Cloned cell strain for determining biological activity of teriparatide
阅读说明：本技术 一种用于测定特立帕肽生物学活性的克隆化细胞株 (Cloned cell strain for determining biological activity of teriparatide ) 是由 王盛 邵泓 陈钢 王灿 王自强 段徐华 郑璐侠 王鸣人 董闪闪 刘荔桢 于 2021-08-18 设计创作，主要内容包括：本发明构建了一种能够稳定表达cAMP反应元件-荧光素酶报告基因的大鼠骨肉瘤细胞株,上述细胞株在给予甲状旁腺素受体激动剂特立帕肽刺激的情况下可稳定表达荧光素酶,通过检测该细胞株所表达的荧光素酶的活性可以评估样品的生物学活性,本发明所提供的方法测定时间短、操作简单、重复性好。(The invention constructs a rat osteosarcoma cell strain capable of stably expressing cAMP response element-luciferase reporter gene, the cell strain can stably express luciferase under the condition of stimulation of a parathyroid hormone receptor agonist teriparatide, and the biological activity of a sample can be evaluated by detecting the activity of the luciferase expressed by the cell strain.)
1. A method for measuring the biological activity of teriparatide, which is characterized by comprising the following steps:
s1, recovering the cloned cell strain UMR-106-GFP-CRE-Puro, carrying out passage and inoculating to a 96-hole culture plate, and culturing overnight;
s2, preparing a teriparatide standard solution and a sample solution to be detected, respectively adding the teriparatide standard solution and the sample solution to be detected into the 96-hole culture plate, and culturing for a certain time;
s3, uniformly mixing a chemiluminescent substrate and a substrate buffer solution containing cell lysate, adding the mixture into the 96-hole culture plate, and oscillating at room temperature;
s4, performing four-parameter logistic regression by using the logarithm of the concentration of the teriparatide standard solution and the corresponding chemical light intensity value, and fitting a standard curve, thereby evaluating the biological activity of the teriparatide in the sample solution to be detected.
2. The assay of claim 1, wherein said cloned cell line UMR-106-GFP-CRE-Puro is seeded into said 96-well culture plate at 100 μ L/well.
3. The method according to claim 2, wherein the cell concentration of the cloned cell line UMR-106-GFP-CRE-Puro is 5X 105one/mL.
4. The assay method according to claim 1, wherein the teriparatide standard solution or the sample solution to be tested is added to the 96-well culture plate at 100. mu.L/well.
5. The method according to claim 1, wherein the incubation time in step S2 is 1 to 5 hours.
6. A cloned cell line used in the assay method according to any one of claims 1 to 5, wherein the cloned cell line is named UMR-106-GFP-CRE-Puro with the preservation number of CGMCC22358 and the preservation unit is the China general microbiological culture Collection center.
7. A method for constructing the cloned cell line of claim 6, comprising the steps of:
s1-1, adopting lentivirus transfection technology to transfect pLVX-GFP-CRE-Puro plasmid into rat osteosarcoma UMR-106 cells;
s1-2, adding puromycin for pressurized screening, enriching positive cells and carrying out flow-parallel sorting to obtain the cloned cell strain UMR-106-GFP-CRE-Puro.
8. The pLVX-GFP-CRE-Puro plasmid used for the construction method of claim 7, wherein the sequence of the pLVX-GFP-CRE-Puro plasmid is shown as SEQ ID NO. 1.
9. A method of constructing the pLVX-GFP-CRE-Puro plasmid of claim 8, comprising the steps of:
s1-1-1, replacing NF-kB-RE sequence on pNL3.2. NF-kB-RE plasmid with CRE sequence by adopting molecular cloning technology to obtain intermediate plasmid;
s1-1-2, inserting a green fluorescent protein TurboGFP sequence into a lentiviral vector pLVX-Puro to obtain a pLVX-GFP-Puro vector;
s1-1-3, and inserting the CRE sequence and the luciferase gene sequence in the intermediate plasmid into the pLVX-GFP-Puro vector to obtain the pLVX-GFP-CRE-Puro plasmid.
