CN105671079A - Mouse with human nerve growth factor transgenes as well as preparation method and application of mouse - Google Patents
Mouse with human nerve growth factor transgenes as well as preparation method and application of mouse Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/48—Nerve growth factor [NGF]
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Abstract
The invention discloses a mouse with human nerve growth factor transgenes as well as a preparation method and application of the mouse. Specifically, a salivary gland specific expression human-derived NGF (Nerve Growth Factor) transgene mouse is obtained by utilizing a transgene method; human-derived NGFs can be secreted through saliva of the transgene mouse prepared by the method; after separation and purification, the human-derived NGFs can be used for treating human diseases. The method comprises the following steps: (1) constructing a transposon carrier, wherein the carrier comprises a human-derived NGF gene sequence, a parotid gland secreted protein gene promoter sequence and 5' and 3' terminal repetition sequences of a transposon piggyBac; (2) injecting the constructed transposon carrier into a male pronucleus of a fertilized ovum of the mouse, and transplanting the injected fertilized ovum of the mouse into a uterus of a female mouse for surrogacy and feeding the female mouse for surrogacy until mice are born; (3) detecting the born mice and screening out transgene mice with human-derived NGF genes capable of being stably propagated in offspring; (4) separating and purifying the human-derived NGFs from the saliva of the mice with the human nerve growth factor transgenes.
Description
Technical field
The present invention relates to animal of being obtained by transgenic method and its preparation method and application, particularly to a kind of mice turning human nerve growth factor gene and its preparation method and application.
Background technology
Nerve growth factor (i.e. NerveGrowthFactor is called for short NGF) is first member of neurotrophins, and it is found in 1954 by Cohen and Levi-Montalcini, and illustrates its function in research subsequently. Therefore Cohen and Levi-Montalcini obtains Nobel's medical science or the physiology prize of 1986. NGF not only propagation and differentiation to neurocyte plays an important role, also to non-neuronal cells, for instance growth and the activity of immunocyte, fibroblast, pancreatic cell and myocardial cell etc. have critical function. Therefore, nerve and non-neuropathic disease are had good potential therapeutical effect by NGF.
Because mouse nerve growth factor and Mus source NGF (mouseNGF, and growth factor of human nerve and people source NGF (humanNGF mNGF), hNGF) on aminoacid sequence, there is the homology of 85%, and many researchs are it have been reported that the various nerve injury for the treatment of or degenerative disease are had obvious effect by Mus source NGF, the Mus source NGF come from the submaxillary gland purification of male mice was used for treating people's nerve injury and degeneration relevant disease by the approval of Bureau of Drugs Supervision of China in 2003. At present, on China's drug market, the price of Mus source NGF is about 1000 yuan/milligram, and the total sales volume of 2015 is about 5,000,000 milligrams, and yield is far from sufficient for the market demand. And, up-to-date research finds that Mus source NGF and people source NGF exists significant difference in physiology, biochemistry and biological nature, and the biological activity of Mus source NGF is substantially less than people source NGF in people's cell. Additionally, due to people is a kind of foreign protein by Mus source NGF, may result in patient produce immunoreation using Mus source NGF to carry out treatment, less safe.
For the problems referred to above brought when eliminating Mus source NGF for human treatment, having had much research to attempt by escherichia coli, yeast, insect cell, the expression system such as mammalian cell produces people source NGF. But the yield that these cell systems express people source NGF is generally relatively low, and its synthesized people source NGF cannot be provided correct post translational modification by some of them system, affects the medicinal effects of people source NGF. Meanwhile, existing team attempts the mammary gland utilizing transgene rabbit and produces people source NGF as bioreactor. But mammary gland has following defects that 1 as bioreactor) only after sexual maturity and be in the jenny of age of sucking could lactation; 2) galactopoiesis amount is limited; 3) milk contains substantial amounts of intrinsic protein, is unfavorable for being purified into the foreign protein of institute's specifically expressing and people source NGF from milk.
