CN109628487A - A method of growth factor of human nerve is prepared using transgene pig salivary gland - Google Patents

Abstract

The present invention by transgenic method preparation can in the parotid gland specifically expressing source of people NGF transgene pig, and acquire the saliva of transgene pig secretion, isolate and purify treatment of the source of people NGF for human diseases, the especially treatment of human nerve's property disease has remarkable result.And the NGF drug for having developed and having put into clinical use currently on the market is the NGF of mouse, there are mouse salivary amount, few and endogenous NGF secretory volume is few, and the few reason of the amount of isolating and purifying causes the market price high；Meanwhile although source of mouse NGF is high with source of people homology, still there may be Immunogenicities by source of mouse NGF, cause the adverse reactions such as allergy.Therefore the method for invention, the efficient expression source of people NGF albumen of high yield in obtained transgene pig saliva, had not only solved the problems, such as source of mouse NGF low output, but also solve the problems, such as homology.

Description

A method of growth factor of human nerve is prepared using transgene pig salivary gland

Technical field

The present invention relates to the animal and preparation method thereof obtained by transgenic method, in particular to it is raw to turn people's nerve for one kind Long factor gene pig and preparation method thereof.

Background technique

Nerve growth factor (i.e. Nerve Growth Factor, abbreviation NGF) is a kind of prominent with neurotrophic and rush A kind of nerve growth regulatory factor for playing growth, is first member of neurotrophins, it is to maincenter and surrounding Each physiology course of neuron has important regulating and controlling effect.It is earliest by Levi-Montalcini in discovery in 1954, then Cohen also studies it, illustrates its physiological function.Therefore Cohen and Levi-Montalcini is obtained 1986 Nobel Prize in Physiology or Medicine.NGF not only plays an important role to the proliferation of nerve cell and differentiation, also to non-neuronal cells, such as The growth of immunocyte, fibroblast, pancreatic cell and cardiac muscle cell etc. and activity have important booster action.Therefore, NGF There is good potential treatment to act on nerve and non-neuropathic disease.

Current developed NGF drug is mainly derived from male mice salivary gland i.e. source of mouse nerve growth factor (mouse NGF, mNGF), because high with people's neural factor (human NGF, hNGF) homology, to the various neurotrosises for the treatment of or degenerative disease It has obvious effects on, therefore granted for clinical treatment in 2003.Price of the drug at present in Chinese market is about 1 Wan Yuan/mg, expensive, demand is big, but current yield is also far from meeting the market demand.In addition source of mouse NGF still may be used There can be Immunogenicity, cause the adverse reactions such as allergy, and source of people NGF then can avoid this problem, and there are some researches prove Its physiological activity is also above source of mouse NGF.Many researchs have attempted a variety of methods and have obtained hNGF, as Escherichia coli, yeast, lactation are dynamic Object cell etc., but all there is the problems such as such as low output, posttranslational modification are not perfect, at high cost.

To solve the above-mentioned problems, have team to attempt to make bioreactor production source of people using galactophore of transgenic animal NGF, yield is considerable, but existing defects, and if any the limitation in gender and period, milk intrinsic protein type is unfavorable for purifying etc. more. Biologically active hNGF is produced as bioreactor using transgenic mice salivary gland for this this laboratory, and It applies for a patent, number of patent application 201610109183.X.But it is unfavorable for largely collecting since mouse salivary amount is less, purifies HNGF albumen, so a kind of method for preparing growth factor of human nerve using transgene pig salivary gland of invention.

Summary of the invention

In view of the above-mentioned deficiencies in the prior art, it is an object of the present invention to provide a kind of more efficient, safer preparation people's mind Method through growth factor.

The present invention by transgenic method preparation can in the parotid gland specifically expressing source of people NGF transgene pig, and acquire should The saliva of transgene pig secretion, isolates and purifies controlling for treatment of the source of people NGF for human diseases, especially human nerve's property disease It treats, there is remarkable result.And the NGF drug for having developed and having put into clinical use currently on the market is the NGF of mouse, there are mouse Saliva amount is less and endogenous NGF secretory volume is few, and the few reason of the amount of isolating and purifying causes the market price high；Meanwhile though source of mouse NGF It is so high with source of people homology, but still there may be Immunogenicities by source of mouse NGF, cause the adverse reactions such as allergy.Therefore originally The method of patented invention, the efficient expression source of people NGF albumen of high yield in obtained transgene pig saliva, had both solved source of mouse NGF The problem of low output, and solve the problems, such as homology.

According to an aspect of the invention, there is provided a kind of prepare growth factor of human nerve using transgene pig salivary gland Method, method includes the following steps:

A, the transposon vector containing source of people ngf gene is constructed；

B, turn the preparation of human nerve growth factor gene pig；

C, the measurement to hNGF protein concentration in hNGF gene pig saliva is turned；

D, the hNGF gene copy number for turning hNGF gene pig is analyzed；

E, hNGF albumen is extracted and purified from transgene pig.

Further, in step a, the method for transposon vector of the building containing source of people ngf gene includes the following steps, benefit The mouse parotid secretion protein gene promoter sequence of 12.2kb long is amplified from mouse gene group DNA with PCR；By 726bp The signal peptide sequence of 54bp long is replaced with the pig PSP signal peptide sequence of 60bp long in long hNGF coded sequence, after recombination The hNGF coded sequence of 732bp long is artificial synthesized；And the artificial synthesized terminal repeat of piggyBac 5 ' and 3 ', and packet One piece of the sequence of polyA containing bGH, CMV promoter sequence, Neo-2A-EGFP fusion gene sequence and bGH polyA sequence Disconnected sequence；It replaces and is located at after these artificial synthesized sequences are connect with recombination hNGF coded sequence and mouse PSP promoter sequence The pmPSP-hNGF that sequence between XhoI the and Not I restriction enzyme site of pGEM-T carrier just generates 20.14kb long contains source of people The transposon vector of ngf gene.

Further, in step b, the preparation method for turning human nerve growth factor gene pig includes the following steps, by 10 μ g PmPSP-hNGF plasmid mixed with the pmPB plasmid of 3 μ g after cotransfection enter about 1 × 10⁶In a porcine fetus fibroblasts, Even be layered in culture dish is cultivated, and density is that average 1~2 cell is advisable in the visual field, and appropriate G418 screening is added in incubation, When cell regimental commander is to diameter about 5mm, there is the cell mass of luciferase expression with cell clone ring picking, then individually culture amplification is thin It is frozen after born of the same parents' amount for use, the cell clone sifted out group is subjected to somatic cell clone as nuclear donor, obtains transgene clone pig.

Further, in step c, the measurement for turning hNGF protein concentration in hNGF gene pig saliva includes the following steps, point Not it is primary turn hNGF gene cloning pig be born 12 days within, 2 monthly ages, six-month-old cotton balls or cotton swab acquisition transgene pig saliva Liquid, and be put into centrifuge tube centrifugation removal impurity, and appropriate extension rate, using hNGF-β gene ELISA kit, The hNGF concentration in hNGF gene pig saliva that turns of acquisition is measured

Further, in step d, the method for turning the hNGF gene copy number analysis of hNGF gene pig includes the following steps, Select two suitable pig genomes high frequency restriction enzyme site, by the primary DNA for turning hNGF gene cloning pig to birth into The copy number of row Southern blot analysis hNGF gene integration.

Further, in step e, the method extracted from transgene pig and purify hNGF albumen includes the following steps, adopts The saliva that transgene pig is collected with operation method, the hNGF egg for being 13.5kD by molecular size range using molecular exclusion chromatography It is white to be purified.

Further, Southern blot analysis of high frequency enzyme is carried out to the primary DNA for turning hNGF gene cloning pig of birth Enzyme site is StuI and EcoRV.

