcaag?gcttgaccga?caattgcatg?aagaatctgc???180

ttagggttag?gcgttttgcg?ctgcttcgcg?atgtacgggc?cagatatacg?cgttgacatt???240

gattattgac?tagttattaa?tagtaatcaa?ttacggggtc?attagttcat?agcccatata???300

tggagttccg?cgttacataa?cttacggtaa?atggcccgcc?tggctgaccg?cccaacgacc???360

cccgcccatt?gacgtcaata?atgacgtatg?ttcccatagt?aacgccaata?gggactttcc???420

attgacgtca?atgggtggag?tatttacggt?aaactgccca?cttggcagta?catcaagtgt???480

atcatatgcc?aagtacgccc?cctattgacg?tcaatgacgg?taaatggccc?gcctggcatt???540

atgcccagta?catgacctta?tgggactttc?ctacttggca?gtacatctac?gtattagtca???600

tcgctattac?catggtgatg?cggttttggc?agtacatcaa?tgggcgtgga?tagcggtttg???660

actcacgggg?atttccaagt?ctccacccca?ttgacgtcaa?tgggagtttg?ttttggcacc???720

aaaatcaacg?ggactttcca?aaatgtcgta?acaactccgc?cccattgacg?caaatgggcg???780

gtaggcgtgt?acggtgggag?gtctatataa?gcagagctct?ctggctaact?agagaaccca???840

ctgcttactg?gcttatcgaa?attaatacga?ctcactatag?ggagacccaa?gctggctagc???900

gtttaaactt?aagcttggta?ccgagctcgg?atccactagt?aacggccgcc?agtgtgctgg???960

aattcggcta?tggctggacc?tgccacccag?agccccatga?agctgatggc?cctgcagctg??1020

ctgctgtggc?acagtgcact?ctggacagtg?caggaagcca?cccccctggg?ccctgccagc??1080

tccctgcccc?agagcttcct?gctcaagtgc?ttagagcaag?tgaggaagat?ccagggcgat??1140

ggcgcagcgc?tccaggagaa?gctgtgtgcc?acctacaagc?tgtgccaccc?cgaggagctg??1200

gtgctgctcg?gacactctct?gggcatcccc?tgggctcccc?tgagcagctg?ccccagccag??1260

gccctgcagc?tggcaggctg?cttgagccaa?ctccatagcg?gccttttcct?ctaccagggg??1320

ctcctgcagg?ccctggaagg?gatctccccc?gagttgggtc?ccaccttgga?cacactgcag??1380

ctggacgtcg?ccgactttgc?caccaccatc?tggcagcaga?tggaagaact?gggaatggcc??1440

cctgccctgc?agcccaccca?gggtgccatg?ccggccttcg?cctctgcttt?ccagcgccgg??1500

gcaggagggg?tcctggttgc?ctcccatctg?cagagcttcc?tggaggtgtc?gtaccgcgtt??1560

ctacgccacc?ttgcccagcc?ctgaagccga?attctgcaga?tatccatcac?actggcggcc??1620

gctcgagtct?agagggcccg?tttaaacccg?ctgatcagcc?tcgactgtgc?cttctagttg??1680

ccagccatct?gttgtttgcc?cctcccccgt?gccttccttg?accctggaag?gtgccactcc??1740

cactgtcctt?tcctaataaa?atgaggaaat?tgcatcgcat?tgtctgagta?ggtgtcattc??1800

tattctgggg?ggtggggtgg?ggcaggacag?caagggggag?gattgggaag?acaatagcag??1860

gcatgctggg?gatgcggtgg?gctctatggc?ttctgaggcg?gaaagaacca?gctggggctc??1920

tagggggtat?ccccacgcgc?cctgtagcgg?cgcattaagc?gcggcgggtg?tggtggttac??1980

gcgcagcgtg?accgctacac?ttgccagcgc?cctagcgccc?gctcctttcg?ctttcttccc??2040

ttcctttctc?gccacgttcg?ccggctttcc?ccgtcaagct?ctaaatcggg?ggctcccttt??2100

agggttccga?tttagtgctt?tacggcacct?cgaccccaaa?aaacttgatt?agggtgatgg??2160

ttcacgtagt?gggccatcgc?cctgatagac?ggtttttcgc?cctttgacgt?tggagtccac??2220

gttctttaat?agtggactct?tgttccaaac?tggaacaaca?ctcaacccta?tctcggtcta??2280

ttcttttgat?ttataaggga?ttttgccgat?ttcggcctat?tggttaaaaa?atgagctgat??2340

ttaacaaaaa?tttaacgcga?attaattctg?tggaatgtgt?gtcagttagg?gtgtggaaag??2400

tccccaggct?ccccagcagg?cagaagtatg?caaagcatgc?atctcaatta?gtcagcaacc??2460

aggtgtggaa?agtccccagg?ctccccagca?ggcagaagta?tgcaaagcat?gcatctcaat??2520

tagtcagcaa?ccatagtccc?gcccctaact?ccgcccatcc?cgcccctaac?tccgcccagt??2580