Summary of the invention
It is an object of the invention to provide a kind of mice turning human nerve growth factor gene, by transgenic method, people source NGF sequence is inserted on No. 5 chromosome of mice, and obtain and can stablize heredity people source ngf gene sequence on No. 5 chromosome and there is at salivary organization's specifically expressing the mice turning human nerve growth factor gene of bioactive people source NGF, and utilize this to turn the mouse salivary glands of human nerve growth factor gene as bioreactor production people source NGF medicine, and separation is purified into people source NGF from the saliva of the mice secretion turning human nerve growth factor gene, and people source NGF is applied to the treatment of human diseases. owing to mouse salivary secretory volume is big, the people source NGF of generation is also many, and in saliva, intrinsic protein is less, it is simple to the separation purification of people source NGF, and NGF can be produced correct post translational modification by salivary gland, the NGF of generation is humanized, and it will not produce immunoreation when human disease treatment, safer, therefore can solve the problems referred to above.
According to an aspect of the present invention, provide the mice turning human nerve growth factor gene, this turn human nerve growth factor gene mice genome in comprise people source ngf gene sequence, and this turns the mouse salivary glands specifically expressing people source NGF of human nerve growth factor gene. Thus, this mice turning human nerve growth factor gene can produce people source NGF by salivation, then people source NGF in saliva is easily separated purification, obtains the people source NGF after purification and can be used for the treatment of human diseases. This mice turning human nerve growth factor gene can by breeding the family obtaining substantial amounts, and produce saliva endlessly, thus obtaining more people source NGF, and correct post translational modification can be carried out in salivary gland, and the less separation purification being easy to people source NGF of intrinsic protein in saliva. And this people source NGF is humanized, immunoreation will not be produced when for human disease treatment, safer.
In some embodiments, this human nerve growth factor gene's sequence is such as shown in SEQIDNO:1.
In some embodiments, this human nerve growth factor gene's sequence is incorporated on No. 5 chromosome of the mice turning human nerve growth factor gene.
In some embodiments, this human nerve growth factor gene's sequence is incorporated into No. 5 chromosomal quaninenucleotide-bindingproteinsubunitalpha-12 gene of the mice turning human nerve growth factor gene with " TTAA " site on the intergenic non-coding intervening sequence of caspaserecruitmentdomain-containingprotein11, and quaninenucleotide-bindingproteinsubunitalpha-12 and caspaserecruitmentdomain-containingprotein11 gene is the known openly gene on No. 5 chromosome of mice.Thus, people source ngf gene sequence is incorporated into No. 5 chromosome of the mice turning human nerve growth factor gene and pinpoints in " TTAA " site inserted on No. 5 chromosome quaninenucleotide-bindingproteinsubunitalpha-12 gene and the intergenic non-coding intervening sequence of caspaserecruitmentdomain-containingprotein11, this turns the mice of human nerve growth factor gene specifically expressing people source NGF and separate the people source NGF medicine of purification turn the mouse salivary of human nerve growth factor gene from this and have better biological activity in salivary gland, the treatment of mankind's relevant disease had better effect.
According to another aspect of the present invention, the preparation method providing the mice turning human nerve growth factor gene, people source NGF is incorporated in mouse genome by this mice turning human nerve growth factor gene by transgenic technology, and heredity that can be stable in the mice progeny turn human nerve growth factor gene. therefore, the mice turning human nerve growth factor gene comprising people source ngf gene sequence that can stablize heredity in offspring can be obtained by the method, and this turns the salivary gland of the mice special secretion people source NGF of human nerve growth factor gene, saliva can be used for after the people source separated purification of NGF of secretion the treatment of human diseases, and by the mice turning human nerve growth factor gene that the method obtains, and the people source NGF of purification is separated by this mice turning human nerve growth factor gene, yield is high, the mankind will not be produced immunoreation safer, the method comprises the steps:
(1) building transposon vector, this transposon vector comprises the people source ngf gene sequence as shown in SEQIDNO:1, parotid secretion protein gene promoter sequence as shown in SEQIDNO:5, the piggyBac transposon 5 ' terminal repeat as shown in SEQIDNO:6 and the piggyBac transposon as shown in SEQIDNO:7 3 ' terminal repeat;
(2) transposon vector built is injected to the male pronucleus of mouse fertilized egg, then the mouse fertilized egg after injection is migrated to the intrauterine of replace-conceive dams, raise replace-conceive dams and be born to mice;
(3) mice of birth is detected, filter out people source ngf gene and can stablize the mice turning human nerve growth factor gene of heredity in offspring.