Further, the operation method for collecting the saliva of transgene pig is parotid gland intubation.

Compared with the existing prior art, the invention has the advantages that and the utility model has the advantages that

(1) because the saliva of pig is more compared with what mouse secreted, the saliva that can be collected is more, isolates and purifies and obtains hNGF production It measures higher；

(2) NGF that the technology of the present invention obtains is source of people NGF, and when treating human diseases, there is no immunogenicities to ask Topic, causes the adverse reactions such as allergy, pharmaceutical activity is more preferable.

Detailed description of the invention

Fig. 1 is transposon vector structural schematic diagram.

Fig. 2 is the monoclonal cell group luciferase expression situation filtered out, and a left side is taken pictures for fluorescence, and the right side is taken pictures for natural light.

Fig. 3 is the pig data statistics of PCR cloning PCR prepare transgenosis.

Fig. 4 turns hNGF gene pig luciferase expression qualification result figure (nose, hoof and viscera tissue) to be primary, and " TG " is indicated Turn people's neural factor gene pig, " WT " indicates wild type pig.

Fig. 5 is that the primary hNGF gene pig PCR that turns detects electrophoresis result figure, and " N " indicates blank control in figure, and " WT " indicates wild Raw type pig, " P " indicate that positive control, " M " indicate that DNA Marker label, " 1-9 " indicate the primary pig of transgene clone.

Fig. 6 is the primary testing result for turning hNGF protein concentration in hNGF gene pig saliva, and " TG " expression turns hNGF gene Pig, " CLONE " indicate clone non-transgenic pig, and " WT " indicates wild type pig.

Fig. 7 is the primary Southern blot result figure for turning hNGF gene pig, and " M " indicates Marker label, " P " in figure Indicate that positive control, " WT " indicate wild type pig, " 1,3,4,5,6 " indicate the primary pig of transgene clone.

Specific embodiment

The invention will now be described in further detail with reference to the accompanying drawings.

SEQ ID NO:2 gene order involved in the present invention derives from GenBank accession number:NM_ 002506.2；SEQ ID NO:3 gene order derives from GenBank accession number:NM_002506.2；SEQ ID NO:4 gene order derives from GenBank accession number:X01697.1；SEQ ID NO:5 gene order derives from Golovan SP et al,Transgenic mice expressing bacterial phytase as a model for Phosphorus pollution control, Nature Publishing Group, 2001 articles report；SEQ ID NO: 6, SEQ ID NO:7, bGH polyA sequence, CMV promoter sequence, Neo-2A-EGFP fusion gene sequence, bGH polyA sequence Column derive from zhenfang Wu et al, Co-expression oftwo fibrolytic enzyme genes in CHO Cells and transgenic mice, Transgenic Research, 2013 articles report.

1, the transposon vector containing source of people ngf gene is constructed.

The mouse parotid secretion albumen (Parotid of 12.2kb long is amplified from mouse gene group DNA using PCR Secretory Protein, PSP) gene promoter sequence (SEQ ID NO:5).By the hNGF coded sequence (SEQ of 726bp long ID NO:2) in 54bp long signal peptide sequence (SEQ ID NO:3) with the pig PSP signal peptide sequence of 60bp long (SEQ ID NO: 4) it replaces, the hNGF coded sequence (SEQ ID NO:1) of 732bp long after recombination is artificial synthesized.In addition, also artificial synthesized PiggyBac5 ' terminal repeat (SEQ ID NO:6) and 3 ' terminal repeats (SEQ ID NO:7), and include bGH One fragment sequence of polyA sequence, CMV promoter sequence, Neo-2A-EGFP fusion gene sequence and bGH polyA sequence. Replacement is located at pGEM-T and carries after these artificial synthesized sequences are connect with recombination hNGF coded sequence and mouse PSP promoter sequence Sequence between XhoI the and Not I restriction enzyme site of body just generate 20.14kb long pmPSP-hNGF carrier (SEQ ID NO: 8), by the verifying such as sequencing and digestion, the Transposon plasmid of hNGF expression casette and EGFP marker gene expression cassette is carried PmPSP-hNGF is successfully constructed, and structure is shown in Fig. 1.

2, turn the preparation and identification of human nerve growth factor gene pig

Cotransfection enters about 1 × 10 after the pmPSP-hNGF plasmid of 10 μ g is mixed with the pmPB plasmid of 3 μ g⁶A pig fetus at It in fibrocyte, is uniformly layered in culture dish and cultivates, density is that average 1~2 cell is advisable in the visual field, is added in incubation Appropriate G418 screening, when cell regimental commander is to diameter about 5mm, the cell mass for thering is green fluorescence to express with cell clone ring picking, Then cell clone group luciferase expression situation such as Fig. 2 individually freezes after culture amplifying cells amount stand-by.

The cell clone sifted out group is subjected to somatic cell clone as nuclear donor, obtains transgene clone pig, data statistics As a result such as Fig. 3.It using fluorescent lamp, observes the transgene clone pig fluorescent protein expression situation of birth and takes pictures, as a result such as Fig. 4.

It acquires primary transgene clone piglet ear sample and DNA is extracted by phenol chloroform extraction method.Pig β-actin reference gene, HNGF, enhanced green fluorescence protein (EGFP), PB transposon gene sequence PCR amplification primer drawn respectively using following four Duis Object is completed.Primer 1: β-actin-F (5 '-CACTGCCGCATCCTCTTCCT-3 ')+β-actin-R (5 '- CTCCTGCTTGCTGATCCACATC-3')；Primer 2: hNGF-F (5 '-GAACTCATATTGTACCACGACT-3 ')+R (5 '- CCCCAGAATAGAATGACACC-3')；Primer 3:EGFP-F (5 '-GCCACAAGTTCAGCGTGTCC-3 ')+EGFP-R (5 '- CATGTGATCGCGCTTCTCGTT-3')；Primer 4:PB-F (5 '-CAGATGCCTGAGGATGGA C-3 ')+PB-R (5 '- CGTTGTGGCTGTAGATGATGA-3').PCR electroresis appraisal result such as Fig. 5 has found have 4 primary transgene pigs to contain in 9 HNGF and EGFP gene segment, and be free of PB transposon gene segment.Meanwhile it being produced containing the PCR of hNGF gene and EGFP gene Object determines that it expands the correctness of aim sequence by sequencing.

3, the measurement to hNGF protein concentration in hNGF gene pig saliva is turned

Respectively it is primary turn hNGF gene cloning pig be born 12 days within, 2 monthly ages, six-month-old cotton balls or cotton swab acquisition turn The saliva of gene pig, and it is put into centrifugation removal impurity in centrifuge tube, and appropriate extension rate, utilize people's nerve growth of doctor's moral Factor-beta ELISA kit is measured the hNGF concentration in hNGF gene pig saliva that turns of acquisition, as a result such as Fig. 6.

4, the hNGF gene copy number for turning hNGF gene pig is analyzed

The high frequency restriction enzyme site (StuI, EcoRV) of two suitable pig genomes of selection and probe (position appropriate As shown in Figure 1), hNGF gene is analyzed by carrying out Southern blot to the primary DNA for turning hNGF gene cloning pig of birth The copy number of integration, as a result such as Fig. 7.

5, the extraction and purifying of hNGF albumen

The saliva of transgene pig is collected using surgical parotid gland intubation, the daily salivary secretion amount of every pig is 15L About 3L is collected in left and right every time, will be divided using molecular-exclusion chromatography (size-exclusion chromatography-based) method The hNGF protein purification that son amount size is 13.5kD comes out, and can purify the hNGF albumen of about 2.1mg/3L every time.And relative to Mouse, daily collectable salivary secretion amount is about 1.1376ml, purifiable daily if preparing hNGF by transgenic mice HNGF protein content is about 0.79 μ g.

