CN107287236B - Construction method of mouse model for human acute pancreatitis - Google Patents

Abstract

The invention discloses a method for constructing a mouse model of human acute pancreatitis. Constructing a targeting vector plasmid containing PRSS1 gene, and electrically transferring the targeting vector plasmid containing PRSS1 gene into mouse embryonic stem cells to obtain the mouse embryonic stem cells carrying recombinant PRSS1 gene; microinjecting the mouse embryonic stem cells carrying the recombinant PRSSS1 gene into albino mouse blastocyst, transferring the injected blastocyst into a pseudopregnant female mouse uterus for future birth, and obtaining a chimeric mouse after transplantation; the male chimera mouse with the chimera rate of more than 50% is combined with wild C57BL/6J female mouse to breed the next generation mouse, and the mouse carrying PRSS1 gene is selected. The phenotype of the model mouse is very consistent with the pathogenesis process of human pancreatitis, so that a specific animal model which completely imitates human pancreatitis from gene to phenotype is obtained, and the model can be further widely used for researches on pathogenic causes, pathological mechanisms and the like of human PRSS1, and screening of pancreatitis treatment medicines, gene treatment experiments and the like.

Description

Construction method of mouse model for human acute pancreatitis

The technical field is as follows:

the invention belongs to the field of animal model construction, and particularly relates to a construction method of a mouse model for human acute pancreatitis.

Background art:

acute Pancreatitis (AP) is a clinically common and potentially fatal risk of acute abdomen. Despite the great progress made in the diagnosis and treatment of AP, the mortality rate is still around 10%, and the rate of severe cases reaches 50%. The most important reason is that the specific mechanism of initialization of AP is not fully understood, and there is no way to guide the development of new, more effective drugs for the treatment of pancreatitis. Over the last 20 years, a number of studies based on experimental animal models of pancreatitis have found that premature activation of trypsinogen (trypsingen) within acinar cells plays a critical role in the process of AP initialization. However, the specific mechanism of trypsingen activation and its role in the development of pancreatitis have not been clearly explained. The important reason for the lack of research delay in the field of pancreatitis at present is that: there is a lack of ideal experimental animal models to perform in vivo experiments.

At present, several animal models of experimental acute pancreatitis are widely used, including methods of ranophanin injection, pancreatic duct ligation, bile salt pancreatic duct retrograde perfusion, ethylene thiochloric acid diet, arginine injection, and the like. These animal models have largely deepened our understanding of the mechanism of acinar injury in the early stage of acute pancreatitis. However, these animal models often produce non-specific manifestations in the course of inducing pancreatitis, rather than specific activation for trypsinogen. And over 70 clinical trials over the past 40 years have found no clear experimental evidence: inhibition of Trypsin (produced by trypsinen activation) activation may improve the pathogenesis of pancreatitis. The greatest possibility of inconsistency between clinical and animal experimental results is that the existing animal models are not suitable for researching the action of activation of the endoplasminogen in the initial pathogenesis stage of pancreatitis and detecting the action of potential drugs on treatment and protection of pancreatitis. Therefore, the novel animal model needs to be constructed to research the action of trypsin in the initial process of pancreatitis, and transgenic animals bring possibility for basic research and development of new drugs.

Saluja et al established a transgenic mouse in which a trypsingen heterobody expression gene (a mouse gene) was knocked out by a gene knock-out method, and conducted preliminary studies. Unlike the traditional view, they consider: activation of trypsinogen within the acinus results in necrosis of the AP early acinus, and these lesions account for 50% of the AP lesions. However, local and systemic inflammatory responses of AP, as well as the progression of chronic pancreatitis, do not require activation of trypsingen.

N et al generated transgenic mice expressing the human wild-type PRSS1 gene and they considered: expression of the PRSS1 gene promotes apoptosis of mouse acinar cells and can contribute to the development of spontaneous pancreatitis. The two experimental conclusions described above are partly contradictory, and the reason for this analysis is that the transgenic mice used have defects: the promoters used for expressing the target genes are all elastase of rat origin, but not elastase of human origin, so the credibility of the experimental results is basically questioned. In addition to the targeted expression of the human cationic trypsinogen gene in pancreatic acinar cells, the ideal AP transgenic animal model must use human elastase as a promoter.

The invention content is as follows:

the invention aims to provide a method for constructing a human acute pancreatitis mouse model, the human acute pancreatitis mouse model constructed by the method accords with the positioning expression of human cationic trypsinogen genes in pancreatic acinar cells, simultaneously human elastase is used as a promoter, and the phenotype of the human cationic trypsinogen gene is very similar to the pathogenesis process of human acute pancreatitis, so the human acute pancreatitis mouse model is a very good disease model of human acute pancreatitis.

The method for constructing the mouse model of the human acute pancreatitis is characterized by comprising the following steps of:

constructing a targeting vector plasmid containing PRSS1 gene (human cationic trypsin protogene), and electrically transferring the targeting vector plasmid containing PRSS1 gene into mouse embryonic stem cells to obtain the mouse embryonic stem cells carrying the recombinant PRSS1 gene; microinjecting the mouse embryonic stem cells carrying the recombinant PRSSS1 gene into albino mouse blastocyst, transferring the injected blastocyst into a pseudopregnant female mouse uterus for future birth, and obtaining a chimeric mouse after transplantation; the male chimera mouse with the chimerism rate of more than 50 percent and a wild C57BL/6J female mouse are combined into a cage, a next generation mouse is generated through propagation, and a mouse carrying PRSS1 gene is screened, namely the mouse model simulating the human acute pancreatitis.

The nucleotide sequence of the targeting vector plasmid containing PRSS1 gene (human cationic trypsin gene) is preferably shown in SEQ ID NO. 1.

Furthermore, the construction method of the targeting vector plasmid containing PRSS1 gene (human cationic trypsin gene, GenBank Accession Number: NM-002769.4) is as follows:

using PRSS1-BAC plasmid as a template, carrying out PCR amplification under the action of primers PCR1F and PCR1R, PCR2F and PCR2R, and PCR3F and PCR3R to respectively obtain an amplification product PRSS1-PCR1, an amplification product PRSS1-PCR2 and an amplification product PRSS1-PCR 3; PRSS1-PCR1 and a vector PL253 are connected to form a plasmid PRSS1-PCR1-PL253, an amplification product PRSS1-PCR2 and a plasmid PRSS1-PCR1-PL253 are connected after double enzyme digestion to form a plasmid PRSS1-PCR1-PCR2-PL253, and then the amplification product PRSS1-PCR3 and a plasmid PRSS1-PCR1-PCR2-PL253 are connected after double enzyme digestion to form a plasmid PRSS1-PCR1-PCR2-PCR3-PL 253.

The method for constructing the mouse embryonic stem cell carrying the recombinant PRSS1 gene comprises the following steps:

(1) treating the targeting vector plasmid containing PRSS1 gene with NotI to linearize it and regulate the DNA concentration of the plasmid vector to about 1-5. mu.g/. mu.l;

(2) preparation of fertilized eggs: female C57BL/6J mice were screened for 3-4 weeks and injected with pregnant horse serum (PMSG) and chorionic gonadotropin (hcg) separately, with a time interval of 46-48 h. After HCG injection, female mice were mated with adult fertile adult male mice to fertilize the female mice. After euthanizing the female rat the next day, collecting fertilized eggs from the oviduct, and culturing in an incubator for later use.

(3) Prokaryotic microinjection: microinjection injection needles and fixing needles were prepared. The linearized and purified targeting vector plasmid containing PRSS1 gene was diluted to 1-5ng/ul injection solution, which was loaded into a microinjection needle. And screening fertilized eggs with normal shapes, and placing the fertilized eggs in an injection dish for microinjection. Under an inverted microscope of 200-fold and 400-fold, an exogenous DNA solution (injection) is injected into the nucleus of a fertilized egg cell by means of microinjection. Transferring the injected fertilized eggs into an incubator, and transplanting after culturing for 1 h; or cultured to 2 cells for transplantation the next day.

(4) Preparing a surrogate mouse and transplanting an embryo:

preparing a pseudopregnant mother mouse: and (3) mating fertile male mice with proper age with the sterilized male mice after the spermaduct ligation, and stimulating female mice to generate a series of pregnancy changes to obtain pseudopregnant female mice which are used as surrogate pregnant mice after the fertilized eggs are transgenic. The fertilized egg injected with the exogenous gene is transplanted into the egg duct of the surrogate mother mouse on the day of the insertion. And (4) putting the surrogate mother mouse into a clean cage box, preserving heat, and putting the surrogate mother mouse back to the cage for breeding after the surrogate mother mouse is clear. After successful oviduct transplantation, the female mouse will normally farrow 19-20 days after the operation. After the mouse is born for 1 week, the mouse can be subjected to paw shearing numbering and PCR identification; mice were housed independently 3 weeks after birth in cages.

(5) Identification of born F0 mice: collecting 1-2 weeks old young mouse tissue (tail or toe tissue), lysing the tissue and extracting genome. And carrying out PCR amplification and electrophoresis detection by using a specific primer aiming at the target gene, and screening out the offspring integrated with the exogenous gene. The mouse with integration is called the first building mouse (fountain), can be passaged and established, and needs to be identified by the protein expression level.

(6) Passage and establishment of transgenic mouse, mating and passage of mouse with exogenous gene and mouse without transgene; each first-established mouse needs to be passaged independently. The F1 mouse was born and was identified as F0 that was normally germline transmitted, with a 50% probability of the progeny carrying the gene of interest integrated. The resulting F1 positive mice can be used for experiments and continued passaging. If homozygotes are to be obtained, the positive F1, which may be of the same origin, is mated siblings, and the F2 mice are born with a 25% probability of being homozygotes. And (4) establishing stable passage of the screened mice with the expressed target genes, and recording the passage condition and the pedigree. This resulted in a PRSS1 knock-in mouse, a mouse model of human acute pancreatitis.

Phenotypic identification of PRSS1 knock-in mice: the PRSS1 gene constructed by the invention can show the expression of acute pancreatitis after being knocked into a mouse (12-16 weeks) and injected with ranulin for 24 hours; chronic pancreatitis can be manifested after repeated injections of ranolanin for 4 weeks. So far, no information about the knock-in mouse of the wild type PRSS1 gene is found in domestic and foreign literature reports, patent databases and animal model catalogues including the Jackson institute of the largest mouse resource center in the world. Therefore, the mouse model can be widely used for researching pathogenic causes, pathological mechanisms and the like of the acute pancreatitis and the chronic pancreatitis of the human body.