In some embodiments, the sequence of transposon vector is such as shown in SEQIDNO:8.
In some embodiments, transposon vector be incorporated into No. 5 chromosomal quaninenucleotide-bindingproteinsubunitalpha-12 gene of the mice turning human nerve growth factor gene with in " TTAA " site on the intergenic non-coding intervening sequence of caspaserecruitmentdomain-containingprotein11.
According to another aspect of the present invention, provide the method preparing growth factor of human nerve medicine, by setting up the method that the salivary gland utilizing the mice turning human nerve growth factor gene produces people source NGF medicine as bioreactor, obtain the mice turning human nerve growth factor gene at salivary organization's specifically expressing with bioactive people source NGF, and separation is purified into people source NGF from the saliva of the mice secretion turning human nerve growth factor gene, the people source NGF yield obtained by the method is higher, biological safety better, and the method comprises the steps:
1) cultivate the mice turning human nerve growth factor gene according to said method, this mice turning human nerve growth factor gene is the mice turning human nerve growth factor gene according to preceding claim;
2) from step 1) described in turn human nerve growth factor gene mice saliva separate be purified into people source NGF medicine.
In some embodiments, this people source NGF medicine can promote that NGF recipient cell is bred.
In some embodiments, this people source NGF medicine can make the preparation treatment for human diseases.
Accompanying drawing explanation
Fig. 1 is transposon vector structural representation.
Fig. 2 is that in transgenic primary mice PCR detection electrophoresis result figure, figure, " N " represents blank, and " WT " represents wild-type mice, and " P " represents positive control, and " M " represents Marker labelling, and " 557-569 " represents transgenic primary mice.
Fig. 3 is the fluorescence results figure of transgenic primary mice.
Fig. 4 is the Southernblot result figure of transgenic primary mice, in figure, " M " represents Marker labelling, " P (3C) " represents positive control (3 copies), " P (5C) " represents positive control (5 copies), " WT " represents wild-type mice, and " 557-569 " represents transgenic primary mice.
Fig. 5 is the relative expression quantity testing result of hNGFmRNA in the transgenic primary mice parotid gland.
Fig. 6 turns the Concentration Testing result of salivation NGF in the mice family of human nerve growth factor gene.
Fig. 7 is that the transposon vector sequence comprising people source ngf gene sequence is incorporated into 553 familys and turns the comparison result analysis chart on No. 5 chromosome of mice of human nerve growth factor gene.
Fig. 8 turns hNGF testing result figure in the different tissues of mice of human nerve growth factor gene, in figure, " M " represents Marker labelling, " TG " represents the mice turning human nerve growth factor gene, " Pa " represents the parotid gland, " Sm " represents submaxillary gland, " Sl " represents sublingual gland, " Muscle " represents muscle, " Liver " represents liver, " Lung " represents lung, and " Fat " represents fat, and " Testis " represents testis tissue, " N " represents blank, and " WTPa " represents the wild-type mice parotid gland.
Fig. 9 turns hNGFmRNA relative expression quantity testing result figure in 3 salivary organizations of mice of human nerve growth factor gene.
Figure 10 is the mice and the wild-type mice photographic result under white light and fluorescence respectively that turn human nerve growth factor gene, and in figure, " TG " represents the mice turning human nerve growth factor gene, and " WT " represents wild-type mice.
Figure 11 turns mNGF detection of expression result figure in the mice of human nerve growth factor gene and 3 salivary organizations of wild-type mice, in figure, " WTmale " represents wild type public affairs Mus, " WTfemale " represents wild type dams, " TGmale " represents transgenic public affairs Mus, " TGfemale " represents transgenic dams, and " Pa " represents the parotid gland, and " Sm " represents submaxillary gland, " Sl " represents sublingual gland, and " M " represents Marker labelling.