Above-described is only some embodiments of the present invention.For those of ordinary skill in the art, not Under the premise of being detached from the invention design, various modifications and improvements can be made, these belong to the protection scope of invention.

SEQUENCE LISTING

<110>Agricultural University Of South China

<120>a kind of method for preparing growth factor of human nerve using transgene pig salivary gland

<130> 2018

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tccacaagaa tggttctatt gactgagaaa taatgttcag gataaagatc caggaagaaa 5100

agatcaggga taagtaaaat actaaactct tttgcaaagt acatagaccc tctttcataa 5160

caatgggttc tattgactga caagcactgc tcaggagttg ggaaagagtc tagcataagc 5220

acgatagcct ggagactcta gtgaggtcta gtcttacaga cagcaaaaat caccaggtta 5280

caaactacat tcatttccag ttttctgatc aggcacaggt atgaatccct tctgttgaag 5340

agaaaagtcc atgtgtttaa aatatctggt ttctccagtg ctattagcga gaagacttga 5400

gccctataca actcccacct ggagtgacat cctgtcttca tggtatatta catacctaga 5460

cacgctcatc tcacagactt aggactttgt cttctgatct ccatttctga tcccacttcc 5520

acctttgcct tgatagtgtc attttcttca ctgccttggt gacaaccatg ttatcctctg 5580

tgtatttgag tgttaccatt ttcagatttt acctgtatgc aagatcacac agtctttgtc 5640

tttctgtctg gatgcatgct aatctctaca caacaaccct tccccgtcac tcagatcttc 5700

ctccattaac acatacatgg tgctgaagag gctagggagc ttcccttcag tggggagcta 5760

gctggctatt gggccttttt gactgtccag gaaggccccc aattgctgag acaagaactt 5820

agattcttca ttattgactc taactcatgt atcaagcaga agctaatgaa tagttatcaa 5880

caggatcaga ggttccagtg taagacactt tgacatgaaa gaacggagga aggacagatg 5940

gatgcataaa agcaggacca ctgccccagg aaggtcctgg aaactgatgc agggcaaagg 6000

acaggttata aaccaaatct tagggagtca ggaagagcac agaggagctc aaccaactga 6060

ccactgctta ggggctacca acccaatcct ccctgtggga acagctaagc tatcagccaa 6120

gggtaataaa caggcaggac ctgtggatga catggagagc atagggaccc tgggtccagc 6180

ctttagcacc tgcactctca ggatactcca ccattgtgtc ttagagagcc tagggatact 6240

gggtccagcc tttggtacct tcactctcag ggtaccccat cactgtgtct tggagagcct 6300

aggcaccctg ggtccagcct tcagtacctg cgctctcagg acaccccacc attgtctctt 6360

gccccgtctc ttcttcctct tcctcccttt cattgtctct tctctgtttc tttcttgact 6420

ctcctttccc ctcacaccct cactctagtt ctccccttcc ctctctgcat caccctattc 6480

tctctgtggt ccctccactt tcctttatct ctcatgcttc tctcctccct caaatacttg 6540

tcacccacta tacttcaggg gccagctcta gtgacaaagc tgttaatagc aagactctca 6600

gatctccaac ggctcagagg agccagaccc accaagaact ctctccaggt ccaatttcag 6660

gttccttcga aagctttcag caaatgctca gggaacatgc cactaacaag aagatgcaaa 6720

ttccagttga gagtgggaaa ggcccttgcg taggtcccat cttccaggcc aaggtcagag 6780

gggctctgtg taatccggat tgacagggct cagaacaatg ttttgttttt aaggtttatt 6840

tattttaggt gttagtgtct ttgcttgcat gaccttatgt gcatcatgtg tgtgcaggtt 6900

cctgatgaca gtagaggagg gctttgaatc cctggggata ggaagttaca ggaaattata 6960

agctgctttg tgggtcttct agctttccca acagaagtga atgctcttca ccactgagcc 7020

atctctctag gcccaagaga cattgcttta tggatataat tgtgtgtgtg tgtcaacatt 7080

gaggaaaggg aaataaaaaa aaaacttcag ccgctaaggt tgtacagttt cactaattgc 7140

tacttttagt tgtgataaaa tggcaggtgc ttcaacattt atatatacaa aaacttccct 7200

gctggtggtt caactgtgag aactggggta agtgggtgag ttctcttttt ctgtctctgt 7260

ctctgtctct ctccttccat tctttcttaa aggaaataaa cattgcagct gggttatagc 7320

tcatcaatat ggaagttaca gaagtgaaaa aaggcattgc cttggtgggt ggtgttacca 7380

gctgattttt ggttgtcctg caaggaggtc tggggactgg ctgctctgtc tctgtctgta 7440

tgagtgaggg aagtctgggg agcagattcc ctaaccttca gcctggcctg gttcctgagt 7500

gaacccagcc tctctggtcc tagtagcttt ttccaaacag gaatctgagt ggtgacaggg 7560

aacaagtacc agcccattgc ttaagtgcca gggttagtga gggcaggaag ctgccatagc 7620

tgggattagt agttgtattg gatgtaggaa gtcctatcct gggacagcta atccttaatg 7680

cttcactgga gattttcaat gagaaattta tcccacggcc catatggccc catccttttg 7740

tctccaacag ccaagtattt tccattagag gagacttcct gtacacttga tggatgctca 7800

tttcaaggtg acttggggca gtcagtacag acttgggatg acctctgaca gcctaacctc 7860

tccccaacaa gggccctcta tgtttgctat gtaatgtaat gtcagacatt gtcaggagtg 7920

tccgcagcac agcctgccca gtgtgagggc tctcataggt ttcccactgt cttatctaca 7980

cagggataac gaggaggtaa gctgcagttc ccagtctcac ttcacagagg aagagataac 8040

cccatcccag gtcatgtagc cagcagtgga aagaatgagg atttgaactc aggtcttcca 8100

agtcccattg atagcatctc ctcacaagtc ccttgccacc ctcacgatgc cttagacact 8160

tgcctgccct ttatactaag gagatgcagg tacaaggggt ttacccatgt agcagctgag 8220

gcagctgggg atagatacca gcagcaggcc tgatgtcacc actctaactc cagcatcccc 8280

agtctgtgtt cctggagtgt gaaaatccct acttaacaag attgtgcaac agtccttggc 8340

tctgtgaccc atagctggaa acaggattct cattgatttg tggaacatgg tggcagccag 8400

ccaaaaagag ggtctgcata cagaagacag ctgtggcaag gccacagcag actctgacta 8460

ccttagctta cagaattaca aggtcataat gtcctctgct ttggtcacct catgttaagg 8520

acaggcccta atgaagatgg ggcagaagac tgaaggaatg gccaaccaat aactggccca 8580

acttgagacc catcctacag gcaagcatca attcctgaca ctactaatga tactctgtta 8640

tgcttgcaga cagaagccta gcataactat cctccgagag gtccacccag caactgactg 8700

aaacagaaaa agatatccac aggcaaacag tggatggagg tcagggacta ttatgggaga 8760

gctgtgggaa ggattaaaaa ccctgaaggg gataggaacc ccacaggaag accaacagag 8820

tcaactaaga gacctgtggg agctctcaga gactgagcca ccaaccaaag agcatacaca 8880

ggccggtccg aggcacctgg cacgtgtgaa gcagacatgc agctcagtct ccatgtaggt 8940

cctccaataa gcggtagcct gactgcagta tccaatccct aacagggctg cacagtctgg 9000

cctcagtggg ggagggtgcc cctaatcctg cagagacttg atgagtggag agctatccag 9060

ggggaaccca ccctctctga gaagggaatg gggatggggg agggactctg tgaagagggg 9120

acaaggacaa acaagaacct caaataggtc aggccctaaa ggcttgctaa gtagcagtgg 9180

cccagctctg tcctgttcct cagcccaagg ctcagctccc acctgtttct gtgtttttct 9240

ggcttttcat gggcctagga cttggtggcc agttcaaaca atggggcctg tggaagacac 9300

aatatacaag actagggaca ttcctgttct gctgactatc cacagcctga tgtaggtgga 9360

aggacccaat cactggattt ctacccttgc gcaaccttga cagctgaggg cctctcagaa 9420

acctatttct tccactgaaa aatgagactc tcaaatgaac gtcctgacaa tcatcaggct 9480

tattaaagag gtgtatctaa cctgaatggc aagcagacag caggcaaatg tctgtatcaa 9540

cctctaggaa ggacaagaac tgctcactgc tgccccccag gaggccattt gctgaaacag 9600

ctgctctcct gctggtgcac aggccctgcc ttctcattgc agctacagcc ccttcctgtc 9660

tgaacctcct gtcaggtcac tgggaaacag atcaagatgg aacaggacag ctcctgatgg 9720

taaataaaaa acagtggtca tggctattca taggggttta tgcttcttca gtccacactg 9780

tgaagagctg tgggcatgaa ccacagtgtt cgaggtagag ttggggttct gaaattcaca 9840

gtggggtgag ctcagtaaat gtgagctgga ggtcactcgt gagacacaca gtcctgctgc 9900

ttctgttccc aatatcctga ggagacgaca catctacttt gttcagaggc cacagtctag 9960

ttgacctgag agttaccagt ttcttatttg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 10020