The invention has the beneficial effects that: the invention utilizes gene targeting technology to generate and screen the first international PRSS1 gene knock-in mouse strain. The phenotype of the model mouse is very consistent with the pathogenesis process of human pancreatitis, so that a specific animal model which completely imitates human pancreatitis from gene to phenotype is obtained, and the model can be further widely used for researches on pathogenic causes, pathological mechanisms and the like of human PRSS1, and screening of pancreatitis treatment medicines, gene treatment experiments and the like.

Description of the drawings:

FIG. 1 is a schematic diagram of the construction of a targeting vector plasmid containing PRSS1 gene.

FIG. 2 is an electrophoretogram of the amplification products of primers F2/R2 and F3/R3.

FIG. 3 is an electrophoretogram of the cleavage products of the enzyme DraIII and the enzyme NruI.

FIG. 4 is an electrophoretogram of the cleavage product of the enzyme Bg 1I.

FIG. 5 shows the results of PCR identification of PRSS1 transgene in PRSS1 knock-in mice (primers F1 and R1), PRSS1 knock-in mice 10, Marker M, positive control, WT wild type mice, and water 10.

FIG. 6 shows the results of PCR identification of PRSS1 transgene in PRSS1 knock-in mice (primers F2 and R2), PRSS1 knock-in mice 10, Marker M, positive control, WT wild type, and water 10.

FIG. 7 shows the results of PCR identification of PRSS1 transgene in PRSS1 knock-in mice (primers F3 and R3), PRSS1 knock-in mice 10, Marker M, positive control, WT wild type, and water 10.

FIG. 8 shows the results of PCR identification of PRSS1 transgene in PRSS1 knock-in mice (primers F4 and R4), PRSS1 knock-in mice 10, Marker M, positive control, WT wild type, and water 10.

FIG. 9 shows the results of PCR identification of PRSS1 transgene in PRSS1 knock-in mice (primers F5 and R5), PRSS1 knock-in mice 10, Marker M, positive control, WT wild type, and water 10.

FIG. 10 shows the expression of PRSS1 protein in pancreatic tissue of mice knocked in the PRSS1 gene. In Wild Type (WT) mice, PRSS1 is hardly expressed, while the expression level of PRSS1 protein in PRSS1 knock-in mice accounts for almost 50% of that of normal people, which shows that the transgenic mice are comparable to people and can be used for simulating the pathogenesis of acute pancreatitis of people.

FIG. 11 is a photograph showing the phenotype of Acute Pancreatitis (AP) induced by injecting ranulin (cerulein) into PRSS1 knock-in mice (24 hours later) and the HE staining of pancreatic tissue. Compared with wild type mice, the PRSS1 gene knock-in mice have obvious pancreatic tissue edema with inflammatory cell infiltration after cerulein injection.

FIG. 12 is a graph showing that pancreatic amylase, tissue score, degree of tissue edema, and Trypsin activity were significantly increased 24h after the injection of ranolanin into PRSS1 knock-in mice, compared to wild-type mice. The PRSS1 can well simulate the pathogenesis of the human acute pancreatitis.

FIG. 13 shows that the activity of Microsome enzyme was significantly increased after repeated injections of ranulin (cerulein) into PRSS1 knock-in mice (after 40 min), while the activity of Zymogen and Lysome was not significantly changed. None of these changes were significantly altered in wild type mice.

FIG. 14 is a photograph showing the phenotype of Chronic Pancreatitis (CP) induced by repeated injections of ranulin (cerulein) into PRSS1 knock-in mice (after 4 weeks), and staining of HE and Mason in pancreatic tissues. PRSS1 mice can also well simulate the pathogenesis of chronic pancreatitis, i.e., acute pancreatitis with repeated attacks can be evolved into chronic pancreatitis.

FIG. 15 is a comparison of the markers associated with chronic pancreatitis in wild-type mice and PRSS1 knock-in mice. It is evident that PRSS1 mice showed significant changes in pancreatic specific gravity, tissue score, and blood glucose after the injection of ranophanin.

The specific implementation mode is as follows:

the following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Breeding and breeding of mice: c57BL/6J was purchased from Jackson laboratories (USA) and maintained in the SPF-rated animal house of the southern Hospital animal institute of southern medical university. All mouse manipulations were approved and supervised by the southern university of medical institute animal care committee.

Example 1 construction of targeting vector plasmid containing PRSS1 Gene

Firstly, constructing a vector: the schematic construction is shown in FIG. 1.

1. Constructing an intermediate vector, and constructing a target fragment into a pStart-K framework;

2. amplifying a fragment to be recombined;

3. carrying out homologous recombination on the amplified fragment and BAC;

4. screening correct clones;

5. amplifying the target fragment on the BAC and sending the amplified target fragment to a detection device;

6. the correctly sequenced BACs were subjected to cycle sequencing.

7. And verifying the correctness of the obtained carrier.

8. The carrier plasmid (namely the targeting carrier plasmid containing PRSS1 gene, the nucleotide sequence of which is shown in SEQ ID NO. 1) is transformed into escherichia coli to obtain the escherichia coli liquid transformed with the carrier plasmid.

II, plasmid preparation

1. Plate-streaking the bacterial solution, and picking out the monoclonal

Carrying out plate streaking on the escherichia coli liquid transformed with the vector plasmid in the step one, and then randomly selecting 8 clones for PCR verification

The primers used were:

F2:GGAAGACTGATTTTTGCATATCATG

R2:GGTTCTTTAATCTAACATATCCCCT

the amplification product was 314 bp.

F3:AGCACATAGAGACAAAATATGC

R3:GGTCATCAGTATATCGAACTATTCT

The amplification product is 364 bp.

If both amplification products were amplified, the clone was a positive clone, designated E.coli harboring a targeting vector plasmid containing the PRSS1 gene. The electrophoretogram of the PCR product is shown in FIG. 2.

2. Selecting one from the positive clones for shake culture

Escherichia coli carrying the plasmid containing PRSS1 gene as a targeting vector was inoculated into 100mL of LB medium supplemented with antibiotics, and cultured in a shaking incubator (Shanghai Zhichu apparatus Co., Ltd., product number: ZQLY-300S) at 37 ℃ at 225rpm/min for 16 hours.

3. Plasmid extraction

After the time for shaking the bacteria, plasmid extraction was performed with QIAGEN plasmid DNA purification kit (QIAGEN corporation, Shanghai, Inc., Cat. No.: 12145) to obtain plasmid, the specific method was as follows:

a) the overnight LB-cultured bacteria were harvested and centrifuged at 6000g at 4 ℃ for 15 min.

b) 10ml Buffer P1 was added to resuspend the bacteria.

c) Adding 10ml Buffer P2, shaking the centrifuge tube 4-6 times to mix thoroughly, and standing at room temperature for 5 min. If Lyseblue reagent is used, the solution should become uniformly blue.

d) During incubation, the seal of the needle opening is opened and the filter is placed on the appropriate rack.

e) Add 10ml of ice bath Buffer P3 to the lysate and mix it by vigorously shaking the tube 4-6 times. If Lyseblue reagent is added, mix the reagents until there is no color.

f) The lysate was poured into a filter sealed at the needle and left at room temperature for 10min without the insertion of a piston. The needle seal was opened and the piston gently squeezed to filter the lysate into a new 50ml centrifuge tube.

g) 2.5ml Buffer ER was added to the filtered lysate, mixed well 10 times and incubated on ice for 30 min.

h) A column of QIAGEN-tip 500 was hung from another centrifuge tube, 10ml of Buffer QBT was added, and the solution in the tube was emptied by gravity drip.

i) The lysate incubated on ice from step 7 was added to a QIAGEN-tip column and allowed to slowly drip through the resin.

j)10 the QIAGEN-tip column was washed with 2X 30ml Buffer QC.

k)11 DNA was eluted with 15ml Buffer QN and the DNA eluate was collected in a centrifuge tube.

l)12 Add 10.5ml of isopropanol to the eluate to precipitate the DNA and mix well. Greater than 15000 Xg, centrifuged at 4 ℃ for 30min and carefully decanted supernatant after centrifugation.

m)13 washing of DNA with 5ml endotoxin free-70% ethanol (40 ml 96% -100% ethanol added to endotoxin free water in the kit), 15000g, centrifugation at 4 ℃ for 10 min. Carefully pour out the supernatant without encountering the plasmid.

n)14 drying the plasmid at the bottom of the centrifuge tube for 5-10 min. The DNA was re-solubilized using an appropriate volume of Buffer PA.

o)15 the concentration of the DNA plasmid was determined and recorded.

4. Detection of plasmids

Carrying out enzyme digestion verification on the plasmid extracted in the step 3, wherein the enzyme digestion time is 1h

Enzyme digestion system: 20uL

DNA 500ng

Buffer 2uL

Enzyme (DraIII or NruI) 1uL

Replenishing water to 20uL

The enzyme digestion scheme is as follows:

DraIII 12.514/9.181/6.336/3.557/3.132/1.740 (Kbp)

NruI 36.460 (Kbp)

the electrophoretogram of the cleavage product is shown in FIG. 3. Thus obtaining the targeting vector plasmid containing the PRSS1 gene, and the nucleotide sequence is shown as SEQ ID NO.1 after sequencing.

5. Linearization of plasmids

The targeting vector plasmid containing the PRSS1 gene is subjected to linear enzyme digestion, and an enzyme digestion system

DNA 30ug

Enzyme (NruI) 70U

Buffer 25uL

Replenishing water to 250uL

The enzyme digestion time is 16h at 37 DEG C

Restriction enzyme cleavage scheme NruI 36.460Kbp

This gave a linearized targeting vector plasmid containing the PRSS1 gene.