tccgcccatt?ctccgcccca?tggctgacta?atttttttta?tttatgcaga?ggccgaggcc??2640

gcctctgcct?ctgagctatt?ccagaagtag?tgaggaggct?tttttggagg?cctaggcttt??2700

tgcaaaaagc?tcccgggagc?ttgtatatcc?attttcggat?ctgatcagca?cgtgatgaaa??2760

aagcctgaac?tcaccgcgac?gtctgtcgag?aagtttctga?tcgaaaagtt?cgacagcgtc??2820

tccgacctga?tgcagctctc?ggagggcgaa?gaatctcgtg?ctttcagctt?cgatgtagga??2880

gggcgtggat?atgtcctgcg?ggtaaatagc?tgcgccgatg?gtttctacaa?agatcgttat??2940

gtttatcggc?actttgcatc?ggccgcgctc?ccgattccgg?aagtgcttga?cattggggaa??3000

ttcagcgaga?gcctgaccta?ttgcatctcc?cgccgtgcac?agggtgtcac?gttgcaagac??3060

ctgcctgaaa?ccgaactgcc?cgctgttctg?cagccggtcg?cggaggccat?ggatgcgatc??3120

gctgcggccg?atcttagcca?gacgagcggg?ttcggcccat?tcggaccgca?aggaatcggt??3180

caatacacta?catggcgtga?tttcatatgc?gcgattgctg?atccccatgt?gtatcactgg??3240

caaactgtga?tggacgacac?cgtcagtgcg?tccgtcgcgc?aggctctcga?tgagctgatg??3300

ctttgggccg?aggactgccc?cgaagtccgg?cacctcgtgc?acgcggattt?cggctccaac??3360

aatgtcctga?cggacaatgg?ccgcataaca?gcggtcattg?actggagcga?ggcgatgttc??3420

ggggattccc?aatacgaggt?cgccaacatc?ttcttctgga?ggccgtggtt?ggcttgtatg??3480

gagcagcaga?cgcgctactt?cgagcggagg?catccggagc?ttgcaggatc?gccgcggctc??3540

cgggcgtata?tgctccgcat?tggtcttgac?caactctatc?agagcttggt?tgacggcaat??3600

ttcgatgatg?cagcttgggc?gcagggtcga?tgcgacgcaa?tcgtccgatc?cggagccggg??3660

actgtcgggc?gtacacaaat?cgcccgcaga?agcgcggccg?tctggaccga?tggctgtgta??3720

gaagtactcg?ccgatagtgg?aaaccgacgc?cccagcactc?gtccgagggc?aaaggaatag??3780

cacgtgctac?gagatttcga?ttccaccgcc?gccttctatg?aaaggttggg?cttcggaatc??3840

gttttccggg?acgccggctg?gatgatcctc?cagcgcgggg?atctcatgct?ggagttcttc??3900

gcccacccca?acttgtttat?tgcagcttat?aatggttaca?aataaagcaa?tagcatcaca??3960

aatttcacaa?ataaagcatt?tttttcactg?cattctagtt?gtggtttgtc?caaactcatc??4020

aatgtatctt?atcatgtctg?tataccgtcg?acctctagct?agagcttggc?gtaatcatgg??4080

tcatagctgt?ttcctgtgtg?aaattgttat?ccgctcacaa?ttccacacaa?catacgagcc??4140

ggaagcataa?agtgtaaagc?ctggggtgcc?taatgagtga?gctaactcac?attaattgcg??4200

ttgcgctcac?tgcccgcttt?ccagtcggga?aacctgtcgt?gccagctgca?ttaatgaatc??4260

ggccaacgcg?cggggagagg?cggtttgcgt?attgggcgct?cttccgcttc?ctcgctcact??4320

gactcgctgc?gctcggtcgt?tcggctgcgg?cgagcggtat?cagctcactc?aaaggcggta??4380

atacggttat?ccacagaatc?aggggataac?gcaggaaaga?acatgtgagc?aaaaggccag??4440

caaaaggcca?ggaaccgtaa?aaaggccgcg?ttgctggcgt?ttttccatag?gctccgcccc??4500

cctgacgagc?atcacaaaaa?tcgacgctca?agtcagaggt?ggcgaaaccc?gacaggacta??4560

taaagatacc?aggcgtttcc?ccctggaagc?tccctcgtgc?gctctcctgt?tccgaccctg??4620

ccgcttaccg?gatacctgtc?cgcctttctc?ccttcgggaa?gcgtggcgct?ttctcatagc??4680

tcacgctgta?ggtatctcag?ttcggtgtag?gtcgttcgct?ccaagctggg?ctgtgtgcac??4740

gaaccccccg?ttcagcccga?ccgctgcgcc?ttatccggta?actatcgtct?tgagtccaac??4800

ccggtaagac?acgacttatc?gccactggca?gcagccactg?gtaacaggat?tagcagagcg??4860

aggtatgtag?gcggtgctac?agagttcttg?aagtggtggc?ctaactacgg?ctacactaga??4920

agaacagtat?ttggtatctg?cgctctgctg?aagccagtta?ccttcggaaa?aagagttggt??4980