tgtgtgttgt tcgtgtgtga gtgcaggtgc acatatgata gcgtacacgt tgaggtcaga 10080

ggataactat caggcgttgt cccctcctac ttttcctcgg actctggaga acaaacatgg 10140

gtccttattc caggggagca agtgcctgtt ggctgacaca tcttgctcac atacatttta 10200

cctagacaat ggagcctcca tcagagtatt actttagctc ctcaccgatg gcaatgcacc 10260

acctctctac ccacatagga gttgggtctc cacacacccc cacaccccct tcaccaaaac 10320

gttttcagtt actttatctg gtaaagttca tcagagaatg aagccagtat taagaacatg 10380

gaatcatttg ggaacctgga tctagcaata ccccacccta gatggagttg ctgagttttc 10440

acctcagatt ataattcccc cctagcttct atggtttatt ctgaaaccag gggaactcga 10500

ttcctccctt tggaccacag acatcctggc ttgtgaattc acatgtcatc tactgctaat 10560

ccattggtag tatgtggctc acagagacac actacagtca tggccaatgt caaggtagga 10620

cagatgtgaa tcattccccc agtcctgctg ttttcatgac taaccctcct cagcacagtg 10680

accatgaacc tacttttccc ctccttttat ttttagaatt gctggaattt tctattttga 10740

gaaataatag ccttggggca gcattaaaca aaatcatcta gaaagctggt ttaaaataca 10800

gatggttgag tcagtgaaag agtgaggaat gtcattattg gcccctcaca gaggctggct 10860

cactccagca gaggtggttg aagctcttgg acacgggtca ggtgcatagg aagggtggtc 10920

tgggacacct gagaaccaca attgaacaaa cagaagctgc tggctttttt ttttttaaat 10980

gagttctcaa aaaatgactg ggctagctta ggcaaatact tcgagccaac ccaacagaac 11040

attcttccat tgattcattc tggatcttct ttctagacaa tactgaactg accccttgtt 11100

ggcagtctca agtttgacaa catagggctt tgaacttggc acaaggtcca tcactgtcac 11160

ccaagcatcc tgggtgacct ttgggttgga atatcttggc taaccttaga tattttcttt 11220

ggagtatctt tagaacatcc aggaaatagg gcttgattct catcctggga ccacaatata 11280

agtcacccta gaatcccagg agatcgtgca gagaaacaag gatctctctc gtgtgcatcc 11340

ttcttcaaag cagtgagtag tgactccact aaactgagtt cccatctgag agtccacagg 11400

aggctttggg gcaagaagca gagggaaggc actgtttgtg ttggtaaagt tttgactcta 11460

acaaatttga agacatagat gacattgtgt cagactaaca acaacctaga ctcatgtggg 11520

ttctgtttag ggatcagatt ttattcatca atgacttgtc ttagtgtata gagaaaggct 11580

tcctactgga gtgtaggctc aataatgaca gaagagatag ctatttcccc tagggactgt 11640

gctgctccaa gtttggtgga gaaaggcagt ggggaaccta gatgtgctct ctggggaggg 11700

ggtctgaagc tggcttcata gaaggtgtga agttttgctg aaacatctaa acagaattat 11760

agcttaggaa agtgagcagg caaggcaggg aatgtgttgc atatgtatat gtacatgaat 11820

atattatgtt atagatacac acacatttga acctcatttg cagatgacag aaaataggtt 11880

attttgcctc tcttaactgc taagcacaat gacttccagt tccatccatt tcctgaaatg 11940

ccacaatttc atttttcatt gtggctgaat aaaattccat tgcagactgg gccctacttc 12000

atccactcct gagggcaggc atatcccctg gctccatttc ttacctattg tgaagagaag 12060

tgcaactgtc ttgttgaaag gcaagcgtga gagaggcagg cactaattgt gggtttttgt 12120

ttcttcttcc tgctatgact ctccatttgt c 12151

<210> 6

<211> 303

<212> DNA

<213>unknown

<400> 6

ttaaccctag aaagatagtc tgcgtaaaat tgacgcatgc attcttgaaa tattgctctc 60

tctttctaaa tagcgcgaat ccgtcgctgt gcatttagga catctcagtc gccgcttgga 120

gctcccgtga ggcgtgcttg tcaatgcggt aagtgtcact gattttgaac tataacgacc 180

gcgtgagtca aaatgacgca tgattatctt ttacgtgact tttaagattt aactcatacg 240

ataattatat tgttatttca tgttctactt acgtgataac ttattatata tatattttct 300

tgt 303

<210> 7

<211> 265

<212> DNA

<213>unknown

<400> 7

gagaggtccc tcgacatact agttaaaagt tttgttactt tatagaagaa attttgagtt 60

tttgtttttt tttaataaat aaataaacat aaataaattg tttgttgaat ttattattag 120

tatgtaagtg taaatataat aaaacttaat atctattcaa attaataaat aaacctcgat 180

atacagaccg ataaaacaca tgcgtcaatt ttacgcatga ttatctttaa cgtacgtcac 240

aatatgatta tctttctagg gttaa 265

<210> 8

<211> 20124

<212> DNA

<213>artificial synthesized

<400> 8

ggccgcgcgc gccattctag attaacccta gaaagatagt ctgcgtaaaa ttgacgcatg 60

cattcttgaa atattgctct ctctttctaa atagcgcgaa tccgtcgctg tgcatttagg 120

acatctcagt cgccgcttgg agctcccgtg aggcgtgctt gtcaatgcgg taagtgtcac 180

tgattttgaa ctataacgac cgcgtgagtc aaaatgacgc atgattatct tttacgtgac 240

ttttaagatt taactcatac gataattata ttgttatttc atgttctact tacgtgataa 300

cttattatat atatattttc ttgttataga tatcaactag aatgctagca ccggttgcca 360

aagaccaaag acttgttctc cattagaaat gacagctggc ttgacccgac agcataataa 420

gcagagtgta ctctgattgg agaactttaa tgtgtttcat tcagtattat aaaaggacag 480

tattacagat tttgttgtac actgctgtta catgtggggc agtgtgtctt taagtagggt 540

aaagtactct ttaaaaatgg gtcctagata ttttttcctt taactcaagt ctcttactgt 600

ttaaatgatt tttattttgt ttaatatgga ggaaaaagaa gcgtaaatgg acaatatata 660

tttagagaaa gatggttagc tgtcagaaaa atatgcaaat caaaatcaca ccaagactgc 720

agcacacccc tgtcagatgg ctgtgatcaa gaaaataaat gacaatgagt ggtggtgaag 780

atgtactaaa gggaaacaca cacacacaca cacacacaca cacacacaca cactggagca 840

accactgtgg aaatcagtat gaatggtcct caaaaacctg aagatagagc ggggcgtggt 900

ggcatacact tttattccca gcactgggga ggcagaggca ggtggatctc tgagttccag 960

gccagcctgg tctatagcac aggttctagg acagccaggg ctacacagaa aaaccctgcc 1020

ttgattaaac caaaccaaac caaaccaaac caaaccaaac caaaccaaac caaaccaaac 1080

caaaccagac caaaccaaaa cactgaagat agaacttcag tattccattc ctagatatat 1140

acccaatgga gactaagtca gcaagacacc tgcacagcca tgttcactac tacactgttc 1200

accacagcca ggctgtggaa ccagcctgag tgtccatgat aaatgaatgg ataggtaact 1260

ttcaaggtaa atggactctg ctgtgtacat gcctcacatt ctgtttattc atttttcttt 1320

atgaggtgtc cattcaggag tcacatggta gttctatttt cagtcttctg aagatactac 1380

actggtcccc acagtttaca cttttatcag cagtgaataa gggttcctct atccttacca 1440

tcatttgttg taatttttct tgatgaccct ctttctgaca gggataggat gtaatatcag 1500

tgtgaggaag tacaacttgt tttctaagta tttattggcc ccttgcattt cttcttttga 1560