Technical Field
The invention relates to the technical field of biochemical detection, in particular to a cloned cell strain for determining the biological activity of teriparatide.
Background
Parathyroid hormone receptor (PTHR) belongs to a G protein-coupled receptor that activates adenylate cyclase/cAMP signaling pathway, highly expressed in bone cells and kidney cells. Parathyroid hormone (PTH) is an endogenous agonist thereof, and is an endocrine hormone that regulates the levels of calcium and inorganic phosphate in blood by acting on bones and kidneys after activating the receptor, promoting bone formation. Teriparatide (PTH (1-34)) is a synthetic polypeptide hormone, the 1-34 amino acid fragment of human parathyroid hormone PTH. It has the same biological activity as intact endogenous parathyroid hormone PTH containing 84 amino acids, and can increase the calcium concentration in serum by activating parathyroid hormone receptor, and the bone can partially increase the calcium absorption accompanying the increase of calcium concentration, and activate osteoblast, thereby stimulating bone formation and bone absorption, and can be used for treating osteoporosis with high fracture risk in men and women at menopause. At present, the in vitro biological activity of parathyroid hormone receptor agonist teriparatide is determined by using cell factor determination method, enzyme-linked immunosorbent ELISA kit, and cAMP detection by antigen and antibody specificity combination for quantitative analysis. The method has the defects of complicated operation, unstable result and the like, and a new detection method is needed to overcome the defects, wherein the transcription factor reporter gene is a stable and effective detection technology.
The transcription factor reporter gene method is to express a DNA binding region of a specific signal transcription factor, generally a promoter region, in an effector cell and insert luciferase (luciferase) at the downstream, wherein the activation of an upstream receptor stimulates the expression of the luciferase, and the expression amount is in direct proportion to the action intensity of the transcription factor. Adding a specific substrate, reacting the luciferase with the substrate to generate fluorescence, and detecting the intensity of the fluorescence to quantify the activity of the luciferase, so as to judge the relative amount of transcription factors in cells and indirectly judge the relative biological activity of the combination of the ligand and the receptor.
In the case of teriparatide, the effector cells were selected from rat osteosarcoma UMR-106 cells that highly express PTH receptor, which respond well to PTH without the need for exogenous introduction into the receptor. Since PTH receptor activation generates cAMP second messenger, DNA binding domain we select a CRE (cAMP response element) response element. The general strategy is to stably express CRE-luciferase element plasmid in UMR-106 cells by adopting a lentivirus infection mode and screen cloning stable transformants.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a cloned cell line for measuring the biological activity of teriparatide.
In order to achieve the purpose, the invention adopts the technical scheme that:
the first aspect of the invention provides a method for measuring the biological activity of teriparatide, which comprises the following steps:
s1, recovering the cloned cell strain UMR-106-GFP-CRE-Puro, carrying out passage and inoculating to a 96-hole culture plate, and culturing overnight;
s2, preparing a teriparatide standard solution and a sample solution to be detected, respectively adding the teriparatide standard solution and the sample solution to be detected into the 96-hole culture plate, and culturing for a certain time;
s3, uniformly mixing a chemiluminescent substrate and a substrate buffer solution containing cell lysate, adding the mixture into the 96-hole culture plate, and oscillating at room temperature;
s4, performing four-parameter logistic regression by using the logarithm of the concentration of the teriparatide standard solution and the corresponding chemical light intensity value, and fitting a standard curve, thereby evaluating the biological activity of the teriparatide in the sample solution to be detected.
Preferably, the cloned cell line UMR-106-GFP-CRE-Puro is seeded into the 96-well culture plate at 100. mu.L/well.
Preferably, the cell concentration of the cloned cell strain UMR-106-GFP-CRE-Puro is 5X 105one/mL.
Preferably, the teriparatide standard solution or the sample solution to be tested is added to the 96-well culture plate at 100. mu.L/well.
Preferably, the culturing time in step S2 is 1-5 hours.