Figure 12 is the hNGF protein expression analysis in the mice turning human nerve growth factor gene and the wild-type mice salivary organization of 553 familys and the hNGF analysis of protein result figure of purification in saliva thereof, in figure, " TG " represents the mice turning human nerve growth factor gene of 553 familys, " WT " represents wild-type mice family, " S " represents saliva, " P " represents the parotid gland, " SL " represents sublingual gland, " SM " represents submaxillary gland, and " PurifiedhNGF " represents the hNGF of purification from the mouse salivary turning human nerve growth factor gene.
Figure 13 turns the hNGF albumen of purification and the mNGF medicine comparative result figure to people's TF1 cel l proliferation the mouse salivary of human nerve growth factor gene from 553 familys, in figure, " Negative " represents negative control, " mNGF " represents mNGF medicine, and " PurifiedhNGF " represents the hNGF albumen of purification the mouse salivary turning human nerve growth factor gene from 553 familys.
Detailed description of the invention
Below in conjunction with accompanying drawing, the present invention is further detailed explanation.
SEQIDNO:2 gene order involved in the present invention derives from GenBankaccessionnumber:NM_002506.2; SEQIDNO:3 gene order derives from GenBankaccessionnumber:NM_002506.2; SEQIDNO:4 gene order derives from GenBankaccessionnumber:X01697.1; SEQIDNO:5 gene order derives from SergueiP.Golovanetal, Transgenicmiceexpressingbacterialphytaseasamodelforphosp horuspollutioncontrol, NaturePublishingGroup, 2001 article report; SEQIDNO:6, SEQIDNO:7, bGHpolyA sequence, CMV promoter sequence, Neo-2A-EGFP fusion gene sequence, bGHpolyA sequence derive from zhenfangWuetal, Co-expressionoftwofibrolyticenzymegenesinCHOcellsandtran sgenicmice, TransgenicResearch, 2013 article reports.
1, the transposon vector containing people source ngf gene is built.
PCR is utilized to amplify mice parotid secretion albumen (ParotidSecretoryProtein, PSP) gene promoter sequence (SEQIDNO:5) of 12.2kb length from mouse gene group DNA. The signal peptide sequence (SEQIDNO:3) of 54bp length in the hNGF gene coded sequence (SEQIDNO:2) of 726bp length is replaced with pig PSP signal peptide sequence (SEQIDNO:4) of 60bp length, by hNGF gene coded sequence (SEQIDNO:1) synthetic of 732bp length after restructuring. In addition, also by synthetic piggyBac5 ' terminal repeat (SEQIDNO:6) and 3 ' terminal repeats (SEQIDNO:7), and comprise bGHpolyA sequence, one fragment sequence of CMV promoter sequence, Neo-2A-EGFP fusion gene sequence and bGHpolyA sequence. The sequence replacing between XhoI and the NotI restriction enzyme site of pGEM-T carrier after being connected with restructuring hNGF coded sequence and mice PSP promoter sequence by these artificial synthesized sequences just produces the pmPSP-hNGF carrier (SEQIDNO:8) of 20.1kb length, through the checking such as order-checking and enzyme action, the Transposon plasmid pmPSP-hNGF carrying hNGF expression casette and EGFP marker gene expression cassette is successfully built, and its structure is shown in Fig. 1.
2, preparation and the qualification thereof of the mice of human nerve growth factor gene are turned.
By the pmPSP-hNGF plasmid that concentration is 9ng/ μ l and the pmPB plasmid that concentration is 3ng/ μ l by the male pronucleus being injected to 110 mouse fertilized eggs after 1:1 volume mixture, each germ cell injection 1-2pL. Embryo transfer after injection, to 7 replace-conceive dams uterus, has 35 mice births.