agctcttgat?ccggcaaaca?aaccaccgct?ggtagcggtg?gtttttttgt?ttgcaagcag??5040

cagattacgc?gcagaaaaaa?aggatctcaa?gaagatcctt?tgatcttttc?tacggggtct??5100

gacgctcagt?ggaacgaaaa?ctcacgttaa?gggattttgg?tcatgagatt?atcaaaaagg??5160

atcttcacct?agatcctttt?aaattaaaaa?tgaagtttta?aatcaatcta?aagtatatat??5220

gagtaaactt?ggtctgacag?ttaccaatgc?ttaatcagtg?aggcacctat?ctcagcgatc??5280

tgtctatttc?gttcatccat?agttgcctga?ctccccgtcg?tgtagataac?tacgatacgg??5340

gagggcttac?catctggccc?cagtgctgca?atgataccgc?gagacccacg?ctcaccggct??5400

ccagatttat?cagcaataaa?ccagccagcc?ggaagggccg?agcgcagaag?tggtcctgca??5460

actttatccg?cctccatcca?gtctattaat?tgttgccggg?aagctagagt?aagtagttcg??5520

ccagttaata?gtttgcgcaa?cgttgttgcc?attgctacag?gcatcgtggt?gtcacgctcg??5580

tcgtttggta?tggcttcatt?cagctccggt?tcccaacgat?caaggcgagt?tacatgatcc??5640

cccatgttgt?gcaaaaaagc?ggttagctcc?ttcggtcctc?cgatcgttgt?cagaagtaag??5700

ttggccgcag?tgttatcact?catggttatg?gcagcactgc?ataattctct?tactgtcatg??5760

ccatccgtaa?gatgcttttc?tgtgactggt?gagtactcaa?ccaagtcatt?ctgagaatag??5820

tgtatgcggc?gaccgagttg?ctcttgcccg?gcgtcaatac?gggataatac?cgcgccacat??5880

agcagaactt?taaaagtgct?catcattgga?aaacgttctt?cggggcgaaa?actctcaagg??5940

atcttaccgc?tgttgagatc?cagttcgatg?taacccactc?gtgcacccaa?ctgatcttca??6000

gcatctttta?ctttcaccag?cgtttctggg?tgagcaaaaa?caggaaggca?aaatgccgca??6060

aaaaagggaa?taagggcgac?acggaaatgt?tgaatactca?tactcttcct?ttttcaatat??6120

tattgaagca?tttatcaggg?ttattgtctc?atgagcggat?acatatttga?atgtatttag??6180

aaaaataaac?aaataggggt?tccgcgcaca?tttccccgaa?aagtgccacc?tgacgtc?????6237

<210>26

<211>9975

<212>DNA

＜213〉artificial

<220>

＜223〉expression vector

<400>26

cgatgtacgg?gccagatata?cgcgttgaca?ttgattattg?actagttatt?aatagtaatc?????60

aattacgggg?tcattagttc?atagcccata?tatggagttc?cgcgttacat?aacttacggt????120

aaatggcccg?cctggctgac?cgcccaacga?cccccgccca?ttgacgtcaa?taatgacgta????180

tgttcccata?gtaacgccaa?tagggacttt?ccattgacgt?caatgggtgg?agtatttacg????240

gtaaactgcc?cacttggcag?tacatcaagt?gtatcatatg?ccaagtacgc?cccctattga????300

cgtcaatgac?ggtaaatggc?ccgcctggca?ttatgcccag?tacatgacct?tatgggactt????360

tcctacttgg?cagtacatct?acgtattagt?catcgctatt?accatggtga?tgcggttttg?????420

gcagtacatc?aatgggcgtg?gatagcggtt?tgactcacgg?ggatttccaa?gtctccaccc?????480

cattgacgtc?aatgggagtt?tgttttggca?ccaaaatcaa?cgggactttc?caaaatgtcg?????540

taacaactcc?gccccattga?cgcaaatggg?cggtaggcgt?gtacggtggg?aggtctatat?????600

aagcagagct?ctctggctaa?ctagagaacc?cactgcttac?tggcttatcg?aaattaatac?????660

gactcactat?agggagaccc?aagctggcta?gcgtttaaac?ttaagcttgg?taccgagctc?????720

ggatccacta?gtccagtgtg?gtggaattct?gcagatatcc?agcacagtgg?cggccgctcg?????780

agatgcaaat?agagctctcc?acctgcttct?ttctgtgcct?tttgcgattc?tgctttagtg?????840

ccaccagaag?atactacctg?ggtgcagtgg?aactgtcatg?ggactatatg?caaagtgatc?????900

tcggtgagct?gcctgtggac?gcaagatttc?ctcctagagt?gccaaaatct?tttccattca?????960

acacctcagt?cgtgtacaaa?aagactctgt?ttgtagaatt?cacggatcac?cttttcaaca????1020

tcgctaagcc?aaggccaccc?tggatgggtc?tgctaggtcc?taccatccag?gctgaggttt????1080