The second aspect of the invention provides a cloned cell strain used for the determination method, which is characterized in that the cloned cell strain is named as UMR-106-GFP-CRE-Puro, the preservation number is CGMCC22358, and the preservation unit is China general microbiological culture Collection center.
A third aspect of the present invention provides a method for constructing a cloned cell line as described above, comprising the steps of:
s1-1, adopting lentivirus transfection technology to transfect pLVX-GFP-CRE-Puro plasmid into rat osteosarcoma UMR-106 cells;
s1-2, adding puromycin for pressurized screening, enriching positive cells and carrying out flow-parallel sorting to obtain the cloned cell strain UMR-106-GFP-CRE-Puro.
The fourth aspect of the invention provides a pLVX-GFP-CRE-Puro plasmid used for the construction method, which is characterized in that the sequence of the pLVX-GFP-CRE-Puro plasmid is shown as SEQ ID NO. 1.
The fifth aspect of the invention provides a method for constructing the pLVX-GFP-CRE-Puro plasmid, which is characterized by comprising the following steps:
s1-1-1, replacing NF-kB-RE sequence on pNL3.2. NF-kB-RE plasmid with CRE sequence by adopting molecular cloning technology to obtain intermediate plasmid;
s1-1-2, inserting a green fluorescent protein TurboGFP sequence into a lentiviral vector pLVX-Puro to obtain a pLVX-GFP-Puro vector;
s1-1-3, and inserting the CRE sequence and the luciferase gene sequence in the intermediate plasmid into the pLVX-GFP-Puro vector to obtain the pLVX-GFP-CRE-Puro plasmid.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
(1) compared to the cytokine ELISA assay: the detection mode is simple, the repeatability is good, and the stability of the detection result is ensured;
(2) the invention takes the activation condition of a cAMP channel as a research object, designs a cell strain capable of stably integrating a cAMP reaction element-luciferase reporter gene, which is closer to the actual condition of the biological activity action of a sample in vivo and has more accurate determination result;
in conclusion, the UMR-106 cell strain capable of stably integrating the cAMP response element-luciferase reporter gene constructed by the invention can stably express luciferase under the stimulation of teriparatide; the invention establishes a simpler and faster method for measuring the in vitro biological activity of teriparatide by using the cell strain, is beneficial to the quality control and clinical application of biologically active medicaments, and has higher application value.
Drawings
The cloned cell strain UMR-106-GFP-CRE-Puro used in the invention is rat osteosarcoma cell UMR-106 which carries a luciferase gene regulated by cAMP and can stably express luciferase and is cultured in vitro, the cell strain is preserved, the preservation number is CGMCC22358, the preservation date is 2021 year, 7 months and 7 days, the preservation unit is the China general microbiological culture Collection center, and the preservation unit address is the institute of microbiology of China academy of sciences No. 3 of North Chen West Lu No.1 of North Yang, in Beijing.
FIG. 1 shows the results of the identification of luciferase activities of different cloned cell lines UMR-106-GFP-CRE-Puro in example 1 of the present invention; wherein, No.1-No.10 are numbers of different clone cells; PTH (1-34) is a teriparatide sample stimulant; forskolin (Fosk) is a PTH receptor downstream adenylate cyclase agonist as a positive control;
FIG. 2 shows the results of the identification of luciferase activities stimulated by clone No. 3 at different cell densities on teriparatide samples at different concentrations;
FIG. 3 shows the results of identifying the luciferase activity stimulated by clone No. 3 with different generations on teriparatide samples with different concentrations;
FIG. 4 shows the results of the identification of luciferase activities stimulated by clone No. 3 with respect to teriparatide samples and standards at different concentrations.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
The invention is further described with reference to the following drawings and specific examples, which are not intended to be limiting.