The genomic DNA of the mousetail tissue turning human nerve growth factor gene by 35 is obtained by TissueDNAextractionkit (Omega, GA, USA) test kit extracting. The pcr amplification of hNGF, EGFP and Rgs7 gene uses following three pairs of primers to complete respectively. Primer pair 1:P1 (5 '-GAACTCATATTGTACCACGACT-3 ')+P2 (5 '-CCCCAGAATAGAATGACACC-3 '); Primer pair 2:EGFP-F (5 '-TTGATGCCGTTCTTCTGCTTG-3 ')+EGFP-R (5 '-ACGTGCTGGTTGTTGTGCTGT-3 '); Primer pair 3:Rgs7-F (5 '-CAACCACTTACAAGAGACCCGTA-3 ')+Rgs7-R (5 '-GAGCCCTTAGAAATAACGTTCACC-3 '). PCR electroresis appraisal result is shown in Fig. 2, and PCR primer is all passed through order-checking and determined the correctness of its amplification aim sequence.Simultaneously, the whole mice turning human nerve growth factor gene is passed through living body fluorescent albumen viewing system LivingOrganism ' sFluorescentProteinObservationSystem (Model:FBL, BLSLtd., Hungary) EGFP expression is observed, and the EGFP of the fore paw tissue of the mice turning human nerve growth factor gene is expressed by by fluorescence microscope and qualification of taking pictures, qualification result is shown in Fig. 3.
Tail genomic DNA by 35 Primary mouse of pcr analysis, it has been found that wherein 18 are carried hNGF and EGFP gene (result is shown in Fig. 2). These 18 mices through the PCR primary human nerve growth factor gene of turning identified express the EGFP (result is shown in Fig. 3) of varying level in its fore paw tissue, and this is likely to EGFP gene is integrated in every primary mouse genome turning human nerve growth factor gene position and copy number are relevant.
3, exogenous gene turns the integration mode in the mice family of human nerve growth factor gene and expression compares in difference.
PCR is accredited as 18 each histioid total proteins of mice extracting turning human nerve growth factor gene of the positive, measures the protein concentration of each sample again through Bradford method. For each transgenic sample and corresponding wild-type samples, identical total protein (15-30 μ g) is carried out electrophoresis by the SDS-PAGE glue being loaded to 10%. After electrophoretic separation, albumen is transferred on pvdf membrane from glue. HNGF on film detects analysis and utilization rat anti-human NGF monoclonal primary antibodie (#MAB2562, R&Dsystems), the mountain goat anti rat two of HRP conjugation resists (JacksonImmunoResearch) and chemiluminescence developing agent SuperSignalWestPicoChemiluminentSubstrates (ThermoFisherScientific) to complete. All operations carries out according to the description of antibody and reagent. The Southernblot of the mouse gene group DNA of the primary human nerve growth factor gene of turning is analyzed, and Fig. 1 is seen in cleavage site and the EGFP probe position on pmPSP-hNGF plasmid of the HindIII enzyme that Southernblot analysis uses. Southernblot analyzes result and shows that the mice of every primary human nerve growth factor gene of turning carries the exogenous gene (result is shown in Fig. 4) of about 1-5 copy. Although the primary mice 558 turning human nerve growth factor gene, the Southernblot result of 566 and 567 only shows a band, but their Band signal ratio 553,556,568 and the last 569, and its Band signal intensity is close to the positive control (result is shown in Fig. 4) of 3 copies, so 558, the 566 and 567 actual exogenous genes carrying more than 1 copy.