atgatacagt?ggtcattaca?cttaagaaca?tggcttccca?tcctgtcagt?cttcatgctg????1140

ttggtgtatc?ctactggaaa?gcttctgagg?gagctgaata?tgatgatcag?accagtcaaa????1200

gggagaaaga?agatgataaa?gtcttccctg?gtggaagcca?tacatatgtc?tggcaggtcc????1260

tgaaagagaa?tggtccaatg?gcctctgacc?cactgtgcct?tacctactca?tatctttctc????1320

atgtggacct?ggtaaaagac?ttgaattcag?gcctcattgg?agccctacta?gtatgtagag????1380

aagggagtct?ggccaaggaa?aagacacaga?ccttgcacaa?atttatacta?ctttttgctg????1440

tatttgatga?agggaaaagt?tggcactcag?aaacaaagaa?ctccttgatg?caggataggg????1500

atgctgcatc?tgctcgggcc?tggcctaaaa?tgcacacagt?caatggttat?gtaaacaggt????1560

ctctgccagg?tctgattgga?tgccacagga?aatcagtcta?ttggcatgtg?attggaatgg????1620

gcaccactcc?tgaagtgcac?tcaatattcc?tcgaaggtca?cacatttctt?gtgaggaacc????1680

atcgccaggc?gtccttggaa?atctcgccaa?taactttcct?tactgctcaa?acactcttga????1740

tggaccttgg?acagtttcta?ctgttttgtc?atatctcttc?ccaccaacat?gatggcatgg????1800

aagcttatgt?caaagtagac?agctgtccag?aggaacccca?actacgaatg?aaaaataatg????1860

aagaagcgga?agactatgat?gatgatctta?ctgattctga?aatggatgtg?gtcaggtttg????1920

atgatgacaa?ctctccttcc?tttatccaaa?ttcgctcagt?tgccaagaag?catcctaaaa????1980

cttgggtaca?ttacattgct?gctgaagagg?aggactggga?ctatgctccc?ttagtcctcg????2040

cccccgatga?cagaagttat?aaaagtcaat?atttgaacaa?tggccctcag?cggattggta????2100

ggaagtacaa?aaaagtccga?tttatggcat?acacagatga?aacctttaag?actcgtgaag????2160

ctattcagca?tgaatcagga?atcttgggac?ctttacttta?tggggaagtt?ggagacacac????2220

tgttgattat?atttaagaat?caagcaagca?gaccatataa?catctaccct?cacggaatca????2280

ctgatgtccg?tcctttgtat?tcaaggagat?taccaaaagg?tgtaaaacat?ttgaaggatt????2340

ttccaattct?gccaggagaa?atattcaaat?ataaatggac?agtgactgta?gaagatgggc????2400

caactaaatc?agatcctcgg?tgcctgaccc?gctattactc?tagtttcgtt?aatatggaga????2460

gagatctagc?ttcaggactc?attggccctc?tcctcatctg?ctacaaagaa?tctgtagatc????2520

aaagaggaaa?ccagataatg?tcagacaaga?ggaatgtcat?cctgttttct?gtatttgatg????2580

agaaccgaag?ctggtacctc?acagagaata?tacaacgctt?tctccccaat?ccagctggag????2640

tgcagcttga?ggatccagag?ttccaagcct?ccaacatcat?gcacagcatc?aatggctatg????2700

tttttgatag?tttgcagttg?tcagtttgtt?tgcatgaggt?ggcatactgg?tacattctaa????2760

gcattggagc?acagactgac?ttcctttctg?tcttcttctc?tggatatacc?ttcaaacaca????2820

aaatggtcta?tgaagacaca?ctcaccctat?tcccattctc?aggagaaact?gtcttcatgt????2880

cgatggaaaa?cccaggtcta?tggattctgg?ggtgccacaa?ctcagacttt?cggaacagag????2940

gcatgaccgc?cttactgaag?gtttctagtt?gtgacaagaa?cactggtgat?tattacgagg????3000

acagttatga?agatatttca?gcatacttgc?tgagtaaaaa?caatgccatt?gaaccaagaa????3060

gcttctccca?gaattcaaga?catcaagctt?atcgataccg?tcgaggggaa?ataactcgta????3120

ctactcttca?gtcagatcaa?gaggaaattg?actatgatga?taccatatca?gttgaaatga????3180

agaaggaaga?ttttgacatt?tatgatgagg?atgaaaatca?gagcccccgc?agctttcaaa????3240

agaaaacacg?acactatttt?attgctgcag?tggagaggct?ctgggattat?gggatgagta????3300

gctccccaca?tgttctaaga?aacagggctc?agagtggcag?tgtccctcag?ttcaagaaag????3360

ttgttttcca?ggaatttact?gatggctcct?ttactcagcc?cttataccgt?ggagaactaa????3420