Example 1: construction of cloned cell line UMR-106-GFP-CRE-Puro
(1) Determination of the optimal selection concentration of puromycin: inoculating rat osteosarcoma cell UMR-106 into a 24-hole cell culture plate, adding puromycin (the concentration range is 0.1 mu g/mL-10 mu g/mL) with different screening concentrations, culturing for 2-3 days, observing the growth form of cells, and determining the optimal screening concentration of puromycin to be 2 mu g/mL;
(2) constructing a plasmid: replacing NF-kB-RE elements on pNL3.2. NF-kB-RE [ NlucP NF-kB-RE Hygro ] plasmids with cAMP-RE (namely CRE) elements, and then inserting TurboGFP elements and CRE-NlucP elements into pLVX-Puro vectors to obtain pLVX-GFP-CRE-Puro plasmids;
(3) collecting lentiviruses: HEK-293T cell plates overnight; transfecting pLVX-GFP-CRE-Puro plasmids, pCMV-dR8.2 dvpr plasmids and pCMV-VSV-G plasmids into HEK-293T cells by using a Lipofectamine 3000 reagent, placing the HEK-293T cells in a carbon dioxide incubator for culturing for 6 hours, changing the liquid, continuously culturing for 48 hours, collecting the supernatant, filtering and concentrating to obtain the high-titer lentivirus;
(4) cells were transfected and expanded: UMR-106 cell plates overnight; adding lentiviruses with different titers and polybrene serving as an auxiliary infection reagent, placing the mixture in a carbon dioxide incubator for culturing for 24 hours, changing the culture solution, continuing culturing for 48 hours, adding puromycin for screening for 2-3 days, sorting drug-resistant clones by a flow type GFP signal, and performing enlarged culture;
(5) identification of cloned cell luciferase activity: taking the drug-resistant clone cells to be planted on a 96-hole culture plate one day before detection, and culturing the cells in a carbon dioxide incubator overnight; on the detection day, the culture supernatant is removed by suction, teriparatide samples are added into the plate by serum-free medium gradient dilution, the concentrations are respectively 0nM, 0.01nM, 0.1nM, 1nM, 10nM, 100nM and 1000nM, meanwhile, 10 mu M Fosk positive control is added, the culture is carried out for 3 hours in a carbon dioxide incubator, and a luminescent substrate (Promega, Nano-Glo) is added for incubation for 5min for luminescence;
the calculation method comprises the following steps: the negative control group at 0 concentration was normalized to about 1, and other relative activities were also normalized; relative luminous intensity is experimental group luminous intensity/control group luminous intensity; as a result, as shown in FIG. 1, 10 cloned cells each had significant luciferase activity.
Example 2: verification of the activity and stability of the No. 3 cloned cell strain UMR-106-GFP-CRE-Puro
(I) Verifying cell density and reporter gene signal detection:
(1) cell subculture and plating: subculturing the stable cell strain in a 5% carbon dioxide incubator at 37 ℃ by using a complete culture solution, changing the culture solution once every 3 days, collecting the stable cell strain into a sterile centrifuge tube, centrifuging the stable cell strain for 2 minutes at 1000rpm, removing supernatant, re-suspending cell precipitates by using a fresh complete culture solution, transferring the cell precipitates into a cell culture bottle, and continuing culturing; plating P2 secondary cells with good growth state, centrifuging at 1000rpm for 2min, discarding supernatant, adding maintenance culture solution to resuspend cells, and adjusting cell concentration to 2 × 1055X 10 units/mL5one/mL and 1X 106Adding the cells/mL into a 96-well culture plate at a rate of 100 mu L/well, and culturing overnight;
(2) sample adding: the culture supernatant was aspirated off, and teriparatide samples were added to the plate at concentrations of 0nM, 0.01nM, 0.03nM, 0.1nM, 0.3nM, 1nM, 3nM, 10nM, 30nM, 100nM, 300nM, and 1000nM, respectively, at a gradient dilution in serum-free medium; each 100. mu.L of the sample solution was added to a 96-well plate with different cell densities and cultured at 37 ℃ under 5% carbon dioxide for 3 hours.