6. Recovery of DNA fragment (linearized targeting vector plasmid containing PRSS1 Gene)

The linearized targeting vector plasmid reaction solution containing the PRSS1 gene of step 5 was recovered in agarose Gel using the QIAquick Gel Extraction Kit (250) with the cat No. 28706 by the following method:

a) a fragment containing DNA of about 36.460Kbp in size was carefully excised under an ultraviolet lamp. And the excess gel was removed as much as possible.

b) Weighing the empty centrifuge tube, cutting off the gel with the target fragment, loading in a 1.5ml centrifuge tube, weighing, determining the weight of the gel block, determining the volume approximately, adding 700. mu.l Buffer QG, and incubating at 50 deg.C for 10 min.

c) After 10min, the gel block is completely dissolved, and the color of the mixed solution is closely observed whether the mixed solution is yellow or not, if the mixed solution is yellow, the next operation is continued.

d) To the mixed solution was added 230. mu.l of isopropyl alcohol and mixed well.

e) QIAquick spin columns (QIAquick spin columns) were packed in a clean 2ml collection tube.

f) The resulting solution from (c) was placed in a QIAquick adsorption column and centrifuged at 13,000rpm for 1 min. The waste was discarded and the QIAquick spin column was replaced in a 2ml collection tube.

g) 0.5ml of Buffer QG was added to the column and centrifuged at 13,000rpm for 1 min.

h) The column was mounted on a clean 1.5ml centrifuge tube, 50. mu.l of eluent or sterile water column membrane was added and centrifuged at 10,000 Xg for 1min, the solution in the centrifuge tube being the purified recovered DNA product and stored at-20 ℃ for use in enzyme applications. 0.75ml of Buffer PE was added again to the spin column and centrifuged at 13,000rpm for 1 min.

i) The liquid was discarded and centrifuged again at 13,000rpm for 1 min.

j) The column was removed and placed in a clean 1.5ml microcentrifuge tube.

k) Mu.l Buffer EB (10mM Tris. Cl, pH 8.5) was added to the middle of the adsorption column, left in the greenhouse for 1min, centrifuged again at 13,000rpm for 1min, and the supernatant was collected and stored at-20 ℃ for further use. This gave a linearized targeting vector plasmid containing the PRSS1 gene.

7. Identification of recovered fragments

The above-mentioned recovered fragment (linearized targeting vector plasmid containing PRSS1 gene) was subjected to enzyme digestion and identified,

enzyme digestion system: 20uL

DNA 500ng

Buffer 2uL

Enzyme 1uL

Replenishing water to 20uL

The enzyme digestion scheme is as follows: BglI 11/10/6.4/5/3.2/0.8

The electrophoretogram of the cleavage product is shown in FIG. 4, whereby a linearized targeting vector plasmid containing PRSS1 gene was obtained.

8. Delivery downstream

After the detection is finished, the linearized targeting vector plasmid containing the PRSS1 gene is labeled and distributed to downstream for injection.

Secondly, preparation of fertilized ovum

1. Preparation of fertilized eggs: c57BL/6 female mice were screened for 3-4 weeks and injected with Pregnant Mare Serum (PMSG) and chorionic gonadotropin (hcg) separately, with a time interval of 46-48 h.

2. After HCG injection, female mice were mated with adult fertile adult male mice to fertilize the female mice.

3. After euthanizing female mice with carbon dioxide at 9 am the next day, a fertilized egg culture dish (2 ml of M2 (reagent manufacturer SIGMA) culture medium was placed in a 37-degree preheating table and preheated for half an hour) was prepared, the mouse ovaries were taken out and placed in the preheated culture dish, 60 μ l of hyaluronic acid (reagent manufacturer SIGMA) was added, the fertilized eggs were collected from the oviduct, and the fertilized eggs were taken out and placed in a (balanced 12-hour M16 (reagent manufacturer SIGMA) culture dish) C02 incubator for culture and standby.

Third, pronucleus microinjection

1. Microinjection injection needles and fixing needles were prepared.

2. The linearized and purified plasmid (linearized targeting vector plasmid containing PRSS1 gene) was diluted to 1-5ng/ul of injection solution, which was loaded into a microneedle using a sample loader.

3. Selecting normal fertilized eggs, making M2 injection drops, placing the selected fertilized eggs into the injection drops, covering with paraffin oil (SIGMA, reagent manufacturer), and placing under a microscope for injection.

4. Under an inverted microscope of 200-fold and 400-fold, an exogenous DNA solution (linearized targeting vector plasmid containing PRSS1 gene) is injected into the nucleus of a fertilized egg cell by microinjection. The gas for injection is nitrogen, and the gas pressure is adjusted to expand the nuclear region by about 2 to 3 times before injection.

5. After the injection is finished, the injection dish is moved into an incubator and is transplanted after being cultured for 1 h; or transferring the fertilized eggs to an M16 culture medium for culture to 2 cells, and transplanting the fertilized eggs on the next day.

Preparation of surrogate pregnant mouse and embryo transplantation

1. Preparing a pseudopregnant mother mouse: and (3) mating fertile male mice with proper age with the sterilized male mice after the spermaduct ligation, and stimulating female mice to generate a series of pregnancy changes to obtain pseudopregnant female mice which are used as surrogate pregnant mice after the fertilized eggs are transgenic.

2. Before transplantation, the surrogate mouse is numbed by using an anesthetic (abdominal cavity injection is carried out by testing proper anesthetic dosage every time), then a port of 1cm is opened on the right back, an alcohol cotton ball is used for wiping the opening part before opening, the experimental equipment scissors and tweezers with high temperature and high pressure sterilization are used for pulling out the oviduct and exposing the oviduct in the visual field, the fertilized ovum injected with the exogenous gene (linearized targeting vector plasmid containing PRSS1 gene) is transplanted into the oviduct, and stainless steel wound clips are used for suturing.

3. And (4) putting the surrogate mother mouse into a clean cage box, preserving heat, and putting the surrogate mother mouse back to the cage for breeding after the surrogate mother mouse is clear. The following day, the mice subjected to the test were observed for abnormalities.

4. After successful oviduct transplantation, the female mouse will normally farrow 19-20 days after the operation.

5. After the mouse is born for 1 week, the mouse can be subjected to paw shearing numbering and PCR identification; mice were housed independently 3 weeks after birth in cages.

Fifth, mouse identification

1: collected tissues (tail or toe tissues) of 1-2 weeks old pups were lysed and genomes were extracted.

2. PCR amplification and electrophoresis detection are carried out by using specific primers aiming at the target gene (PRSS1 gene), and offspring integrated by the exogenous gene are screened out.

The primers used were as follows:

transgene PCR primer F1: GAGCGGATTTGAACGTTGTG

Transgene PCR primer R1: TACTTGAAGAGATTTGGCGG

Internal control PCR primer (control primer) F: CTATCAGGGATACTCCTCTTTGCC

Internal control PCR primer R: GATACAGGAATGACAAGCTCATGGT

Annealing temp. 60 deg.C

Expected PCR product (Expected product):

transgene PCR product size 356bp

Internal control PCR product size 507bp

Transgene PCR primer F2: GGAAGACTGATTTTTGCATATCATG

Transgene PCR primer R2: GGTTCTTTAATCTAACATATCCCCT

Internal control PCR primer (control primer) F: CTATCAGGGATACTCCTCTTTGCC

Internal control PCR primer R: GATACAGGAATGACAAGCTCATGGT

Annealing temp. 60 deg.C

Expected PCR product (Expected product):

transgene PCR product size 314bp

Internal control PCR product size 507bp

Transgene PCR primer F3: AGCACATAGAGACAAAATATGC

Transgene PCR primer R3: GGTCATCAGTATATCGAACTATTCT

Internal control PCR primer (control primer) F: CTATCAGGGATACTCCTCTTTGCC

Internal control PCR primer R: GATACAGGAATGACAAGCTCATGGT

Annealing temp. 60 deg.C

Expected PCR product (Expected product):

transgene PCR product size 364bp

Internal control PCR product size 507bp

Transgene PCR primer F4: GGTGATGGCTGTGCCCAGAA

Transgene PCR primer R4: CCCCTTTCTCCCAAGCTTCT

Internal control PCR primer (control primer) F: CTATCAGGGATACTCCTCTTTGCC

Internal control PCR primer R: GATACAGGAATGACAAGCTCATGGT

Annealing temp. 60 deg.C

Expected PCR product (Expected product):

transgene PCR product size 339bp

Internal control PCR product size 507bp

Transgene PCR primer F5: TGCTGGCACCAAGTTCCTCATCTC

Transgene PCR primer R5: GCAAGAGATTACGCGCAGACCAA

Internal control PCR primer (control primer) F: GCAGAAGAGGACAGATACATTCAT

Internal control PCR primer R: CCTACTGAAGAATCTATCCCACAG

Annealing temp. 60 deg.C

Expected PCR product (Expected product):

transgene PCR product size 398bp

689bp of Internal control PCR product size

The electrophoretograms of the amplified products obtained by the five PCR primers are shown in FIGS. 5 to 9, and it can be seen from the above that the mice, which can amplify the desired fragment by the five PCR primers, are offspring integrated with PRSS1 gene. The mouse with integration is called the first building mouse (fountain), can be passaged and established, and needs to be identified by the protein expression level.

Sixth, passage and establishment of transgenic mice:

mating and passaging a mouse (a first-built mouse) with an exogenous gene (PRSS1 gene) and a mouse which is not subjected to transgenosis; each first-established mouse needs to be passaged independently. The F1 mouse was born and identified (same as step five) as F0 that was normally germline transmitted, with 50% of its progeny carrying the integrated gene of interest. The resulting F1 positive mice can be used for experiments and continued passaging. If homozygotes are to be obtained, the positive F1, which may be of the same origin, is mated siblings, and the F2 mice are born with a 25% probability of being homozygotes. And (4) establishing stable passage of the screened mice with the expressed target genes, and recording the passage condition and the pedigree. This resulted in a PRSS1 knock-in mouse, a mouse model of human acute pancreatitis.

Seventhly, phenotypic identification of PRSS1 knock-in mice:

1. detecting the expression of PRSS1 protein in mouse model tissue of human acute pancreatitis. As shown in fig. 10, PRSS1 was hardly expressed in wild type (WT, control) mice, while PRSS1 knock-in mice (Transgene) PRSS1 protein expressed in an amount of almost 50% of that of normal humans (Human), indicating that the transgenic mice are comparable to humans and can be used to simulate the onset of Human acute pancreatitis.