atgaacattt?gggactcctg?gggccatata?taagagcaga?agttgaagat?aatatcatgg????3480

taactttcag?aaatcaggcc?tctcgtccct?attccttcta?ttctagcctt?atttcttatg????3540

aggaagatca?gaggcaagga?gcagaaccta?gaaaaaactt?tgtcaagcct?aatgaaacca????3600

aaacttactt?ttggaaagtg?caacatcata?tggcacccac?taaagatgag?tttgactgca????3660

aagcctgggc?ttatttctct?gatgttgacc?tggaaaaaga?tgtgcactca?ggcctgattg????3720

gaccccttct?ggtctgccac?actaacacac?tgaaccctgc?tcatgggaga?caagtgacag????3780

tacaggaatt?tgctctgttt?ttcaccatct?ttgatgagac?caaaagctgg?tacttcactg????3840

aaaatatgga?aagaaactgc?agggctccct?gcaatatcca?gatggaagat?cccactttta????3900

aagagaatta?tcgcttccat?gcaatcaatg?gctacataat?ggatacacta?cctggcttag????3960

taatggctca?ggatcaaagg?attcgatggt?atctgctcag?catgggcagc?aatgaaaaca????4020

tccattctat?tcatttcagt?ggacatgtgt?tcactgtacg?aaaaaaagag?gagtataaaa????4080

tggcactgta?caatctctat?ccaggtgttt?ttgagacagt?ggaaatgtta?ccatccaaag????4140

ctggaatttg?gcgggtggaa?tgccttattg?gcgagcatct?acatgctggg?atgagcacac????4200

tttttctggt?gtacagcaat?aagtgtcaga?ctcccctggg?aatggcttct?ggacacatta????4260

gagattttca?gattacagct?tcaggacaat?atggacagtg?ggccccaaag?ctggccagac????4320

ttcattattc?cggatcaatc?aatgcctgga?gcaccaagga?gcccttttct?tggatcaagg????4380

tggatctgtt?ggcaccaatg?attattcacg?gcatcaagac?ccagggtgcc?cgtcagaagt????4440

tctccagcct?ctacatctct?cagtttatca?tcatgtatag?tcttgatggg?aagaagtggc????4500

agacttatcg?aggaaattcc?actggaacct?taatggtctt?ctttggcaat?gtggattcat????4560

ctgggataaa?acacaatatt?tttaaccctc?caattattgc?tcgatacatc?cgtttgcacc????4620

caactcatta?tagcattcgc?agcactcttc?gcatggagtt?gatgggctgt?gatttaaata????4680

gttgcagcat?gccattggga?atggagagta?aagcaatatc?agatgcacag?attactgctt????4740

catcctactt?taccaatatg?tttgccacct?ggtctccttc?aaaagctcga?cttcacctcc????4800

aagggaggag?taatgcctgg?agacctcagg?tgaataatcc?aaaagagtgg?ctgcaagtgg????4860

acttccagaa?gacaatgaaa?gtcacaggag?taactactca?gggagtaaaa?tctctgctta????4920

ccagcatgta?tgtgaaggag?ttcctcatct?ccagcagtca?agatggccat?cagtggaccc????4980

tcttttttca?gaatggcaaa?gtaaaggttt?ttcagggaaa?tcaagactcc?ttcacacctg????5040

tggtgaactc?tctagaccca?ccgttactga?ctcgctacct?tcgaattcac?ccccagagtt????5100

gggtgcacca?gattgccctg?aggatggagg?ttctgggctg?cgaggcacag?gacctctact????5160

gagcggcccg?tttaaacccg?ctgatcagcc?tcgactgtgc?cttctagttg?ccagccatct????5220

gttgtttgcc?cctcccccgt?gccttccttg?accctggaag?gtgccactcc?cactgtcctt????5280

tcctaataaa?atgaggaaat?tgcatcgcat?tgtctgagta?ggtgtcattc?tattctgggg????5340

ggtggggtgg?ggcaggacag?caagggggag?gattgggaag?acaatagcag?gcatgctggg????5400

gatgcggtgg?gctctatggc?ttctgaggcg?gaaagaacca?gctggggctc?tagggggtat????5460

ccccacgcgc?cctgtagcgg?cgcattaagc?gcggcgggtg?tggtggttac?gcgcagcgtg????5520

accgctacac?ttgccagcgc?cctagcgccc?gctcctttcg?ctttcttccc?ttcctttctc????5580

gccacgttcg?ccggctttcc?ccgtcaagct?ctaaatcggg?ggctcccttt?agggttccga????5640

tttagtgctt?tacggcacct?cgaccccaaa?aaacttgatt?agggtgatgg?ttcacgtagt????5700

gggccatcgc?cctgatagac?ggtttttcgc?cctttgacgt?tggagtccac?gttctttaat????5760