(3) Color rendering and results calculation: sucking 50 mu L of supernatant per hole, adding 50 mu L of Nano-Glo luminescent substrate, shaking for 5 minutes at room temperature, and reading on an enzyme-linked immunosorbent assay (ELISA) instrument; performing four-parameter logistic regression on the corresponding chemical light intensity (RFU) value according to the logarithm of the concentration of the sample solution, and fitting a standard curve; 5X 104The relative luciferase activity of a negative control group with the cell density of one/mL is normalized to be about 1, and other relative activities are normalized; the results are shown in FIG. 2, which shows a good positive correlation between cell concentration and RFU value.
(II) verifying cell generation and reporter gene signal detection:
(1) cell subculture and plating: subculturing the stably transformed cell strain in a 5 percent carbon dioxide incubator at 37 ℃ by using a complete culture solution, subculturing once every 3 days, collecting the stably transformed cell strain into a sterile centrifuge tube, centrifuging for 2 minutes at 1000rpm, discarding supernatant, re-suspending cell precipitates by using a fresh complete culture solution, transferring the cell precipitates into a cell culture bottle, and continuously culturing; plating P2, P6 and P10 secondary cells with good growth state, centrifuging at 1000rpm for 2min, discarding supernatant, adding maintenance culture solution to resuspend cells, and adjusting cell concentration to 5 × 105one/mL, 100. mu.L/well into 96-well plates, and incubated overnight.
(2) Sample adding: the culture supernatant was aspirated off, and teriparatide samples were added to the plate at concentrations of 0nM, 0.01nM, 0.03nM, 0.1nM, 0.3nM, 1nM, 3nM, 10nM, 30nM, 100nM, 300nM, and 1000nM, respectively, at a gradient dilution in serum-free medium; each 100. mu.L of the sample solution was added to a 96-well plate with different cell densities and cultured at 37 ℃ under 5% carbon dioxide for 3 hours.
(3) Color rendering and results calculation: sucking 50 mu L of supernatant per hole, adding 50 mu L of Nano-Glo luminescent substrate, shaking for 5 minutes at room temperature, and reading on an enzyme-linked immunosorbent assay (ELISA) instrument; performing four-parameter logistic regression on the corresponding chemical light intensity (RFU) value according to the logarithm of the concentration of the sample solution, and fitting a standard curve; the P2 generation cell negative control group is normalized to the luciferase activity by about 1, and other relative activities are also normalized; the results are shown in FIG. 3, and the RFU values of different cell generations are relatively stable.
Example 3
This example provides a method for determining the biological activity of teriparatide, comprising the steps of:
culturing cells with complete culture solution (taking 10mL of heat inactivated fetal bovine serum FBS, adding 90mL of DMEM culture solution, 10 mu L of 20mg/mL puromycin, mixing uniformly, storing at 4 ℃) at 37 ℃ under 5% carbon dioxide, and taking cells in good growth state for test; under aseptic conditions, cells were trypsinized, the complete medium neutralized and resuspended cells, centrifuged at 1000rpm for 2 minutes, the supernatant discarded, resuspended with complete medium and adjusted to 5X 10 cell concentration5Inoculating 100 mu L of the strain/mL into a 96-well plate, and culturing overnight; the supernatant was aspirated the next day, and DMEM diluted standard solutions and sample solutions of different concentrations were added to each well at 100. mu.L, 37 ℃ and 5% CO2Culturing for 3 hours under the condition; sucking 50 mu L of supernatant, and adding 50 mu L of luminescent substrate; performing four-parameter logistic regression on the corresponding chemical light intensity (RFU) value by using the logarithm of the concentration of the standard solution, and fitting a standard curve; the relative luciferase activity of the cell negative control group of the standard substance group is standardized to be about 1, and other relative activities are also standardized; the results are shown in FIG. 4, where the sample has a dose-response curve similar to that of the standard, sample EC50Standard EC50Is 101.4 percent and meets the requirement of model parameters.
In conclusion, the rat osteosarcoma cell strain capable of stably expressing the cAMP response element-luciferase reporter gene is constructed, the cell strain can stably express luciferase under the condition of being stimulated by a parathyroid hormone receptor agonist teriparatide, and the biological activity of a sample can be evaluated by detecting the activity of the luciferase expressed by the cell strain.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.