2. As shown in fig. 11, fig. 11 is a graph showing that PRSS1 knock-in mice induced Acute Pancreatitis (AP) phenotype (after 24 h) after injection of ranulin (cerulein) and HE staining of pancreatic tissue. Compared with wild type mice, the PRSS1 mice injected with cerulein had significant pancreatic tissue edema with inflammatory cell infiltration.

3. FIG. 12 shows that pancreatic amylase, tissue score, degree of tissue edema, and Trypsin activity were significantly increased in PRSS1 gene knock-in mice 24h after the injection of ranulin (cerulein) compared to wild-type mice in PRSS1 gene knock-in mice 24h after the injection of ranulin. The PRSS1 can well simulate the pathogenesis of the human acute pancreatitis.

4. FIG. 13 shows that the activity of Microsome enzyme was significantly increased after repeated injections of ranulin (cerulein) into PRSS1 knock-in mice (after 40 min), while the activity of Zymogen and Lysome was not significantly changed. None of these changes were significantly altered in wild type mice.

5. FIG. 14 is a photograph showing the phenotype of Chronic Pancreatitis (CP) induced by repeated injections of ranulin (cerulein) into PRSS1 knock-in mice (after 4 weeks), and staining of HE and Mason in pancreatic tissues. The PRSS1 knock-in mouse can also well simulate the pathogenesis process of chronic pancreatitis, namely, repeatedly-occurred acute pancreatitis can be evolved into chronic pancreatitis.

6. FIG. 15 comparison of wild type mice with PRSS1 mice for chronic pancreatitis-associated markers. It is evident that PRSS1 mice showed significant changes in pancreatic specific gravity, tissue score, and blood glucose after the injection of ranophanin.

In conclusion, after the PRSS1 gene constructed by the invention is knocked into a mouse (12-16 weeks) and is injected with ranulin for 24 hours, the acute pancreatitis can be expressed; chronic pancreatitis can be manifested after repeated injections of ranolanin for 4 weeks. So far, no information about the knock-in mouse of the wild type PRSS1 gene is found in domestic and foreign literature reports, patent databases and animal model catalogues including the Jackson institute of the largest mouse resource center in the world. Therefore, the mouse model can be widely used for researching pathogenic causes, pathological mechanisms and the like of the acute pancreatitis and the chronic pancreatitis of the human body.

The PRSS1 mouse obtained by the invention can be widely used for research on pathogenic causes and pathological mechanisms of human acute pancreatitis, screening of acute pancreatitis treatment drugs, gene therapy tests and the like through phenotype verification.

In addition to the above embodiments, the present invention may have other embodiments. All technical solutions formed by adopting equivalent substitutions or equivalent transformations fall within the protection scope of the claims of the present invention.