Meanwhile, PCR is accredited as the total serum IgE of 18 mice parotid gland tissues samples turning human nerve growth factor gene of the positive by test kit E.Z.N.A.TMTotalRNAKitI (OMEGA) extracting. CDNA passes through test kitRTreagentKitWithgDNAEraser (TaKaRa) synthesizes. HNGF, mNGF and GAPDH gene obtains respective cDNA respectively through following three pairs of primers through reverse transcriptional PCR amplification. Primer pair 4:P3 (5 '-GACCCAAATCCCGTTGACAGC-3 ')+P4 (5 '-ATGGCTGGCAACTAGAAGGCACA-3 '); Primer pair 5:mNGF-F (5 '-TCCAATCCTGTTGAGAGTGGGTG-3 ')+mNGF-R (5 '-GCCTGCTTCTCATCTGTTGTCAAC-3 '); Primer pair 6:GAPDH-F (5 '-CTCCCACTCTTCCACCTTCG-3 ')+GAPDH-R (5 '-CCACCACCCTGTTGCTGTAG-3 ').The cDNA obtained carries out quantitative PCR reaction again, and quantitative PCR passes through instrument EcoTMReal-timePCRsystem (Illumine) and reagentPremixExTaq (TaKaRa) expands, and relative gene expression amount is by 2-ΔΔCtMethod calculates. Quantitative PCR result shows that the hNGFmRNA relative expression quantity in 18 primary mice parotid gland tissues turning human nerve growth factor gene is different, and result is shown in Fig. 5.
Further, the mice family turning human nerve growth factor gene that hNGF albumen is the highest is expressed in order to filter out, by the hNGF protein concentration in 18 primary mouse salivary turning human nerve growth factor gene by hNGFELISA kit measurement (Catalogno:E0105hEIAabScienceCo., Ltd, Wuhan, China). Endogenous mNGF protein concentration in mouse salivary by mNGFELISA kit measurement (Catalogno:E0105m,EIAabScienceCo., Ltd, Wuhan, China). Concrete operations carry out according to the description of test kit. The hNGF protein concentration in the mouse salivary turning human nerve growth factor gene of the family (including 558,559,560,561,562,566,552,553 and 567) of the hNGFmRNA that 9 expressions are of a relatively high by elisa assay. Result shows that in the mouse salivary turning human nerve growth factor gene of 553 familys, hNGF protein concentration reaches 1.36 ± 0.06 μ g/ml, in all detection familys the highest (result is shown in Fig. 6). Turn and the mouse salivary of human nerve growth factor gene is also secreted low-level endogenous mNGF, its concentration (0.04-0.06 μ g/ml) and the endogenous mNGF concentration (0.07 ± 0.02 μ g/ml) similar (result is shown in Fig. 6) in wild-type mice saliva. Owing in the mouse salivary turning human nerve growth factor gene of 553 familys, the hNGF concentration of secretion is the highest, and Southernblot result shows that 553 familys only carry the exogenous gene (result is shown in Fig. 4) of 1 copy, and this makes exogenous gene that segregation phenomenon will not occur in succeeding generations. Therefore, select the mice turning human nerve growth factor gene of 553 familys for follow-up test.
4, exogenous gene turns the analysis integrating position in the mouse genome of human nerve growth factor gene in 553 familys.
Further, in order to determine that single copy exogenous gene turns the integration position in the mouse genome of human nerve growth factor gene in 553 familys, we analyze the genomic DNA of the primary mice 553 turning human nerve growth factor gene with inverse PCR, the primer used is P5 (5 '-CACTTCGCCCAATAGCAG-3 ') and P6 (5 '-ATACAGACCGATAAAACACATG-3 '), and Fig. 1 is seen in its position on pmPSP-hNGF plasmid. by Inverse PCR products is checked order and to sequencing result respectively with pmPSP-hNGF plasmid and mouse genome sequences comparison, we have found that the list copy exogenous gene that this mice 553 turning human nerve growth factor gene primary is carried is to be incorporated in " TTAA " site on No. 5 chromosomal quaninenucleotide-bindingproteinsubunitalpha-12 gene and the intergenic non-coding intervening sequence of caspaserecruitmentdomain-containingprotein11 by piggyBac swivel base mode, and quaninenucleotide-bindingproteinsubunitalpha-12 and caspaserecruitmentdomain-containingprotein11 gene is the known open gene on No. 5 chromosome of mice, qualification result is shown in Fig. 7.
5, hNGF turns the expression pattern analysis in the mice of human nerve growth factor gene in 553 familys.