Sequence listing
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<120> a cloned cell line for determining the biological activity of teriparatide
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gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa 4680
tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat 4740
aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg 4800
gaggtttttt aaagcaagta aaacctctac aaatgtggta ggatcccgcg actctagata 4860
attctaccgg gtaggggagg cgcttttccc aaggcagtct ggagcatgcg ctttagcagc 4920
cccgctgggc acttggcgct acacaagtgg cctctggcct cgcacacatt ccacatccac 4980
cggtaggcgc caaccggctc cgttctttgg tggccccttc gcgccacctt ctactcctcc 5040
cctagtcagg aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga 5100
agtagcacgt ctcactagtc tcgtgcagat ggacagcacc gctgagcaat ggaagcgggt 5160
aggcctttgg ggcagcggcc aatagcagct ttgctccttc gctttctggg ctcagaggct 5220
gggaaggggt gggtccgggg gcgggctcag gggcgggctc aggggcgggg cgggcgcccg 5280
aaggtcctcc ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt 5340
ctcctcttcc tcatctccgg gcctttcgac ctgcagccca agcttaccat gaccgagtac 5400
aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc 5460
gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag 5520
cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg 5580
tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg 5640
ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc 5700
gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc 5760
ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg 5820
ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg 5880
ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg 5940
cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgacc gcgtctggaa 6000
caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc 6060
tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg 6120
tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt 6180
gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac 6240
tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc 6300
tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct 6360
gttgggcact gacaattccg tggtgttgtc ggggaagctg acgtcctttc catggctgct 6420
cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct 6480
caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct 6540
tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc ctggaattaa 6600
ttctgcagtc gagacctaga aaaacatgga gcaatcacaa gtagcaatac agcagctacc 6660
aatgctgatt gtgcctggct agaagcacaa gaggaggagg aggtgggttt tccagtcaca 6720
cctcaggtac ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta 6780
aaagaaaaga ggggactgga agggctaatt cactcccaac gaagacaaga tatccttgat 6840
ctgtggatct accacacaca aggctacttc cctgattagc agaactacac accagggcca 6900
ggggtcagat atccactgac ctttggatgg tgctacaagc tagtaccagt tgagccagat 6960
aaggtagaag aggccaataa aggagagaac accagcttgt tacaccctgt gagcctgcat 7020
gggatggatg acccggagag agaagtgtta gagtggaggt ttgacagccg cctagcattt 7080
catcacgtgg cccgagagct gcatccggag tacttcaaga actgctgata tcgagcttgc 7140
tacaagggac tttccgctgg ggactttcca gggaggcgtg gcctgggcgg gactggggag 7200
tggcgagccc tcagatcctg catataagca gctgcttttt gcctgtactg ggtctctctg 7260
gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc 7320
tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 7380
taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtagtagttc 7440
atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag 7500
gccttgacat tgctagcgtt ttaccgtcga cctctagcta gagcttggcg taatcatggt 7560
catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg 7620
gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt 7680
tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg 7740
gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg 7800
actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 7860
tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 7920
aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 7980
ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 8040
aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 8100
cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 8160
cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 8220
aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 8280
cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 8340
ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 8400
gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 8460
gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 8520
agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 8580
acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 8640
tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg 8700
agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct 8760
gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg 8820
agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc 8880
cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa 8940
ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc 9000
cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt 9060
cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc 9120
ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt 9180
tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc 9240
catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt 9300
gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata 9360
gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 9420
tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag 9480
catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa 9540
aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt 9600
attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 9660
aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg 9720
gatcgggaga tcaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc 9780
acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc 9840
atcaatgtat cttatcatgt ctggatcaac tggataactc aagctaacca aaatcatccc 9900
aaacttccca ccccataccc tattaccact gccaattacc tgtggtttca tttactctaa 9960
acctgtgatt cctctgaatt attttcattt taaagaaatt gtatttgtta aatatgtact 10020
acaaacttag tagtttttaa agaaattgta tttgttaaat atgtactaca aacttagtag 10080
t 10081