Sequence listing

<110> southern hospital of southern medical university

<120> construction method of mouse model for human acute pancreatitis

<160>1

<210>1

<211>36500

<212>DNA

<213> Artificial sequence

<400>1

cgtcgacatg aagaattaac tctcaacacc tctccactgc ctttggcaca gaacaagtag 60

gtaactatac ccacagtagc ataataatat atttttattt gaagaaattg agcagcccct 120

gaaaagagac ttacaaatac tgatatttca gtgtccctta cgaaacaacc agttccccgc 180

caaatctctt caagtaaaat ctcctagcaa acaagtacca tccatattac caggctttca 240

gtcaggtttt tagtgcttta ctcttaaaaa tcaatataca atcaaagatc actagtatac 300

aaagtttcta acatagatag aaaggaaaag gatacagaca ttaaaagaaa gaagagaaaa 360

agaaaataaa aaacaagaaa cagaaaaagg aaatgaaatt aaataaatag tgggaacaaa 420

gaaaattgac tcaagcatat aaaataaaga aaaaaagtgg acttctaagt agtaatcatg 480

taaattcttg tttctatccc agacagatat aagaaacaca tcgtccttaa aacagaataa 540

gataaagggg agaaacttta agaaagtcaa aataaatttt taaaataaga gtcaaaagaa 600

aactcgatat aacatcccag aaaatagaac aaaaagttca agaagttaaa taacagaaaa 660

catgaaagaa aatataagtg acttgaaggg tgaacaaagg agagtcaact gtccaagaga 720

aactccagaa aaaagaaaaa aaaaattttt aattatcaga gaaattatac cctaaaaatt 780

agttcccaga aatgaaaaaa aattagcctc catattttaa aatgcctgct gttggctaat 840

taatgtcaga caactgaaat gaaggcatat cttcatgatt tcagaacact ggggattaaa 900

tgattctaaa ccacaaaaag acaaagaaaa tgttaaaaat caaaattata cttagagaaa 960

agtgataaat aaatgcaaaa gtggaatgac atcacatttc tacacagcaa cgctgaattc 1020

cagaaaaaaa ggtgcaagaa atatcaataa aattctgata gaaaatcatt ttaatcttag 1080

atttctctac taacccaata tatcaataaa atgcaaataa aaagacattt ttaataatgt 1140

taggatccaa aacatttacc tccatctcaa gctttcttag gaaattactg gctagtatgt 1200

ctcagcaaac tgagaaaaca aaccaaaaag aaaaaaaaaa tggggtccag gaaatagaaa 1260

ttctcattca gaataataag aaagaaaagt gccaggataa aagctgggca gtggaactaa 1320

ttagcaacta gcccagacta aagcagaggg actccagggt tgcattttca aatagtgcca 1380

aaacgtagac tatggatcta ttaaatgata catctgagta actcatccac aattttgtag 1440

tatcagtttg catggcctgt caaaaatcca gatccatcct ggagaaggtg atcccattaa 1500

aaagaatcca gattattcag caatatcaaa tataggtaga aaccaaaaga caaaaatctg 1560

ctgatttttc aaaccattaa aaagattgat gcagttctgg taatggcatc ataattctta 1620

taagacccac gctcccacaa ataacaaata taaactcagg acgaacattt ttaaagccta 1680

tcttaaggga cagcaaagca accaatagcc agtagaaact acagggaagt caacacttgg 1740

atgacaggta agttttctgt ttttgtagct ttttgctgga aagtagttcc cagtcaaata 1800

gaaacccatt aaaaagccag ctgtagagtt aggaaagaca tccaagaagg gaaagtccaa 1860

aaaagaacaa cccaatgttc tccatacaaa ctctgtttga atctctggtt gatgcctaaa 1920

ctatgcatgt gagaggaata ctggaatcag cagtgttaaa cctaacataa ctggatagag 1980

atttcagctg ttgccggcta taggaaacag aatttgaagt ataaatccaa ccaagttaaa 2040

tgcctgttaa aacaaaaatc caaccatcgt tagaaggata aaacaaaatc tagatatatt 2100

gttgacaatg tccaagatac aacgcaaaaa ttaccaggca tatgaagaaa agaaatcaca 2160

tgacctaatt tgaatgaaga ggccattagt ggatacaaac tttgagttta cctggatgct 2220

ggaattacta gactgaaatt taaagtagct attataatca tgtttaagga tataaaagaa 2280

aatgttctta taatgaataa aataggaacg tttataacaa taatgaaacc tattaaaaat 2340

aaatgaaaaa tttaaaattt aaactgcaat atttgaaaat taaataaaat ctcactaaac 2400

gaaatctcta gcagattaga gatgacggaa gaaagattca gaaacttgat gttatatcca 2460

tagaaattac aaaatctaga aaacaggaga aaaaatatga aaaaatagcc tcagtgatct 2520

ttagaaaaat atcaaaaggt acaaaatgtg tgaaattgga gtaaaagtga aagaagagga 2580

gatataaaaa agcatggata aaaaacaata tttttttaaa aaagcctaat taaaaagtat 2640

attgaaagac ataaaaagaa tttcagaaaa ataatttgta gaacacctac ctaggcacat 2700

cacagtcaaa ttgtccaaaa ctagaaaaga agacaaaatc ttcaaacagc cagagaaaaa 2760

tcatacatac caggaacaat gacatgaata atgataattt ttcattttaa aaaagtgaat 2820

accagaagag aagggaactg tagctttaaa gtgctacaag aaatataaaa agtcaaccca 2880

gaatttgata taaaatattc agctaaaaat taaaatacta cagtaaagtt atctgaatat 2940

aaataaaaat taaaatatcc attgacagta gaactacatg atagtaaatc gcaatggaat 3000

tctcttaggt aaaaaagaaa taataccagt tggaagcttg gattgtgagg aagaaatgaa 3060

gagcacaaga agtagtgaaa tgcagtcaga tatattacat atatcatatg taagactagc 3120

taaagtaaaa atttcatcat tgtattatag gatatataaa atatatataa tacagaatga 3180

cactaaaatc taaaggatgg aaataggaaa aacagatcta ttagatacaa atttcttata 3240

ttttaagtaa acttctacaa tatagtaagt agacaaatag ttttaaatgt atcattcttt 3300

attttccaga atagccctga aaatattaga ggtatagcaa aaagccaaca aataaaaatc 3360

aaaacagaat ctaaaaatat tattcaaata tttaaataga gtcaattcta ggagaaagtt 3420

tggtataata ttaaaaaatc aatcaatgta attcatcttg ttaacacatt tttaaaaatc 3480

tatttgatta tacgtacaga tagaaaaaac ttggtaaaat tccaaactca tttataataa 3540

aaacactcag aaaattaggt aaaaaaggga cttcttcaat ctaacaaagg cacctgaaaa 3600

aaaccaacag cttatgtcac acttaatgac agaaatatta aacgtcttcc catttttatt 3660

tcttaaatat taaacatgtt caagatcata aacaagtcaa taatgttcac ccttaccact 3720

tctattccat attgtactga tgcataaaga caagaaaaat actaaaaagc taggtatcaa 3780

aagataaaga aaaccttttt cagagatgaa aggactactt acatgcaaat attaaaagaa 3840

atctatatat taactactca aattagtaag ttcatacatt aatttattat tacaacatat 3900

aaaaattata ttttgtatac taatgataaa caattggata attgaatttt aaaacctgaa 3960

tttatattaa aaaacataaa atatttaaat ttagcaaaag acatgcaaga cttgtgtaca 4020

gaaaactaaa aacagttgct aaaggaaatg aaagacagtt ttaaaatata gaaatagaaa 4080

taatgtcctg ttgcagcctc tgcctcagga gtcccctgca gcctggaaca cctagcaaaa 4140

gaaatgcagg tgcagtgcca gggattggag ctggatcccc acaagtccta gaagcaaacc 4200

ttgtgagggg gccatctgtc tccccgaccc acctcagagc atgcctgtga acaagaaagt 4260

acgagagcca cacaagctgg gtgttagcct agctaccggc cgttactctt aagcgcatct 4320

actggatcac agcacaaact acaacactaa aattaccgtg ctaatatatg caggatgtga 4380

aaccaggtgc aagaattcac ccacatatgc ggattctgta cagagccctg gccctccgaa 4440

agcatccaga aatgaagcca actgaccaca ctcagcttac accacagtta agggaacacc 4500

aaccctacca gatgagaaag aatcagcaca agaactctgg caattcaaaa aaaccacagc 4560

atctccttac atccaaatgg gtcactagct ccctagaaat ggttcttaac cggtctgaaa 4620

tggctgaaat gacaaacaca aaattcagaa tctggataga aaggaagctc attgagattc 4680

agaagaaagt tgaaactcaa tccaaggaat ccaagtaacc cagtaaaatg atccaagaac 4740

tgaaagatga aatagccatt ttgagaaaga atcaaaataa acttctagag ctcaaaaatt 4800

tactacaaga atttaataat gcaataagaa gtattaacag cagaatacac caagctgagg 4860

aaagaatctc agagcttgaa gaccagttct ttgaatcaac ttagatgaaa ataaagaaaa 4920

aagaatttta aaaaatgaac aaaactggcc aggcacggtg gctcacacct gtaatcccag 4980

ccctttggga ggccaaggct ggtggatcac gagttcggga gatcgagacc atcctggcta 5040

acacggtgaa accccgtctc tattaagaat ataaaaaaat tagctgggca tggtggcggg 5100

cacctatagt cccagctact caggaggctg aggcaggaga atggcatgaa cctgggaggc 5160

ggagcttgca gtgagccgag atagtgccac tgcactccag cctgggtgac agagcgagac 5220

tccatctcaa aaaaaaaaaa aaaataagga aataagaaat atagggttat gtaaagatac 5280

caaatctatg cctcatcggc attcccaaaa taggagaaag agtaagcaat ttcaaaaata 5340

tatttgaaaa atatattttt caaaaatata aataatgcct aatataaata taaataatgc 5400

caaaaatcgc ctaatcttgc tagagaggtt gaaattcaat tccaagaaat acagagaacc 5460

ctggctagtt actatatgag acaactatcc ccaagacaca tagtcatcag attcaccaag 5520

gtcaaggtaa aataaaaaaa attctaaagg cagctagagt gaagggtcaa gtcacataca 5580

gaggaaaccc catcaggcta gccacagact tctcagcaga aaacttataa gccagaagag 5640

attgggagac tattttcagc atcctgaaaa aaattcaact aaaaatgtta tatcctgcca 5700

aactaagctt cataaatgaa ggagaaataa aatccttctt agacaagcaa atctgaggaa 5760

atttgtttta agtagaccat ccttacaaaa ggtccttaag gtagtgctaa atatggaatc 5820

taaagaacct gctgccataa aacacttgaa tgcatagccc acaggcacca caaagcaact 5880

acgctatcaa atttacataa caaccagcta acaacatggg tatgagatca aaatcacaca 5940

catcaatact aatcttgaat ataaatgggc taaacatctg cttaaaagac attgagtggc 6000

aaactagata aaaacacaag acccagccat cattgccttc aagagaccca tctcacatgt 6060

aacaacaccc acaggctcaa aacaaagaga ggggaaaata tctatcatgc aaatgaaaaa 6120

caaaaaagga caggagttgc tattcttata tcagataaaa caaacattaa acaatgaagg 6180

gcattactat aatgataaag agtacaatcc aacaagaaga actaacaatg ctaaatatat 6240

atgcatccaa cattagaaca cccatatcca taaaacaagc tcttcttggc ctatgtaaag 6300

atttaaacaa ccaaacaata atagtaggaa acttcaacac cctactgaca gtgttagaca 6360

gatcactggg cagaaaacta acaaagaaat gctgggctta aactcaacat ttaaccagtt 6420

gaaactaata gacaaataca gaacactcca cccaaaagga atgcttatac actgttggtg 6480

gaaacgtaaa ttagttcagg cactgaggaa agcagttgga gatttctcaa ataatttaaa 6540

acagagctac cattccacct agcaatccca ttattgaaga tatatccaaa ggaaactaga 6600

tcattatacc aaaatgcact catatgttca tcaccatgca attcacaata gcaaagacat 6660