agtggactct?tgttccaaac?tggaacaaca?ctcaacccta?tctcggtcta?ttcttttgat????5820

ttataaggga?ttttgccgat?ttcggcctat?tggttaaaaa?atgagctgat?ttaacaaaaa????5880

tttaacgcga?attaattctg?tggaatgtgt?gtcagttagg?gtgtggaaag?tccccaggct????5940

ccccagcagg?cagaagtatg?caaagcatgc?atctcaatta?gtcagcaacc?aggtgtggaa????6000

agtccccagg?ctccccagca?ggcagaagta?tgcaaagcat?gcatctcaat?tagtcagcaa????6060

ccatagtccc?gcccctaact?ccgcccatcc?cgcccctaac?tccgcccagt?tccgcccatt????6120

ctccgcccca?tggctgacta?atttttttta?tttatgcaga?ggccgaggcc?gcctctgcct????6180

ctgagctatt?ccagaagtag?tgaggaggct?tttttggagg?cctaggcttt?tgcaaaaagc????6240

tcccgggagc?ttgtatatcc?attttcggat?ctgatcagca?cgtgatgaaa?aagcctgaac????6300

tcaccgcgac?gtctgtcgag?aagtttctga?tcgaaaagtt?cgacagcgtc?tccgacctga????6360

tgcagctctc?ggagggcgaa?gaatctcgtg?ctttcagctt?cgatgtagga?gggcgtggat????6420

atgtcctgcg?ggtaaatagc?tgcgccgatg?gtttctacaa?agatcgttat?gtttatcggc????6480

actttgcatc?ggccgcgctc?ccgattccgg?aagtgcttga?cattggggaa?ttcagcgaga????6540

gcctgaccta?ttgcatctcc?cgccgtgcac?agggtgtcac?gttgcaagac?ctgcctgaaa????6600

ccgaactgcc?cgctgttctg?cagccggtcg?cggaggccat?ggatgcgatc?gctgcggccg????6660

atcttagcca?gacgagcggg?ttcggcccat?tcggaccgca?aggaatcggt?caatacacta????6720

catggcgtga?tttcatatgc?gcgattgctg?atccccatgt?gtatcactgg?caaactgtga????6780

tggacgacac?cgtcagtgcg?tccgtcgcgc?aggctctcga?tgagctgatg?ctttgggccg????6840

aggactgccc?cgaagtccgg?cacctcgtgc?acgcggattt?cggctccaac?aatgtcctga????6900

cggacaatgg?ccgcataaca?gcggtcattg?actggagcga?ggcgatgttc?ggggattccc????6960

aatacgaggt?cgccaacatc?ttcttctgga?ggccgtggtt?ggcttgtatg?gagcagcaga????7020

cgcgctactt?cgagcggagg?catccggagc?ttgcaggatc?gccgcggctc?cgggcgtata????7080

tgctccgcat?tggtcttgac?caactctatc?agagcttggt?tgacggcaat?ttcgatgatg????7140

cagcttgggc?gcagggtcga?tgcgacgcaa?tcgtccgatc?cggagccggg?actgtcgggc????7200

gtacacaaat?cgcccgcaga?agcgcggccg?tctggaccga?tggctgtgta?gaagtactcg????7260

ccgatagtgg?aaaccgacgc?cccagcactc?gtccgagggc?aaaggaatag?cacgtgctac????7320

gagatttcga?ttccaccgcc?gccttctatg?aaaggttggg?cttcggaatc?gttttccggg????7380

acgccggctg?gatgatcctc?cagcgcgggg?atctcatgct?ggagttcttc?gcccacccca????7440

acttgtttat?tgcagcttat?aatggttaca?aataaagcaa?tagcatcaca?aatttcacaa????7500

ataaagcatt?tttttcactg?cattctagtt?gtggtttgtc?caaactcatc?aatgtatctt????7560

atcatgtctg?tataccgtcg?acctctagct?agagcttggc?gtaatcatgg?tcatagctgt????7620

ttcctgtgtg?aaattgttat?ccgctcacaa?ttccacacaa?catacgagcc?ggaagcataa????7680

agtgtaaagc?ctggggtgcc?taatgagtga?gctaactcac?attaattgcg?ttgcgctcac????7740

tgcccgcttt?ccagtcggga?aacctgtcgt?gccagctgca?ttaatgaatc?ggccaacgcg????7800

cggggagagg?cggtttgcgt?attgggcgct?cttccgcttc?ctcgctcact?gactcgctgc????7860

gctcggtcgt?tcggctgcgg?cgagcggtat?cagctcactc?aaaggcggta?atacggttat????7920

ccacagaatc?aggggataac?gcaggaaaga?acatgtgagc?aaaaggccag?caaaaggcca????7980

ggaaccgtaa?aaaggccgcg?ttgctggcgt?ttttccatag?gctccgcccc?cctgacgagc????8040

atcacaaaaa?tcgacgctca?agtcagaggt?ggcgaaaccc?gacaggacta?taaagatacc????8100