aaactgtcgg ttcctgacat ctgctcaggt attcattgga tgttgtttct ttggtgtttg 1620

agttcttatg aattctagat gttaaatccc tgcctgtggt tctctcccat tctgtaggct 1680

gcctcctcac cctggcaatt gttgtccttg ttttgcagaa acttttgact tcatggaatc 1740

tcatttgtca gttttccctc ctctgctata gcctgagcta atgcactggt ttttacagag 1800

ccctggtcta tgcctttatc ctcctctggc agcttcggag tttcatttct tacatttaga 1860

tctttgatcc actttgaaca agttttggag cagggtgaga gatacgaatc tagttccatt 1920

cttccatatg tgatcctagt ttacatagca tcgttggttg aagaggtttt attttatttt 1980

taaataatgt gtcataaaaa acgaggtggt tgtagcagtg tggatttgtt tctttgtcct 2040

ttgatctaca ggtcttgttt tgtgtcagtc tcatgatgtt ttattgctat ggctctgtca 2100

tacagtctga ggtcaggtat tgtgatatac cttcagtatt gctccctcag actcaggttt 2160

gctttggcca ggagtcatct tactcagtgc tcttagagct cccccagcat gtagctgcta 2220

ctattcttag ttgataaatc aggaaactgg ggctcagaga gattaactgt cttgaactac 2280

ttctggggag gtgaaacgtg gagacactaa actgtgttta ccctgtactg ctccagtagc 2340

tgtcgggtgc tgggctacag caaagcacct atactatata ttactcagga ggtggaaaaa 2400

ctcagcctcc cttggggttc ccaagctccc aggtgtccag tcactgctgg aaacctcatg 2460

gagtctgaaa ggaagggttg agggtacatg gggcagcgat gaggagcctg gggctgggat 2520

ctcccaaaca cctggatatc cagatgccac tgggtcaggg ggagttggga acagagttgg 2580

gatgtccatg gacctgtgac aaggccaggg ccagggggag gataactctg gctttactaa 2640

tttgcgaaag tccttagctt agcagcagtt gtctgggagc acagaggggc cttctgtaag 2700

aggctcaggc agtgccgctc tgtaggcgaa ggtcttctcc atgttcccca tggtggttct 2760

tgatgaaaga gacagtcctt ggctccaaac tggtttattg attgttcatt gtggaaaatg 2820

ggtgcacacc accttctcag ggtggaccag agatcaaata ccttttgcag ggaggaatat 2880

ctgggaaggg acgcttactg gctaaaccct cagggcctct agatacatca ttagcatgga 2940

gaactctgtt ctgggctaca tgaccacagg ccacatttcc acaagccaca tgtgggaagt 3000

gtggcacatg ttctaggcca ggaatctggt agggagcgtg gagccaccta ccatcccagg 3060

tgggtgcctg ggtgccaggg accctgaacc cgctcaacct taccaagttt cctggcaggg 3120

tccactgtcc tacacagaag ctggaggagg tgtgagggtt gtgtctttgt ggaatgtccc 3180

atgctgcttg gggctcagtt tctccacctg tacctcattg gtttgggtat aaaaagtggg 3240

gatactttat tattctctga ctcggtcctg aggaaaaagc atcgtggcag tccaggaacc 3300

acaccctgag gttcctgcac tgaagggact ccctaagtct ctggagtctc tccccttcac 3360

agagctgcca aagtctaggt tcttttgagg ataacagagc catgcttggt aagcagacaa 3420

cagcatttgt ttactcaacc ttcttttgtc agctccctct tcataaacaa gttgagacac 3480

catgctggct tgaggaagac ttctaaagcc agacaactgt gcaaggaaga agaagaaggg 3540

gcaagtggag ttagcctgga tgtagccctc aaagtctcca gagaccagcc atgaaggctc 3600

aagtggaggg caagacctgc agcagccaag catctggcag gagaggatcc tgggaacccc 3660

tctaccatga cacacattct tcctgcaggt cacacttaat aggccatttc ttatttggat 3720

ctatcatggt gttctgtgcg agattaatga ggtgttatgc tgcgaacaga aagttatata 3780

aaaacaagtc cccccccctt gtcactgctg ctaagaatgt agcagaaatt gtctcaagtg 3840

tctctctaat cagaaacaat aaaggtctcc ttggattcaa gccctccagt ttcctccttc 3900

cttgctgagc cttggacaca ccggtccata caaacctcct ggatgctaca gctctgggca 3960

gagactccaa ggtggggaga gactgatggt acaaaagcaa aatacttgtt tgggggtaca 4020

cccactcctc tgcctgtgtg gttcctgcag tcagtcctgc agacaggccc tcagtgggtc 4080

ttccatgggc aacacgcaga gggaggcaat ggatgggaat acccacaccc tggttagttt 4140

accccggcca tgctctctgc tcttcatccc tcctctgccc tctgccacgg ctttctctgc 4200

aggaatcata tcttcatatt ggcccacagg tgttctcctc accctagcta tgatgtttac 4260

tttagagtga ccttagcagg gctggtggga atgagttcta gaaggctcac ggagatgcta 4320

gggaagaaac gtcttctaac tactgaggtt actaagttcc tggtggttgt ctctgccttt 4380

cccttgttaa agtcaccttg aagttagtgc agaagaaatc agagcccagt cacagagtaa 4440

atatggtcct gaagatttcc tttgagtgcc cagaatccat gacatttcaa gagccctctt 4500

tgtaccttaa gtcatttggg gttgtatctt ctgcttgatg tatgtgtgtg tgtttatcaa 4560

agagtgagat ggttacataa gaggtgctct aaaggacaga gaggatttgc aattgtggca 4620

tgtgacatcc tcaggccttg ctctggtgcc aggaggaact gatgcagaaa agagtaagag 4680

gtcatttcct ggaggctgtc actatagagg agatcttaca gtgcattccc tcctccaggc 4740

cctgcctgag gatagacatg tgctgactgc aactgaaaca gaggcttggg atggagagtt 4800

aggttcacag aagggagggt gggagatgga tgcttgctgg gttctgggtc tcatcaccag 4860

ctcctgacca cccggtcagc ccatgtgctt attccatagc tttcttttgc tatgtttact 4920

cagtgtggtg tttgttggga cccagcagaa gccagtccca ggctgacagc tgtggataca 4980

cagggcagca tgagggtcct cagcctgaag cagtcaggct ggcagaagag aaagaccagc 5040

acacattcct tcaaccaact atgtcttgaa aaacaaacat attatatcac atatattgca 5100

tttatgagac agctaaaatg tactcgggta gcatgactcc aggtggggat atctgcaagt 5160

gccatgagtg gcagagggac agccaatgtg aggcaagaag gaattctggc tcaacacagc 5220

ttagctccct ggtgttggtt caaactttga gagtttgacc acaagcactt tatttttgac 5280

atatttaaac agagcacaac tttgggaaaa agttttctta tgaaaattat cacaataaag 5340

cttaaggcat gactacatta aaatgccttt gcaaagtata tgtgccctct tccacaagaa 5400

tggttctatt gactgagaaa taatgttcag gataaagatc caggaagaaa agatcaggga 5460

taagtaaaat actaaactct tttgcaaagt acatagaccc tctttcataa caatgggttc 5520

tattgactga caagcactgc tcaggagttg ggaaagagtc tagcataagc acgatagcct 5580

ggagactcta gtgaggtcta gtcttacaga cagcaaaaat caccaggtta caaactacat 5640

tcatttccag ttttctgatc aggcacaggt atgaatccct tctgttgaag agaaaagtcc 5700

atgtgtttaa aatatctggt ttctccagtg ctattagcga gaagacttga gccctataca 5760

actcccacct ggagtgacat cctgtcttca tggtatatta catacctaga cacgctcatc 5820

tcacagactt aggactttgt cttctgatct ccatttctga tcccacttcc acctttgcct 5880

tgatagtgtc attttcttca ctgccttggt gacaaccatg ttatcctctg tgtatttgag 5940

tgttaccatt ttcagatttt acctgtatgc aagatcacac agtctttgtc tttctgtctg 6000