ggaatcaacc gaggttccca tcaacgatgg attggatgaa gaaaacatat acaccatgga 6720

ataccacaca ggcctaaaaa agaatgaaat caagttgttt gcagcaacat aaatagagct 6780

gaaggccgta gtcctaagta aattaatgca ggaacagaaa accaaatact acatgttctc 6840

acttacaagt gggaactaaa cattgagcgc acatgaacac aaacatggga atgattgaca 6900

ctgagcacta ctcaagggga agagagaggg aggttgacac gggttgaaaa actacctatt 6960

gggtactatg ttcactatct gggtgcagta tacccgtgta acaaaccttc atatgtaccc 7020

cctgtatcta aaataaaagt gagaatttta aaaagactaa atatgttaaa accagtactc 7080

tctaaatttt atctacagtc attaaaatct caattgttat ctcaactgtt tttgttttct 7140

aagaaactga gaagctgatt ctagaaggta tatgaacaca caaagaattt atgggaagtc 7200

ccgctcctgg ttagaagcct atgtatgatg tttcaggcca tagataatta attacaaagg 7260

ctttacctgg ctggtttaca gggtgtggag aggctgttct gcccacccca gacttggtgc 7320

acagccaatg cattgcagtc tttgaaggga gttgcagtca aggaccccct ggctggtcac 7380

gcccttgtgc atatttatat tgccagctca ggatccttca ttcttaggtc tctttttctg 7440

aggcctatgt agaggtcttc ttcaggtgcc tctgtcaagg cctcctaata ggagaggaag 7500

agagagcctt aaagacactt ttctccactc tgagtaccca gtgagtttcc actttctcct 7560

tgcagggctc cccaacagtg agacaactcc atgtctgtgc taatccaact aacacttgac 7620

cagtgccatt tccacaggga aaaacagaac agggggagtc agtaatttct ctggctttag 7680

actcttgctt acactattga aggaggtaat taaagattta actctttcct ttggggcttg 7740

ttgctttaat tggctcacta cctggatgca atatacccat gtaacaaacc tgcatgtgta 7800

ccccctgtat ctaaataaaa gttgaaattt taaaaagaat aaatatgtta aaatgagtac 7860

ctaagtcctc agcactcttt ttcagctatg ctgagctcct gacactctag aatattcaaa 7920

acaatattta gaaagaacac atttggagtc ctttcactgt ctgaccaaga cttactataa 7980

acccacaata atcaaagtaa tgtggtactg gcaaaagggc agacaaataa aagatacaca 8040

ttaaaaccac aatgagatac cgctaaccat ccacataccc actcaagtta aaccacctgg 8100

caacaccaaa tgctggcaaa aatttggagt cccagaactc acatatattg ttgatgaaag 8160

tggataatag tacaaccaat gtggaaaatg ttctaatagt ttcttataaa actaaacccc 8220

tctgatcatc tacccctatg agccagcaat tatactccta agtatttact taagagaaat 8280

aaaggcataa atctacaaaa aaagacttaa actggaatgt ttacagaaag tgaattcatc 8340

attgccaaaa cctggagata tcttgaacgt ccatctacag gaggctggct aaacaaacgg 8400

tagtatatct atatggttac atattccact gccacaaaaa ggaatgaagt actgatacac 8460

acaacttgaa caaatcttaa aaatattatt ctgggtgaaa gaagtaaaca aatcttaaaa 8520

ccattattct gggtgaaaga aactttgcac agaagagcgt atgatgttat tccatttata 8580

taaaattcta gaaaagaata atttataatg aaaaaatcag aacactaatt gcctctaagg 8640

agttagcagt gggaaaaact aaaggcatga gaaaactttg gagataatgg taatattcta 8700

caatttggta ggttacacag tgtatgcatt tgtcaaaact tgtttaacgg tattcaagat 8760

ttgtgtgtgg tgctgtatgc aaattttacc tgaaaaacac cttgaactcc agttaattat 8820

acacgtgctg aactgcttag aagtgaagta tgcctccaac tgacttttca atgaattaaa 8880

aaattagatg gattgatgga tgaatagaag gatggataga tggatagtta tgtggtaaag 8940

cagtaacagt tttatccaga acctaggcgg tgggtacatg catgttcact gtataatagt 9000

ttcagcttct atagatgttt gaaaattctc ataataaaat ggaaaataat ggcatgattc 9060

tgtgtatttt ttaataaaat caactttgaa atatgctgat aagaaccaca ttgctaatta 9120

aagaagtgaa gtacaggaag gtttgggctg tgtgtttctg tgtaaaagaa ggaggagtca 9180

gatttacata aacttgaaca agcaagaact attatttcta agaacatgta gcaaaataat 9240

tacaagactg tctctggaga aggaacttac cttttgttct ctgttataat tgaatcatta 9300

accaggtgca tgtattaatt caataaataa acacattaaa attcaatgag agtttagtat 9360

ccataacatt ccccgagaga aaaatctaga gaccactgca gtttgaaaag catgaaaagg 9420

gagaggaata tgtttgctgt tttttgtttt ttgttttcca aacaaagaga atactgatga 9480

agaaacaaag atttggaatt tagtaaagaa tagggatcat ttgttaattt tcagaagctt 9540

ttaggataaa taaaatgaaa aagatgctgg gttattttgg catagagttt gatacacttg 9600

actaaaagat ttaaaaatat acctacatgt caagaaaaac tgaagtattc tatacctttt 9660

actacttcta cttagcagag tttatcttga gccactgtcc gtggcatgtc caataagttg 9720

taaaggcttc acacacactc ctcatatgaa caagttgctg aaatattgtt aaagtgtcac 9780

attttgtcca ttataaggta aattgttttt cacaattcta ttttctttga aatacgggta 9840

agtcctataa tcaatcttca cctttaatgt ggtagtacta aacaccaccc aaaagtaaca 9900

attagatatt taatagaaca tcttataata gagagaatct cagaatcaaa taaataagat 9960

gggttagttt ttgcaagcct gtctttctgg agtttcctag tattctttag agtgttaggc 10020

ctatcattct cagttaccca ccagactgta ctttactaag gcacaggcaa ctgcaagagt 10080

cagacaatac ttgatagtgt tgatattgac acttgagatt tgtgatcgca taggcattga 10140

tttgagtcct gactctgtta cttactcttg gattatatga acttggatag gttatttaag 10200

gtctctaggt cttatctgta aaatttccta atccgtaaaa atagtgtgat gataataaat 10260

agcctagcca aaaaaataga cttaaataaa tgtatgaaaa gtagagtaag tgctcaaaaa 10320

cttagctctg cttttatgca aaaataaact attttaagca gttacatgat acgagaaatc 10380

agcttgataa tggaaaaaga tcataccatt gtggtcaagt tttacacaaa catgatctgt 10440

ttcgtggcct agactgagtt gaattagaat agaagaaacc atattacaag tattcaggaa 10500

ttaagcaaag acaaaaaaaa atagttatga ctgtgataac tgaaacaagg gaagagatat 10560

aagagacaat gcatttaatt tgtgaattga caaataaggt aggtgaggaa aaaagaggct 10620

tatggaattc ttgaacctaa catgcaagaa gagcagggat gcaatttatt gggagagggg 10680

aattgaggaa taagagcccg tttattggtg tgtgtacgtg ttgggggagg aaggagttca 10740

gtttgggaca accatgaaga cccagcagac agctgaaata ctgtaatgaa gttcccttat 10800

attctaacat attactttta gttctttgaa gtcaattctg caccccactt tttgctctac 10860

acccagacag tagcacagat tggtgttcag taatgacaga tggaaaagtg atgggagaat 10920

ggtggtgagt ggctggtgca tgctgtgaga cttgtgcatg aagactcatc ctgagtgaag 10980

agccacacgg ttccaccagg gggaagactg atttttgcat atcatgcaga aactcaacca 11040

cctgttagag gagggaaaca gagtgggaag gaactacgct gtaggaagga aagatgaact 11100

tgagtttcac ttcttagtgc cttttctcag gggagaggcc atcacttgaa gatgctgagt 11160

cttctgctcc ttctcctggg actaggtatg agccttgtct tttgaggagt ctggggttaa 11220

tgttaacact gggtggaaag gtggaaagac tttctctcca gagagtctga agccaccaga 11280

cagaagatga aggggatatg ttagattaaa gaaccataac agagtaattg ttggcacaat 11340

aaaaaggagg cagttccctg gggtcctttg ggagctgagg tggtattgga gagataaagc 11400

agagaccttc ctgcctgggg agatgtctga ccccaagctt gtcggtcctc atgaatatct 11460

ctctggaact aggctctgtg ttcagtgctg tcatctctca aaagccaagc agggatatct 11520

gtcaacgtgg aacctccctg acgatccagt gtcaagtcga tagccaagtc accatgatgt 11580

tctggtaccg tcagcaacct ggacagagcc tgacactgat cgcaactgca aatcagggct 11640

ctgaggccac atatgagagt ggatttgtca ttgacaagtt tcccatcagc cgcccaaacc 11700

taacattctc aactctgact gtgagcaaca tgagccctga agacagcagc atatatctct 11760

gcagcgttga agacacagtg cggggcacag atcaaagatc tgagcaagaa cctcagctct 11820

ctcctaccca gctcctctca cacgagcctg aaggccctgc caaggtggga cagaaggagg 11880

aaaccacagc ttttgggcag acacagctgt ttctgtgttt gtgggtgggc atgggtatga 11940

atggatagac tggggatgag gagagcatct gtaggactag ctggagcgtt ctatccacag 12000

gcaggaagag tggctcctgg aaagctgagg gtaaaatttc ttctagaagg gtgctcagaa 12060

atacccaata aaaacaatca gatacagttt ccttcaagtg gcggggtcag taatagcctt 12120

taactgtttt atctcaaggc tctttttagc tggttttcta tcataatata ttttggaaga 12180

actttagtca tcttgaaaca aagctggtac attatattaa tgacatctgg ttgttctcag 12240

tgaataggaa tgataaagcc ctctagatgc ttcaatatga caatatgaca catgcatccc 12300

aggccaatat aaaatctgca aagttcagga accttctata ctggtgagtt ttggagtggt 12360

ccagatgtaa gaagtaagct gctgcacctt agatcatgta ccacggagaa agaggtgcaa 12420

tgcttagtct ttggactttg aattaaactt tggatatact gcatttgatt gtgctactct 12480

atcccattta ctatgtaatc tgtaaagctg gggccagtgc aagagagggt tctgtggcag 12540

aagaagccca tgatgaaagc ttcctgctgc ttgggccatg gactaagcag acccagttgt 12600

tcttgaagag cctatggtag atactgatgc cctgtggagc tcctgacatt ccccaatacg 12660

agaatcacaa ggcagtgtgg aacaggtttg ctgtgccctg atttgcactt cagaccattg 12720

catcttgggc tatacttttg ttgctttcca gatcccagag agaaagaatg ctcacacaca 12780

caaattacat gaggagtgtt tattacttac atacaggcag caagggaaaa cagaggccta 12840

agattcaggg aaagccagtc ccactaggct caggaaagct gcccagggtg gatgaaatct 12900

tgtctgtgcc ttccctactt gcactgccac tgagggacct cagagagcag ccaccctggg 12960

ttttatatct tagagcaact tgacatgctt ggcaaagcat tgaaggacat tctatttcta 13020

gaagggactg gaacagagcc ctggctgttc tggccagttc ctccttatct caggatgtta 13080

cagtcccaga acattgtaca gttatttttg agaactacaa gcaagaaata ggggagtact 13140

gagtttgtcc aaggccacct ggagaattgc cctgcagaca ggtgcagagt tttggtgtaa 13200

agccatactc ttaccggcca acgatcccct ttcttttgaa aagttgctcc tggctctata 13260

atggatgcct cataatggca ttcagaacaa ggtattatct gatctgcaaa actgtaaatt 13320

tggctgtgcc cagagcattc tgttattaaa tggaaaatac ataatcaaga ccatacttaa 13380