In order to determine the hNGF tissue specificity expressed in the mice turn human nerve growth factor gene, we analyze the hNGF 3 salivary organizations the mice turning human nerve growth factor gene with reverse transcriptional PCR: the parotid gland (Pa), sublingual gland (Sl) and submaxillary gland (Sm), muscular tissue (Muscle), liver organization (Liver), lung tissue (Lung), the expression of fatty tissue (Fat) and testis tissue (Testis). Result display hNGF only specifically expressing (result is shown in Fig. 8) in 3 salivary organizations, but the expression in the parotid gland (Pa) is more than submaxillary gland (Sm) and sublingual gland (Sl) high (result is shown in Fig. 9). Marker gene EGFP can stably heredity and expression (result is shown in Figure 10) in 553 family succeeding generations. The mice turning human nerve growth factor gene is the same with wild type control mice, only expresses endogenous mNGF in its submaxillary gland (Sm), and result is shown in Figure 11.
6, from the hNGF protein expression analysis in the mouse salivary glandular tissue turning human nerve growth factor gene of 553 familys and the hNGF analysis of protein of purification in saliva thereof.
Analyzed by Westernblot and the parotid gland (Pa) finding to turn mice 553 family of human nerve growth factor gene is expressed high-caliber hNGF albumen, turn human nerve growth factor gene mice saliva in also hNGF albumen containing higher level, and the hNGF mature peptide separating purification from the mouse salivary turning human nerve growth factor gene meets the 13.5kD molecular weight predicted, qualification result is shown in Figure 12.
7, collection and the purity analysis of the mouse salivary of human nerve growth factor gene are turned.
Mice is anaesthetized by lumbar injection " dog sleeps precious " medicine, and injection dosage is determined according to this medicine description and Mouse Weight. After mice is anesthetized, lumbar injection 100 μ g/mL pilocarpine hydrochloride (0.5 μ g/ gram body weight) exciting salivary secretion, then from murine oral, repeatedly draw saliva with the shifting liquid liquid device of the head that carries a gun. In 20 minutes, about can collect 100-200 μ l saliva from every mice. It is stand-by that the saliva gathered puts-80 DEG C of preservations. About 500mL saliva is collected altogether from about 100 mices turning human nerve growth factor gene that grow up. And by Westernblot analyze find turn human nerve growth factor gene mice the parotid gland in express high-caliber hNGF albumen, turn human nerve growth factor gene mice saliva in also hNGF albumen containing higher level, and the hNGF mature peptide separating purification from the mouse salivary turning human nerve growth factor gene meets the 13.5kD molecular weight predicted, qualification result is shown in Figure 12.
8, the hNGF albumen of purification and the comparison to people's TF1 cel l proliferation of the mNGF medicine the mouse salivary of human nerve growth factor gene is turned from 553 familys.
TF1 cell is NGF recipient cell, after it cultivates 1 week in the RPMI-1640 culture fluid containing 10% hyclone and 2ng/mlrhGM-CSF (R&DSystem), cell is eluted, it is resuspended in the RPMI-1640 culture fluid containing 10% hyclone, is inoculated in 96 orifice plates (30ml/ hole) with 420000 cell/ml. Inoculation 1 as a child, removes original fluid, each hole add respectively containing 10% hyclone and 0,1.6,3,6,25, the RPMI-1640 culture fluid 100 μ l of hNGF or the mNGF medicine (Soviet Union's peptide is raw, Beijing SHUTAISHEN company) of 50ng/ml purification.Each process has 3 repeating holes. At 37 DEG C, after 5%CO2 CMC model 40 hours, removing original fluid, the RPMI-1640 culture fluid 50 μ l containing 10% hyclone is added in every hole, adds " CellTiter96AqueousOneSolutionCellProliferation " (Promega) reagent 20 μ l. Culture plate puts 37 DEG C, cultivates 3 hours when 5%CO2, measures each hole OD value at 490nm by microplate reader. Result shows that the propagation of people's TF1 cell is had an obvious facilitation by the hNGF that purification obtains from the mouse salivary turning human nerve growth factor gene, and its biological activity notable (P < 0.05) higher than mNGF medicine, qualification result is shown in Figure 13.