aggcgtttcc?ccctggaagc?tccctcgtgc?gctctcctgt?tccgaccctg?ccgcttaccg????8160

gatacctgtc?cgcctttctc?ccttcgggaa?gcgtggcgct?ttctcatagc?tcacgctgta????8220

ggtatctcag?ttcggtgtag?gtcgttcgct?ccaagctggg?ctgtgtgcac?gaaccccccg????8280

ttcagcccga?ccgctgcgcc?ttatccggta?actatcgtct?tgagtccaac?ccggtaagac????8340

acgacttatc?gccactggca?gcagccactg?gtaacaggat?tagcagagcg?aggtatgtag????8400

gcggtgctac?agagttcttg?aagtggtggc?ctaactacgg?ctacactaga?agaacagtat????8460

ttggtatctg?cgctctgctg?aagccagtta?ccttcggaaa?aagagttggt?agctcttgat????8520

ccggcaaaca?aaccaccgct?ggtagcggtg?gtttttttgt?ttgcaagcag?cagattacgc????8580

gcagaaaaaa?aggatctcaa?gaagatcctt?tgatcttttc?tacggggtct?gacgctcagt????8640

ggaacgaaaa?ctcacgttaa?gggattttgg?tcatgagatt?atcaaaaagg?atcttcacct????8700

agatcctttt?aaattaaaaa?tgaagtttta?aatcaatcta?aagtatatat?gagtaaactt????8760

ggtctgacag?ttaccaatgc?ttaatcagtg?aggcacctat?ctcagcgatc?tgtctatttc????8820

gttcatccat?agttgcctga?ctccccgtcg?tgtagataac?tacgatacgg?gagggcttac????8880

catctggccc?cagtgctgca?atgataccgc?gagacccacg?ctcaccggct?ccagatttat????8940

cagcaataaa?ccagccagcc?ggaagggccg?agcgcagaag?tggtcctgca?actttatccg????9000

cctccatcca?gtctattaat?tgttgccggg?aagctagagt?aagtagttcg?ccagttaata????9060

gtttgcgcaa?cgttgttgcc?attgctacag?gcatcgtggt?gtcacgctcg?tcgtttggta????9120

tggcttcatt?cagctccggt?tcccaacgat?caaggcgagt?tacatgatcc?cccatgttgt????9180

gcaaaaaagc?ggttagctcc?ttcggtcctc?cgatcgttgt?cagaagtaag?ttggccgcag????9240

tgttatcact?catggttatg?gcagcactgc?ataattctct?tactgtcatg?ccatccgtaa????9300

gatgcttttc?tgtgactggt?gagtactcaa?ccaagtcatt?ctgagaatag?tgtatgcggc????9360

gaccgagttg?ctcttgcccg?gcgtcaatac?gggataatac?cgcgccacat?agcagaactt????9420

taaaagtgct?catcattgga?aaacgttctt?cggggcgaaa?actctcaagg?atcttaccgc????9480

tgttgagatc?cagttcgatg?taacccactc?gtgcacccaa?ctgatcttca?gcatctttta????9540

ctttcaccag?cgtttctggg?tgagcaaaaa?caggaaggca?aaatgccgca?aaaaagggaa????9600

taagggcgac?acggaaatgt?tgaatactca?tactcttcct?ttttcaatat?tattgaagca??9660

tttatcaggg?ttattgtctc?atgagcggat?acatatttga?atgtatttag?aaaaataaac??9720

aaataggggt?tccgcgcaca?tttccccgaa?aagtgccacc?tgacgtcgac?ggatcgggag??9780

atctcccgat?cccctatggt?gcactctcag?tacaatctgc?tctgatgccg?catagttaag??9840

ccagtatctg?ctccctgctt?gtgtgttgga?ggtcgctgag?tagtgcgcga?gcaaaattta??9900

agctacaaca?aggcaaggct?tgaccgacaa?ttgcatgaag?aatctgctta?gggttaggcg??9960

ttttgcgctg?cttcg???????????????????????????????????????????????????9975

<210>27

<211>51

<212>DNA

＜213〉artificial

<220>

＜223〉primer

<400>27

ggcaagaatt?cgcagagcag?tcattatggc?caagttgacc?agtgccgttc?c????51

<210>28

<211>21

<212>DNA

＜213〉artificial

<220>

＜223〉primer

<400>28

ctagaggtcc?aggtcatctt?g??????????????????????????????????21

<210>29

<211>21

<212>DNA

＜213〉artificial

<220>

＜223〉primer

<100>29

tcagggaaat?tggggatcct?g??????????????????????????????????21

Claims (17)

1. do not contain the human somatocyte of the immortalization system of prion protein (PrP), wherein, two allelotrope of described PrP gene are rejected fully.