gcacagctgg aaaaaggcac gattcaattt caagagcaca tctcagagtc tcatgctacc 13440

cactcatact tttctcctct taccttcaaa ccacacccaa gtttcatgga aattccatat 13500

ttccagttga gaaacatgag cttatttttt tgatgggtct tcacattatt ctggaaggat 13560

cattacagtc taaccaagta tggttctgaa aggtagtggt gaagggaaat cttctcgggg 13620

ccaaaactta aattggtata cctgattgtt gtccacattt tattgaagaa gagatggcca 13680

gaagtatagc tatatcttat gttcaatttt ctttcatact atcatctagt ctccttacct 13740

attcagtcat atttttcttc tttttgcttc tctatgctac aatctgggta atttctttaa 13800

aactactgtt tagtttacta attcacacct cagctccagt aagcctctaa tttaatcttt 13860

caactgactt tgttcatcta aattatatta atttttattt ctagaaaatc cacttgggtc 13920

atttacaaat atgagtggac atttttgaaa gcatcttgtt ccttgtgtat tttaaaatta 13980

ttttcttttt ataagtatat taagcatcta ctgggaatta ttatctgaca atgccagcat 14040

ttgatgcctt tttgcatctg gctttcagtg gtctgctggc tctcactcag ggtagcctat 14100

ttccttgtac attatgtaag ttaatgtgaa ttcatctttc agaattcttt aaatactgga 14160

gtaaaagtgg gttttctagg aaaattcatg tttgcctctg tcaagaaatt ggggcgctac 14220

agacctaata tcattttatt ttataattct cagtatatat cttcttggtc atagaatgtc 14280

tatctcaaac cccaatgggc tgactgttca gtgaccatga atattcagag gaaatttttt 14340

aaaatccttc acccaaagcc aagaccaaga caaataatat tctttgttaa ttgcttctag 14400

gattagtttt ctagctcatc ctttcactaa ctgtgtatct ctttgtgtac ctcactttaa 14460

aaagcagtga ttcctgtcta gtttctggtc catgtaaggt ctcatgggcc ttctccagtt 14520

tccatgtgta ctaattctcc aggtcacagg aatgcacatg tatacctgtt ttatctcatg 14580

ggctcagtgc tagcttacct tcctggccca tgctcccatt accataggaa gttgacaaat 14640

ttacctacag tgaagccttg gattatgaag caccttccta tattacaaaa ttgcctcttt 14700

aaaagcagta aagcactgtg gttaggaact tgaactttgt caagtgacag acctaagttt 14760

aaaacttggt tcttctgctt taacactagg tgtaacctta acaagttaca caatttctcc 14820

atgccttaat ttctccatag aaaataagaa taatagtgag tttttaaatg taggtttttt 14880

aaaataagaa agtgatacaa ttatctgtat aaaaagcaca tagagacaaa atatgcaatt 14940

agtgtagttg ctacctaaac gtatttctca ctataaaaac atattttacc actggcaatt 15000

ccttcctcaa gaataagtat ggccctctct cagaaaattt tatcattaca attgtttctc 15060

ttttagctat tttaattaaa taaaaaattt gtggtattat aaaatatgca tattatcaaa 15120

ataaaaaagt caaaatcact cagtttaata aaattagatc atacaatatg tagttttaga 15180

atttattccc ttaacccaac aatatttcat gaatatcatt tcatgtcaaa aatattttac 15240

attaacttta ataagaatag ttcgatatac tgatgaccat ccttcataca atcaaaaccc 15300

atgacaaggt atatatccat aggctagatg acagggttat ttaattttaa atagcaattg 15360

tttgtgtgcg ttgaattatt tctgttgcag actcagaagg aaatcatgaa agccctaagt 15420

ctacctgcag atggctggtt cttttttatt caggtgttaa cttaaatgcc acctccccaa 15480

agagaccttt cctgaccact ctacgtaaca gcacttcagc agtcatgcca tattatcact 15540

ttatcctatt ttagtaatat gtactcatca tttactgaaa gtgatgtatg tgcatcccat 15600

ttctatacct cctcatatgt gcataaatgc atgcacatgc acacatgaat gcactgaaaa 15660

caagaacctt ttctgtcttc ttcgttgctg cttccaaata gcctagaaga actcttggaa 15720

tatcactcca ggaattcaat aaatatttgt gagtaaataa atacatttca aagataagag 15780

gaaaatttat acagctgcag tggatttccc tggtggaaaa ctctacaaag gcatttgagt 15840

ttagataatc taagcccata agaaaattaa ctagattatt ctaacaggaa aggaataagc 15900

acttcaaact ttatattttt tattcttgtt ttaaatttat gttttagttg cggggaggat 15960

acaaaaagat taaaggacac aaaattacag ctagatagga ggaataagtt ccagtgtttt 16020

acagaactgt aggaggacta cagataacaa gcattgtatt tgaatagaaa ggtccatatg 16080

tcatgtgcca accccgttct gctgccttac ccaagactgg gaaatggaac agatgatatg 16140

ggttctatag agaggcaaag tgcagaacct gctcaaacac agtggccctg gggtggaaag 16200

ttctcaattg gaattgtgtt ggtgcctgtg tggatggagc attcaattct gagccttagg 16260

agccttttga aggccctcct ggccaagatg atcttgagga aatgactgac ccattggctg 16320

ccttccaaat ggatgacaca gtctagactt ttataaacta catgagtaga aggaacccag 16380

ttgactggat ttatcaccaa acctagcttg tgagcctcag tctagagcta gcataattta 16440

gaaaaataaa tgcatacatc atatattaaa ctctgagtat catgaaacag atcctagatg 16500

ttacaataat aagaacctac atgggtgttt ctcctgcact ctgtcctggc tttcttctgc 16560

ttcttgtcta agggagcctg gactcactta agcagctcta ataggccaaa caagagagtg 16620

gtcaaggagt ggctgaaatg tgaacagaaa gactcaccat tatattagag caatcaatct 16680

atccttaaaa tactgtgggt ttagtgtcaa gtgctagagg cttccccatt tacccataca 16740

tggaagcctg aagaataaac agaactataa acttccctct gggcataaaa cttgcatgtc 16800

ttccatggct tcaagctggg gagcagctgg gcaggcagaa ggaggggcaa gaggcagtca 16860

agaggagggg cgggaggcag gcggggagga atgggctact ccgtttagct gatcagcctt 16920

gaccagttca tgctctggga ctccatctta caagtcagac ttttccgagt cacacagagc 16980

actcccttca ctctccagat tcaatgattc ctgggatgat atagtaacga aaataattcc 17040

ttattggata aagagaaatt gtgctgtgcg ggagataagt agcttagaaa aaaaaatgga 17100

tgaggagagg agctgggagt ttgggggtaa gacagcctaa aatctgacag tctggttggg 17160

gaacgataaa agatactgga acccaaggag gagatcagta agaatgtagg agaaagatct 17220

ggtgggattt ggagaagaaa gtgttaaaaa ataaaattgg aatatgacag tttgcagact 17280

actaggtctg aaagacgcaa ctctgtagca acatgaaggg gaacaaactt tgtctgaata 17340

gggaagtttc tagtatgatc tctgctttcc ttagacaagt aaccctggag aagacacttg 17400

acctctgagc tttagattgt tcatctgtaa aaaggagaga acaatatctg ctgcctacca 17460

cctcagtgag gctgcactaa ggatcagctc agaggaggca aagcagaatg ccctattgca 17520

cagcatggag gccctagagt cggaccacca tacttacatc ccatctttaa aacctcctgg 17580

ctgtgtggcc ttgagcaagc cacttcacct ccagaaagtt catttgatca tctgtaaaat 17640

gggcaatccc ataggtggtt gtgatgataa aatgaaaaaa acatttttaa cttacatgag 17700

ttacatgctg tagagtaagt tatgtggtat atagtaagca cacagaaaag actcactgtt 17760

atcaaaatta aaggaaagtg gtttagtgtg ctaagcacta aatacaagcc agagagatgt 17820

cttgagaagt agacaaagaa atctacaatt cagaatgaac agtaacaaga atgacatgtc 17880

ctccaaattg agctttggca gctggatctg aacctatcct acacatctga gtctacagta 17940

tgaggtaaca gtctccacct atagctatct tctctgcctg taaggtcatt cctttctgaa 18000

gaaagctgac ctcagactat tttgcgatca tttaaggaag agcgcaaaat actggagaag 18060

gctagtcttc aagctctaga tacagaaaat cctcaaattc ttgggtgttc cctttcagac 18120

tgagagtcag agccacaagc actgatccaa aaatcccctt ttccaaaaaa attttccatc 18180

ttctaccaag gttcacctcc ctgtttagtg cacactagta tctacagttg ttagagcagg 18240

agtctctcct tatcctctaa ggcaggtgtt ctgtgaagac cccatgacaa gggacaaaat 18300

gaaacaacat gttcagggac aacaaaggaa aagcccattt tatcaatgct ccaaaacctg 18360

gctacctcag gcttggcaag ttattcgtga gcagagaagt ttgtcatcta catggcagtc 18420

atggcctgga tgggtctcga actgtggcat ggtaatgctc tgagactagg tgccaggaga 18480

aagataaatt caatcatcca tttctaccag gaagaaaagc accaggaagg aatcagatgt 18540

acagtttgtc ctctagggac ctctgtcttg atcctcaata aatctccttt tagatacctc 18600

ctcttagatg aaattcccac acttcacaat gctctccatg gcttctgttg gccttatggt 18660

cagctctgcc aagatctgta ttcttgggca agacagaatc cttctgaatc tcagtctctt 18720

gctctataaa tggaaacaat tttgctctgc acaaattaca attagcatgt ttgtgggaag 18780

ttaaagtgtg aaactcagca taaatgaaaa gtaatgtacc actgttgctt acatgaaaac 18840

tcttgtaaag ttgtcaggca ctgtgcacca tctaagcata gcagtgttag gtatgggccc 18900

attagtgatt agagggggtg cgaggcggag agggggtctc caagcagtgg gtctcctggc 18960

tgtggggaag ctggctcagc agccatacca ggagctgaaa ccgaaggtac tctgtgtgtc 19020

ctctcaacac catgggcccc actttggcat tatttccaat tccctctggt ttccatttgc 19080

ttcctccctc tagccccctg gccaggtccg atttcaacac caagtttctg agcttttccc 19140

attttgtcca ccgtctctag aacatttctg cttcttttac ctttcaaaga atccaaactt 19200

gaactctact tactcctgcc catcaccagc ctgagctcag agttgctgag aagggaaagc 19260

accctcataa ggaagctcct cctttcccta catcattaga cttgagcaca gacagcatct 19320

ccatttccac actgctctgg ccaggttgtg cctcagtcaa ttacacaact gtgtcttagt 19380

agccttggta gggcccaaat caggatattc tctaggaaga ttcactttgt actgagccaa 19440

gcataatctc actgcatcga aaatagaaaa tgatcatctg atcattctgg ttcagactgt 19500

cagccctgag caggtgggtc agtttgcatc acctgagtgc agattgcaag gacaacagtg 19560

aggctgcata aaaagaacct atgacaggat gcacatgaga gagacaaatg tcttcacatt 19620

gaagaagggg aggagtgcgc cattggtttt ccatcctcca gaggcactga gaaagctcct 19680

acagaaactg tgccccctcc ttctttgaaa cacttcccat ccttaagcct tggtaggaag 19740

gagagaaatc tggaagccca gaatcctatg gaatgctgag tctcccttct ccttacccct 19800

tagtgttgga attttcattc cccaaaatct tgcatttgac cttcctgcac actgactgga 19860

gagaagcatc ttcatattca caggaatgag ccttagataa gactttgctc ctctcatgtg 19920

ggccccagga aactgaaatc caagcttttt accaatgctt gcccttcctt aagtacatta 19980

aatacattcc caataggtgg aaagatttgt aaacagctag gccattctcc ctccttctaa 20040

ccctcatctc cagggcctaa gaaagcccag ctctgttatc tggggcttga ctttccctgt 20100