gatgcatgct aatctctaca caacaaccct tccccgtcac tcagatcttc ctccattaac 6060

acatacatgg tgctgaagag gctagggagc ttcccttcag tggggagcta gctggctatt 6120

gggccttttt gactgtccag gaaggccccc aattgctgag acaagaactt agattcttca 6180

ttattgactc taactcatgt atcaagcaga agctaatgaa tagttatcaa caggatcaga 6240

ggttccagtg taagacactt tgacatgaaa gaacggagga aggacagatg gatgcataaa 6300

agcaggacca ctgccccagg aaggtcctgg aaactgatgc agggcaaagg acaggttata 6360

aaccaaatct tagggagtca ggaagagcac agaggagctc aaccaactga ccactgctta 6420

ggggctacca acccaatcct ccctgtggga acagctaagc tatcagccaa gggtaataaa 6480

caggcaggac ctgtggatga catggagagc atagggaccc tgggtccagc ctttagcacc 6540

tgcactctca ggatactcca ccattgtgtc ttagagagcc tagggatact gggtccagcc 6600

tttggtacct tcactctcag ggtaccccat cactgtgtct tggagagcct aggcaccctg 6660

ggtccagcct tcagtacctg cgctctcagg acaccccacc attgtctctt gccccgtctc 6720

ttcttcctct tcctcccttt cattgtctct tctctgtttc tttcttgact ctcctttccc 6780

ctcacaccct cactctagtt ctccccttcc ctctctgcat caccctattc tctctgtggt 6840

ccctccactt tcctttatct ctcatgcttc tctcctccct caaatacttg tcacccacta 6900

tacttcaggg gccagctcta gtgacaaagc tgttaatagc aagactctca gatctccaac 6960

ggctcagagg agccagaccc accaagaact ctctccaggt ccaatttcag gttccttcga 7020

aagctttcag caaatgctca gggaacatgc cactaacaag aagatgcaaa ttccagttga 7080

gagtgggaaa ggcccttgcg taggtcccat cttccaggcc aaggtcagag gggctctgtg 7140

taatccggat tgacagggct cagaacaatg ttttgttttt aaggtttatt tattttaggt 7200

gttagtgtct ttgcttgcat gaccttatgt gcatcatgtg tgtgcaggtt cctgatgaca 7260

gtagaggagg gctttgaatc cctggggata ggaagttaca ggaaattata agctgctttg 7320

tgggtcttct agctttccca acagaagtga atgctcttca ccactgagcc atctctctag 7380

gcccaagaga cattgcttta tggatataat tgtgtgtgtg tgtcaacatt gaggaaaggg 7440

aaataaaaaa aaaacttcag ccgctaaggt tgtacagttt cactaattgc tacttttagt 7500

tgtgataaaa tggcaggtgc ttcaacattt atatatacaa aaacttccct gctggtggtt 7560

caactgtgag aactggggta agtgggtgag ttctcttttt ctgtctctgt ctctgtctct 7620

ctccttccat tctttcttaa aggaaataaa cattgcagct gggttatagc tcatcaatat 7680

ggaagttaca gaagtgaaaa aaggcattgc cttggtgggt ggtgttacca gctgattttt 7740

ggttgtcctg caaggaggtc tggggactgg ctgctctgtc tctgtctgta tgagtgaggg 7800

aagtctgggg agcagattcc ctaaccttca gcctggcctg gttcctgagt gaacccagcc 7860

tctctggtcc tagtagcttt ttccaaacag gaatctgagt ggtgacaggg aacaagtacc 7920

agcccattgc ttaagtgcca gggttagtga gggcaggaag ctgccatagc tgggattagt 7980

agttgtattg gatgtaggaa gtcctatcct gggacagcta atccttaatg cttcactgga 8040

gattttcaat gagaaattta tcccacggcc catatggccc catccttttg tctccaacag 8100

ccaagtattt tccattagag gagacttcct gtacacttga tggatgctca tttcaaggtg 8160

acttggggca gtcagtacag acttgggatg acctctgaca gcctaacctc tccccaacaa 8220

gggccctcta tgtttgctat gtaatgtaat gtcagacatt gtcaggagtg tccgcagcac 8280

agcctgccca gtgtgagggc tctcataggt ttcccactgt cttatctaca cagggataac 8340

gaggaggtaa gctgcagttc ccagtctcac ttcacagagg aagagataac cccatcccag 8400

gtcatgtagc cagcagtgga aagaatgagg atttgaactc aggtcttcca agtcccattg 8460

atagcatctc ctcacaagtc ccttgccacc ctcacgatgc cttagacact tgcctgccct 8520

ttatactaag gagatgcagg tacaaggggt ttacccatgt agcagctgag gcagctgggg 8580

atagatacca gcagcaggcc tgatgtcacc actctaactc cagcatcccc agtctgtgtt 8640

cctggagtgt gaaaatccct acttaacaag attgtgcaac agtccttggc tctgtgaccc 8700

atagctggaa acaggattct cattgatttg tggaacatgg tggcagccag ccaaaaagag 8760

ggtctgcata cagaagacag ctgtggcaag gccacagcag actctgacta ccttagctta 8820

cagaattaca aggtcataat gtcctctgct ttggtcacct catgttaagg acaggcccta 8880

atgaagatgg ggcagaagac tgaaggaatg gccaaccaat aactggccca acttgagacc 8940

catcctacag gcaagcatca attcctgaca ctactaatga tactctgtta tgcttgcaga 9000

cagaagccta gcataactat cctccgagag gtccacccag caactgactg aaacagaaaa 9060

agatatccac aggcaaacag tggatggagg tcagggacta ttatgggaga gctgtgggaa 9120

ggattaaaaa ccctgaaggg gataggaacc ccacaggaag accaacagag tcaactaaga 9180

gacctgtggg agctctcaga gactgagcca ccaaccaaag agcatacaca ggccggtccg 9240

aggcacctgg cacgtgtgaa gcagacatgc agctcagtct ccatgtaggt cctccaataa 9300

gcggtagcct gactgcagta tccaatccct aacagggctg cacagtctgg cctcagtggg 9360

ggagggtgcc cctaatcctg cagagacttg atgagtggag agctatccag ggggaaccca 9420

ccctctctga gaagggaatg gggatggggg agggactctg tgaagagggg acaaggacaa 9480

acaagaacct caaataggtc aggccctaaa ggcttgctaa gtagcagtgg cccagctctg 9540

tcctgttcct cagcccaagg ctcagctccc acctgtttct gtgtttttct ggcttttcat 9600

gggcctagga cttggtggcc agttcaaaca atggggcctg tggaagacac aatatacaag 9660

actagggaca ttcctgttct gctgactatc cacagcctga tgtaggtgga aggacccaat 9720

cactggattt ctacccttgc gcaaccttga cagctgaggg cctctcagaa acctatttct 9780

tccactgaaa aatgagactc tcaaatgaac gtcctgacaa tcatcaggct tattaaagag 9840

gtgtatctaa cctgaatggc aagcagacag caggcaaatg tctgtatcaa cctctaggaa 9900

ggacaagaac tgctcactgc tgccccccag gaggccattt gctgaaacag ctgctctcct 9960

gctggtgcac aggccctgcc ttctcattgc agctacagcc ccttcctgtc tgaacctcct 10020

gtcaggtcac tgggaaacag atcaagatgg aacaggacag ctcctgatgg taaataaaaa 10080

acagtggtca tggctattca taggggttta tgcttcttca gtccacactg tgaagagctg 10140

tgggcatgaa ccacagtgtt cgaggtagag ttggggttct gaaattcaca gtggggtgag 10200

ctcagtaaat gtgagctgga ggtcactcgt gagacacaca gtcctgctgc ttctgttccc 10260

aatatcctga ggagacgaca catctacttt gttcagaggc cacagtctag ttgacctgag 10320

agttaccagt ttcttatttg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgttgt 10380