2. clone as claimed in claim 1, this clone

(i) can be transfected and under serum-free condition, cultivate; And/or

The adenoviral sequence that (ii) in its genome, has integration; And/or

(iii) from being selected from following one group initiating cell: kidney, bladder, liver, lung, cardiac muscle, unstriated muscle, ovary and gastrointestinal cell, preferably, described initiating cell is a human kidney cells system; And/or

(iv) be applicable to the production of recombinant protein.

3. clone as claimed in claim 2, wherein, described nephrocyte is the human fetal nephrocyte, preferably, described human fetal nephrocyte is selected from 293 cells (ATCC CRL-1573; DSM ACC 305), FreeStyle 293 cells (293F cell; Invitrogen R79007) and 293T cell (DSM ACC2494), be preferably 293F cell (Invitrogen R79007).

4. as clone any one among the claim 1-3, wherein, two allelotrope of described PrP gene be by can selecting or the rejecting trap of selectable marker gene carries out homologous recombination and deleted fully with carrying, described like this select or the expression of selective marker is started by endogenous PrP promotor.

5. as clone any one among the claim 1-5, including, but not limited to described final prion-free 293F clone pf293F, and all middle population mixtures with for separating its necessary separating clone.

6. the method that is used for the human somatocyte of the immortalization system of any one the no PrP of production claim 1-3, this method comprises successively by homologous recombination to be utilized some different PrP to reject construct or utilizes identical construct to delete PrP ORF in the corresponding initiating cell, and implements antibiotic-screening with the concentration that increases gradually.

7. method as claimed in claim 6, wherein, but described rejecting construct carries described identical or different no promotor selectable marker gene or selective marker, and its side chain is and two sequences of the intragenic insertion of the PrP of described initiating cell site homologous.

8. method as claimed in claim 7, wherein, described rejecting construct

(i) carry different selectable marker genes; And/or

(ii) also carry one of following function sequence: poly A sequence, recombinase recognition sequence, IRES; As/or

(iii) the length of described homologous sequence is 1-10kb, is preferably about 6kb, and/or is equivalent to the sequence of upstream and downstream of the PrP ORF of described initiating cell system, and more preferably, described homologous sequence is a sequence shown in SEQ ID NOs:2 and 3; And/or

(iv) described selective marker coding positive selectable marker, including, but not limited to Xin Meisu, zeocin, Totomycin; And the described marks packets of selecting is drawn together fluorescent mark, as GFP and Dsred and enzyme, as LacZ.

9. method as claimed in claim 8, wherein, described rejecting construct has the one or more of sequence shown in SEQ ID NOs:1 and 16.

10. the PrP as any definition among the claim 6-9 rejects construct.

11. the human clone of the immortalization of no PrP any among the claim 1-5 is used for people's albumen, or the application of the recombinant production of the no PrP of its antibody or derivatives thereof or its mutant (albumen that target is fixed).

12. one kind is used for people's albumen, or the preparation method of the human cell of the no PrP recombinant production of its antibody or derivatives thereof or its mutant (albumen that target is fixed) system, this method comprises the human host cell of the immortalization system with no PrP any among the transfection carrier transfection claim 1-5, described carrier comprises replication orgin, with the fixed proteic gene of people of the described target of coding, and the fixed proteic gene of people of coding target terminally is connected with promotor in its 5 ', is connected with the polyA signal at its 3 ' end.

13. as the method for claim 12, wherein

(i) described transfection is to carry out under serum-free condition; And/or

(ii) described transfection carrier derives from the pcDNA3.1 carrier that Invitrogen company buys; And/or

The fixed people's albumen of (iii) described target is thrombin, as blood coagulation factor VIII (comprising wt Factor IX or B domain deletion Factor IX), plasma thromboplastin component, factor VII/VIIa, the human growth factor, as G-CSF or GM-CSF, vWF or α-1-antitrypsin (A1AT) or human antibodies.

14. stable transfection, preferably under serum-free condition with the human production clone of the immortalization of the no PrP of claim 12 or 13 defined transfection carrier transfections.

15. one kind is used for the method that institute's target is decided the proteic no PrP recombinant production of people, this method comprises cultivation, preferably cultivates the immortalization mankind production clone as no PrP as described in the claim 14 under serum-free condition.

16. do not contain the immortalized cell line of prion protein (PrP), wherein, two allelotrope of described PrP gene are rejected fully, be selected from HEK 293F or Per.C6 cell (the human fetal retinal cell of immortalization), CHO (Chinese hamster ovary cell) and BHK (baby hamster kidney cell) cell.

17. the immortalized cell line of the described no PrP of claim 16 is used for people's albumen, or the application of the recombinant production of the no PrP of its antibody or derivatives thereof or its mutant (target is decided albumen).

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