It is indicated above, when people source ngf gene sequence is incorporated in " TTAA " site on No. 5 chromosome of mice turning human nerve growth factor gene and fixed point No. 5 chromosome quaninenucleotide-bindingproteinsubunitalpha-12 gene of insertion and the intergenic non-coding intervening sequence of caspaserecruitmentdomain-containingprotein11, turn the people source NGF medicine separating purification the mouse salivary of human nerve growth factor gene from this and there is better biological activity, the treatment of mankind's relevant disease is had better effect.
Above-described is only some embodiments of the present invention. For the person of ordinary skill of the art, without departing from the concept of the premise of the invention, it is also possible to make some deformation and improvement, these broadly fall into the protection domain of invention.
Claims (10)
1. the mice turning human nerve growth factor gene, it is characterized in that, the genome of the mice of the described human nerve growth factor gene of turning comprises human nerve growth factor gene's sequence, described in turn the salivary gland specifically expressing growth factor of human nerve of mice of human nerve growth factor gene.
2. the mice turning human nerve growth factor gene according to claim 1, it is characterised in that described human nerve growth factor gene's sequence is such as shown in SEQIDNO:1.
3. the mice turning human nerve growth factor gene according to claim 2, it is characterised in that described human nerve growth factor gene's sequence be incorporated into described in turn on No. 5 chromosome of mice of human nerve growth factor gene.
4. the mice turning human nerve growth factor gene according to claim 3, it is characterized in that, described human nerve growth factor gene's sequence be incorporated into described in turn human nerve growth factor gene mice No. 5 chromosomal quaninenucleotide-bindingproteinsubunitalpha-12 gene and the intergenic non-coding intervening sequence of caspaserecruitmentdomain-containingprotein11 on " TTAA " site in.
5. the preparation method of the mice turning human nerve growth factor gene, human nerve growth factor gene's sequence is incorporated in mouse genome by transgenic technology by the mice of the described human nerve growth factor gene of turning, and heredity that can be stable in the described mice progeny turning human nerve growth factor gene, it is characterized in that, described method comprises the steps:
(1) building transposon vector, described transposon vector comprises the human nerve growth factor gene's sequence as shown in SEQIDNO:1, parotid secretion protein gene promoter sequence as shown in SEQIDNO:5, the piggyBac transposon 5 ' terminal repeat as shown in SEQIDNO:6 and the piggyBac transposon as shown in SEQIDNO:7 3 ' terminal repeat;
(2) transposon vector built is injected to the male pronucleus of mouse fertilized egg, then the mouse fertilized egg after injection is migrated to the intrauterine of replace-conceive dams, raise replace-conceive dams and be born to mice;
(3) mice of birth is detected, filter out human nerve growth factor gene and can stablize the mice turning human nerve growth factor gene of heredity in offspring.
6. the preparation method of the mice turning human nerve growth factor gene according to claim 5, it is characterised in that the sequence of described transposon vector is such as shown in SEQIDNO:8.
7. the preparation method of the mice turning human nerve growth factor gene according to claim 6, it is characterized in that, described transposon vector be incorporated into described in turn human nerve growth factor gene mice No. 5 chromosomal quaninenucleotide-bindingproteinsubunitalpha-12 gene and the intergenic non-coding intervening sequence of caspaserecruitmentdomain-containingprotein11 on " TTAA " site in.
8. the method preparing growth factor of human nerve medicine, it is characterised in that described method comprises the steps:
1) method according to any one of claim 5-7 cultivates the mice turning human nerve growth factor gene, described in turn the mice of human nerve growth factor gene be the mice turning human nerve growth factor gene according to any one of claim 1-4;
2) from step 1) described in turn human nerve growth factor gene mice saliva separate be purified into growth factor of human nerve medicine.
9. the method preparing growth factor of human nerve medicine according to claim 8, it is characterised in that described growth factor of human nerve medicine can promote trk C cell proliferation.
10. the method preparing growth factor of human nerve medicine according to claim 9, it is characterised in that described growth factor of human nerve medicine can make the preparation treatment for human diseases.
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