tcgtgtgtga gtgcaggtgc acatatgata gcgtacacgt tgaggtcaga ggataactat 10440

caggcgttgt cccctcctac ttttcctcgg actctggaga acaaacatgg gtccttattc 10500

caggggagca agtgcctgtt ggctgacaca tcttgctcac atacatttta cctagacaat 10560

ggagcctcca tcagagtatt actttagctc ctcaccgatg gcaatgcacc acctctctac 10620

ccacatagga gttgggtctc cacacacccc cacaccccct tcaccaaaac gttttcagtt 10680

actttatctg gtaaagttca tcagagaatg aagccagtat taagaacatg gaatcatttg 10740

ggaacctgga tctagcaata ccccacccta gatggagttg ctgagttttc acctcagatt 10800

ataattcccc cctagcttct atggtttatt ctgaaaccag gggaactcga ttcctccctt 10860

tggaccacag acatcctggc ttgtgaattc acatgtcatc tactgctaat ccattggtag 10920

tatgtggctc acagagacac actacagtca tggccaatgt caaggtagga cagatgtgaa 10980

tcattccccc agtcctgctg ttttcatgac taaccctcct cagcacagtg accatgaacc 11040

tacttttccc ctccttttat ttttagaatt gctggaattt tctattttga gaaataatag 11100

ccttggggca gcattaaaca aaatcatcta gaaagctggt ttaaaataca gatggttgag 11160

tcagtgaaag agtgaggaat gtcattattg gcccctcaca gaggctggct cactccagca 11220

gaggtggttg aagctcttgg acacgggtca ggtgcatagg aagggtggtc tgggacacct 11280

gagaaccaca attgaacaaa cagaagctgc tggctttttt ttttttaaat gagttctcaa 11340

aaaatgactg ggctagctta ggcaaatact tcgagccaac ccaacagaac attcttccat 11400

tgattcattc tggatcttct ttctagacaa tactgaactg accccttgtt ggcagtctca 11460

agtttgacaa catagggctt tgaacttggc acaaggtcca tcactgtcac ccaagcatcc 11520

tgggtgacct ttgggttgga atatcttggc taaccttaga tattttcttt ggagtatctt 11580

tagaacatcc aggaaatagg gcttgattct catcctggga ccacaatata agtcacccta 11640

gaatcccagg agatcgtgca gagaaacaag gatctctctc gtgtgcatcc ttcttcaaag 11700

cagtgagtag tgactccact aaactgagtt cccatctgag agtccacagg aggctttggg 11760

gcaagaagca gagggaaggc actgtttgtg ttggtaaagt tttgactcta acaaatttga 11820

agacatagat gacattgtgt cagactaaca acaacctaga ctcatgtggg ttctgtttag 11880

ggatcagatt ttattcatca atgacttgtc ttagtgtata gagaaaggct tcctactgga 11940

gtgtaggctc aataatgaca gaagagatag ctatttcccc tagggactgt gctgctccaa 12000

gtttggtgga gaaaggcagt ggggaaccta gatgtgctct ctggggaggg ggtctgaagc 12060

tggcttcata gaaggtgtga agttttgctg aaacatctaa acagaattat agcttaggaa 12120

agtgagcagg caaggcaggg aatgtgttgc atatgtatat gtacatgaat atattatgtt 12180

atagatacac acacatttga acctcatttg cagatgacag aaaataggtt attttgcctc 12240

tcttaactgc taagcacaat gacttccagt tccatccatt tcctgaaatg ccacaatttc 12300

atttttcatt gtggctgaat aaaattccat tgcagactgg gccctacttc atccactcct 12360

gagggcaggc atatcccctg gctccatttc ttacctattg tgaagagaag tgcaactgtc 12420

ttgttgaaag gcaagcgtga gagaggcagg cactaattgt gggtttttgt ttcttcttcc 12480

tgctatgact ctccatttgt cagggcgcgc cgccaccatg ttccagctgt ggaagctggt 12540

gttcctgtgc ggcctgctga tcggcaccag cgccagcgaa ccacactcag agagcaatgt 12600

ccctgcagga cacaccatcc cccaagccca ctggactaaa cttcagcatt cccttgacac 12660

tgcccttcgc agagcccgca gcgccccggc agcggcgata gctgcacgcg tggcggggca 12720

gacccgcaac attactgtgg accccaggct gtttaaaaag cggcgactcc gttcaccccg 12780

tgtgctgttt agcacccagc ctccccgtga agctgcagac actcaggatc tggacttcga 12840

ggtcggtggt gctgccccct tcaacaggac tcacaggagc aagcggtcat catcccatcc 12900

catcttccac aggggcgaat tctcggtgtg tgacagtgtc agcgtgtggg ttggggataa 12960

gaccaccgcc acagacatca agggcaagga ggtgatggtg ttgggagagg tgaacattaa 13020

caacagtgta ttcaaacagt acttttttga gaccaagtgc cgggacccaa atcccgttga 13080

cagcgggtgc cggggcattg actcaaagca ctggaactca tattgtacca cgactcacac 13140

ctttgtcaag gcgctgacca tggatggcaa gcaggctgcc tggcggttta tccggataga 13200

tacggcctgt gtgtgtgtgc tcagcaggaa ggctgtgaga agagcctgat ctagagggcc 13260

cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg 13320

cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata 13380

aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt 13440

ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt 13500

gggctctatg gcttctgaag atctctcgag gtcgacgatg taggtcacgg tctcgaagcc 13560

gcggtgcggg tgccagggcg tgcccttggg ctccccgggc gcgtactcca cctcacccat 13620

ctggtccatc atgatgaacg ggtcgaggtg gcggtagttg atcccggcga acgcgcggcg 13680

caccgggaag ccctcgccct cgaaaccgct gggcgcggtg gtcacggtga gcacgggacg 13740

tgcgacggcg tcggcgggtg cggatacgcg gggcagcgtc agcgggttct cgacggtcac 13800

ggcgggcatg tcgacggtat cgataagctt gggcccctcg agggcgcgcc gatcaattct 13860

ccgatcaatt ctctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat 13920

ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc 13980

tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg 14040

ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg 14100

gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggttt aaactcgatt 14160

ataacttcgt atagcataca ttatacgaag ttatgatcga tatgaagaat ctgcttaggg 14220

ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat 14280

tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt 14340

tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc 14400

cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac 14460

gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata 14520

tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc 14580

agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta 14640

ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac 14700

ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc 14760

aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc 14820

gtgtacggtg ggaggtctat ataagcagag ctctctggct aactagagaa cccactgctt 14880

actggcttat cgaaattaat acgactcact atagggagac ccaagctggc tagcgtttaa 14940

acttaagctt ggtaccgagc tcggatccac tagtccagtg tggtggaatt cgccaccatg 15000

ggatcggcca ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg 15060

ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg 15120

ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat 15180

gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca 15240

gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg 15300

gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat 15360

gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa 15420

catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg 15480

gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg 15540

cccgacggcg atgatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg 15600

gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat 15660

caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac 15720

cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc 15780

cttcttgacg agttcttcgc catggtgagc aagggcgagg agctgttcac cggggtggtg 15840

cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag 15900

ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag 15960

ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc 16020

cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac 16080

gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg 16140

aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag 16200

gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc 16260

atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag 16320

gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc 16380

gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac 16440

gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc 16500

atggacgagc tgtacaagta atctagaggg cccgtttaaa cccgctgatc agcctcgact 16560

gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg 16620

gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg 16680

agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg 16740

gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga agatctataa 16800

cttcgtatag catacattat acgaagttat ggatctgtcg actcgagagg tccctcgaca 16860

tactagttaa aagttttgtt actttataga agaaattttg agtttttgtt tttttttaat 16920

aaataaataa acataaataa attgtttgtt gaatttatta ttagtatgta agtgtaaata 16980

taataaaact taatatctat tcaaattaat aaataaacct cgatatacag accgataaaa 17040

cacatgcgtc aattttacgc atgattatct ttaacgtacg tcacaatatg attatctttc 17100

tagggttaat ctagtatacg cgtatgcctc gaggatcgca tcccgcggcc atggcggccg 17160

ggagcatgcg acgtcgggcc caattcgccc tatagtgagt cgtattacaa ttcactggcc 17220

gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca 17280

gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc 17340

caacagttgc gcagcctgaa tggcgaatgg acgcgccctg tagcggcgca ttaagcgcgg 17400

cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc 17460

ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa 17520

atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac 17580

ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt 17640

tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca 17700

accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt 17760

taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta 17820

caatttcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatcag 17880

gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 17940

caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 18000

ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 18060

gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 18120

tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 18180

ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 18240

tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 18300

atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 18360

gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 18420

caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 18480

ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 18540

ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 18600

ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 18660

ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 18720

gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 18780

ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 18840

taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 18900

agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 18960

atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 19020

aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 19080

caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 19140

ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc 19200

cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 19260

tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 19320

gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 19380

ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 19440

gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 19500

caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 19560

ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 19620

tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 19680

ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 19740

agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 19800

aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 19860

gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 19920

tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 19980

tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 20040

ccaagctatt taggtgacac tatagaatac tcaagctatg catccaacgc gttgggagct 20100

ctcccatatg gtcgacctgc aggc 20124

Claims (8)

1. a kind of method for preparing growth factor of human nerve using transgene pig salivary gland, it is characterised in that: this method include with Lower step:

A, the transposon vector containing source of people ngf gene is constructed；

B, turn the preparation of human nerve growth factor gene pig；

C, the measurement to hNGF protein concentration in hNGF gene pig saliva is turned；

D, the hNGF gene copy number for turning hNGF gene pig is analyzed；

E, hNGF albumen is extracted and purified from transgene pig.

2. a kind of method for preparing growth factor of human nerve using transgene pig salivary gland according to claim 1, special Sign is: the method for transposon vector of the building described in step a containing source of people ngf gene includes the following steps, utilizes PCR The mouse parotid secretion protein gene promoter sequence of 12.2kb long is amplified from mouse gene group DNA；By 726bp's long The signal peptide sequence of 54bp long is replaced with the pig PSP signal peptide sequence of 60bp long in hNGF coded sequence, by 732bp long after recombination HNGF coded sequence it is artificial synthesized；And the artificial synthesized terminal repeat of piggyBac 5 ' and 3 ', and include bGH One fragment sequence of polyA sequence, CMV promoter sequence, Neo-2A-EGFP fusion gene sequence and bGH polyA sequence； Replacement is located at pGEM-T and carries after these artificial synthesized sequences are connect with recombination hNGF coded sequence and mouse PSP promoter sequence The pmPSP-hNGF that sequence between XhoI the and Not I restriction enzyme site of body just generates 20.14kb long contains source of people ngf gene Transposon vector.

3. a kind of method for preparing growth factor of human nerve using transgene pig salivary gland according to claim 1, special Sign is: the preparation method for turning human nerve growth factor gene pig described in step b includes the following steps, by 10 μ g's Cotransfection enters about 1 × 10 after pmPSP-hNGF plasmid is mixed with the pmPB plasmid of 3 μ g⁶In a porcine fetus fibroblasts, uniformly It being layered in culture dish and cultivates, density is that average 1~2 cell is advisable in the visual field, appropriate G418 screening is added in incubation, to When cell regimental commander is to diameter about 5mm, the cell mass for thering is green fluorescence to express with cell clone ring picking, then individually culture amplification It is frozen after cell concentration for use, the cell clone sifted out group is subjected to somatic cell clone as nuclear donor, obtains transgene clone pig.

4. a kind of method for preparing growth factor of human nerve using transgene pig salivary gland according to claim 1, special Sign is: the measurement for turning hNGF protein concentration in hNGF gene pig saliva described in step c includes the following steps, respectively in original Generation turn hNGF gene cloning pig be born 12 days within, 2 monthly ages, six-month-old cotton balls or cotton swab acquisition transgene pig saliva, and It is put into centrifugation removal impurity in centrifuge tube, and appropriate extension rate, using hNGF-β gene ELISA kit, to acquisition The hNGF concentration turned in hNGF gene pig saliva be measured.

5. a kind of method for preparing growth factor of human nerve using transgene pig salivary gland according to claim 1, special Sign is: the method for turning the hNGF gene copy number analysis of hNGF gene pig described in step d includes the following steps, selects two The high frequency restriction enzyme site of suitable pig genome, by being carried out to the primary DNA for turning hNGF gene cloning pig of birth The copy number of Southern blot analysis hNGF gene integration.

6. a kind of method for preparing growth factor of human nerve using transgene pig salivary gland according to claim 1, special Sign is: the method extracted in slave transgene pig described in step e and purify hNGF albumen includes the following steps, using operation Method collects the saliva of transgene pig, the hNGF protein purification for being 13.5kD by molecular size range using molecular exclusion chromatography Out.

7. a kind of method for preparing growth factor of human nerve using transgene pig salivary gland according to claim 5, special Sign is: the high frequency restriction enzyme site is StuI and EcoRV.

8. a kind of method for preparing growth factor of human nerve using transgene pig salivary gland according to claim 6, special Sign is: the operation method is parotid gland intubation.