cttccccatc ccagtatcct tgcaggaaac agccggtctc gctctctgct ctcagaagga 20160

aagtttcctt atcacctgtg aatcacaaac ccagagagtg gccaaacata gccaggctga 20220

tgcaagaccc tgggaagagg aaagctgcag gtgtgtttgt gctgggagga gtggtgaccc 20280

tcacctcaca gtcacctcct ctctggatcc tcgtgaggta taaagacgag tcctccacca 20340

ccagtcaggc acactctacc accatgaatc cactcctgat ccttaccttt gtggcagctg 20400

ctcgtgagta tcatgccctg cctcaggccc caaccacccc cccgttcctg gccgacaaat 20460

gcccttccat tcttaccacc tctcctcttt tgactgtgct ctgatattcc gtttcctcca 20520

tctggcatat ctccttccca tcctccttgg gctcttttta agtctcacct gttcaccttc 20580

tccttgactt cactcccacc actgtcattc atccatatcc gagttgtggt tggagaagct 20640

gggaaggggg ccaggtgggg ctgtcccacg aaatgaagca gcaggcttca ggcttggctc 20700

caacagcaac agaatagcac cactatagct gctcctaacc tcgaatgcac ctggggaggt 20760

tgaaaaatta ctcatgccag agactcagct ctagaaattc taacttcaat tgtctggggt 20820

agagcttgtg ttctgggctt ttaagcttcc caggtgattt ttaatatttc cagccatgca 20880

gccaaggtta agaattgctg tcctattggc caataacaaa gtctaccttt gttctgccaa 20940

agtgagcctg ggggctgccc tcaactctgc cctgactgca cagatctgag ctatggggga 21000

aggtggtcat ggccaggtct atgcagacag ggggttttcc tagcttggca aaagaatcct 21060

gacaatccag ggcccaaata gccaggggaa gtacacaggt gatgaataaa agagagaagc 21120

actcagtggg agagacaacc acatcccaac tcctatccca ctggaagcat tgtgaggaca 21180

ttccttgcga cttcagcctg gtgaccccag gagagctcgg atcctccgca gggtacctag 21240

ctatgtgccc tgcaggcaca gagacttggg agccacaggc agtgatgatc accaggggtg 21300

gcagagctcc ctcccttgcc tagcctcact gtgcttgtta aggatttcta attagcagaa 21360

agcaatcaca ggctgggagc gccaccccta acatgctatt gacttgcctt ctcccttccc 21420

atctccactc cagttgctgc cccctttgat gatgatgaca agatcgttgg gggctacaac 21480

tgtgaggaga attctgtccc ctaccaggtg tccctgaatt ctggctacca cttctgtggt 21540

ggctccctca tcaacgaaca gtgggtggta tcagcaggcc actgctacaa gtcgtaagtg 21600

tggggccccc gactgcaaag ctcccggcca gtctgcctgg gagagcttgg cttcagccca 21660

gggaactact gaggttgggt aagatggatg ggagaggtgg tggagaagaa aacttgttgg 21720

cagctgcgga ctctccagag cagagagtga acacaagaca ggaacctctc acacccaggc 21780

aaatccatga aacagcaagg gttgtggtca taaaagcagg cagggatgat cttggggtgg 21840

tgagagctag tgagaaaagc aggcaagtat cttttgctgg ttagctacac attaaagcca 21900

actaagaaag actttttaaa aatacagatg cctttgtcct atcccagggc aattaagtca 21960

aaattttcag gaagagggtg tgaatatcag tgagaatttt acactctacc tctgctaact 22020

gtagagtgta tagacagagc tgagaactgc tgcctacacc aagaactctc aaacctgagt 22080

atgcatcaga acgccctgca ggcttgttaa ggcacaaatc actgggcccc ttccccaagg 22140

ttctgatcag taggtggggg taaggaccaa gaattcacat ttctaacaag ttcccaggag 22200

atgctaatgc tatggctacc cttggattag attacacaga agggtggttc tcaccaggcc 22260

aagaatggag ggaggaacag gcactgtgca cagttggcaa aggcctgggg tgaagaacgc 22320

tgggaaaact tcaaggagct ccttgtgccc acagtgctag tgactgtgga gattgtggga 22380

aagagtctgg ggaggcaggt tgaggagcag cctctggtgg gatccctttg actcttcccc 22440

accccactac caccaacctc tggagcagat aggtcctggg tctcatacct tcactgaccc 22500

acatccctct gctgcccatg cgatatggcc acacacccca ccccatgcct ccagagctgt 22560

ccatgagcag agagcttgag gaacctgggg aaggtgggat aggtgccctg gctgtgggag 22620

aaggtcttca ccatgcctgc cctgcccatc agccgcatcc aggtgagact gggagagcac 22680

aacatcgaag tcctggaggg gaatgagcag ttcatcaatg cagccaagat catccgccac 22740

ccccaatacg acaggaagac tctgaacaat gacatcatgt taatcaagct ctcctcacgt 22800

gcagtaatca acgcccgcgt gtccaccatc tctctgccca ccgcccctcc agccactggc 22860

acgaagtgcc tcatctctgg ctggggcaac actgcgagct ctggcggtga gtgggaccct 22920

tagtccttct acttccctcc atcctcacaa tttccagaac aaaccatgcc ccttaacttg 22980

aatcctctca cctccaggct taagacacat ttcgagtgcc cattacacac agactctgca 23040

ctgggcacca gagagatgca aactatcaag gacttggctc ctaaaatcaa gagacaggac 23100

aaatggagaa cttgatatga tcacatcttg ggaggggttc aacaatgatc attctgggaa 23160

ctaaaagcca gagtctcttg ccaggactta tgttctggag tcctctccag gggctgtgtt 23220

cctcttcagt tttccatcca agattattgt ctccttctct ggcctgaccc acatttctac 23280

ttcctttgat ctcttcctga tcctcacagc cgactaccca gacgagctgc agtgcctgga 23340

tgctcctgtg ctgagccagg ctaagtgtga agcctcctac cctggaaaga ttaccagcaa 23400

catgttctgt gtgggcttcc ttgagggagg caaggattca tgtcaggtga tttgaccaac 23460

ccttcccatg ctgaggctcc cactgatacc taggccccac cagggaaaag gatttgaact 23520

caaaaggtgg tggggctgag gaggctccct gcagtgccca catggagaag tgaggaagac 23580

tcccttgggc tgcatcttgt ctgcttagga agaacagaga atgggccacc atgagaagga 23640

catggagcca cagagctggc tggaaagggg tcttttaagg ttcagagtaa atgtagctat 23700

attcctcctc catctctcca tacaacttgt cccttcttcc ccccagggtg attctggtgg 23760

ccctgtggtc tgcaatggac agctccaagg agttgtctcc tggggtgatg gctgtgccca 23820

gaagaacaag cctggagtct acaccaaggt ctacaactat gtgaaatgga ttaagaacac 23880

catagctgcc aatagctaaa gcccccagta tctcttcagt ctctatacca ataaagtgac 23940

cctgttctca ctgtctgtgt ctgtgcctgc tccctctcac tccttcacac tggaaagcat 24000

cctccaattt caggttagac acgactgtcc cctttaaagg taagcagagc ccccatctcc 24060

caaaatgtgt tccatggtac actagattag cacatacaaa cagatggaat ccaaaaataa 24120

gaagaagctt gggagaaagg ggagtactgt tttctagaga tacctgtttc agaaaggtgg 24180

tctttggggt gggggggtgg gtattgattt ttatttgggc ttctcacagt agttagagct 24240

actctgcctt cagaacaatt acagcacaga aaatgtgtca gcatcttcga ggtggcccaa 24300

aaaacttgac cagctgaatc ttcttgctaa aatacaataa taatgacaaa tcctgttggt 24360

gatgacgtgc ctctcccagg aatgtgtcgg caccaaaccc tcgaccaagc cctcccttct 24420

cattcacctg gaaaatcaga tgcaaataaa tctccctggc cgcctaactc ttccctcagt 24480

tccctagttc catctctgtg agcaggctag agagatgttc cacctaccat agcaggagcc 24540

agactgcgac ttgggaatca agcgcagatc tacacactgg gcttgatttt cgtcttcttt 24600

gcctttgggg taggatgcca cagtgaatcc cacagctaac accagctcct cactctgacc 24660

agggaaagaa actagagagg gtcaggattc acctatttga tcaattagct gaggaaggat 24720

tcattttcat aaaacttgct tgactttgag acacttcaag tgagttattt gggattcttt 24780

aaaaggggtg gaaggaaaga tctgaggact gtgacaccac cagccactct gaccacacgt 24840

tggctggctt gaacccactg gatgcagcag agggaggcag gccctgtggc acctctggca 24900

cctgccaaag cctcctcctg gcaattctga gagggcccat gttggggctg tagctcatcc 24960

aagcttggca ccgaagatcc aaggaagctt ctctttgaaa ttccaccttc acctctgtcc 25020

caagtggttg tggacacccc tgggagctgg cactaagggc caggagcagc caagggagac 25080

agacaagttc agagcacatt tccagttaca gggaacagag cacaggcctc caagtgtcca 25140

cagagcagcg tgcaaattgc agggatgagt agaggaaaac ctctacatgg agcacagcgt 25200

tcctggcaaa cacaggggac tgcagtccac atgctgtgga atacacccaa gtatgcatca 25260

gacacttgtt tggtaaacag taaatgtgta agatcaatta ccttgagagg gccatctggg 25320

ctccagatgt gtgacttgtg tgaggagacg gctaccactc actatctcca gaggaaaaca 25380

gggctcaggg ctcgcacaca atggatagat acacacgggt cacccagaac atcaatacat 25440

acaaacatca ctttgttcaa catggatttt gtttttatgg agtccaaacg cagaccgtag 25500

ttcaccttac agccttgggt ttgtctgctt ttggagatat atatccagta gatagataga 25560

cagacaagac agataatttt ttccctttct tactataata tcaatgcctt atctgaatat 25620

catcatcact caagagtcaa gggaagaacc aaacgcttca tataaacagg gctgggctgg 25680

gcagggagtg ttagtttcca ctgttttctg tctccattca gatcatccct atcaaagccc 25740

agctggtcac ctcaagccaa gacacagaca taaggatccc aatggccttc cagatggctg 25800

cttccacctc ccacctgggc cactaagact ctttacacag gaaagtgagt caccccagtg 25860

agaagggttg ccagaagaaa cagggcatga aaatcacaga taaccatggg atttcttcca 25920

agtggtcagt ataaaatata ccaacactac catcaccaaa aacacaaaac aagcaaaaac 25980

atgcttattc aaaatcctag acaaaatact gtgtgagagc caggccacgg ggttgccttt 26040

tcacaggcca cacagctttc ccagtccatg tactcacgtg gaaccgtgag atgtgaaagt 26100

ctgcagggtg tgtgctcagg atcgaggctg gtactgttca cctatgggtc cagaccaaag 26160

ggcaataagg cagaaagctg atatctgtcc accgccacgg cttagggcct ccttctcaca 26220

gaggctccta agagccccca cctcagcctt cacacaaaac atctccttct ggctgaacta 26280

cagccaggct ggagtgccgt ggcatgatct ctgctcacta cagtctccac ctcccgggtt 26340

caagtgattc agcctcccaa gtacctggga ttacaggcac tcgccaccat gcccagctga 26400

ttttgtttgt ttgtttgttt gtatttttag tggagacagg gtttcaccat gttggccagg 26460

ctcattttga actcctgacc tcaagtgatc caaggtactg ggattacagg catgagccac 26520

cacttctggc ccaggaccca ttttttaaaa aaattttcaa cttttatttt agattcaggg 26580

catacgtgtg caggtttgtt acatgggtat attgcgtgat gctgaggttt ggggcacaaa 26640

tgttcttacc actcaggcag tgagaatagt gcccaatagg tagttattca gccctttcca 26700

ccctccttct ctcttccctc tagcagttac agtgtctact gttcccatct ttatatccat 26760

gtgtaccaaa tgctcagctc ccacttatac agaagaacgt gatatttggt tttacatacc 26820

tgtgttaatt tgcttagaat tatggtctcc atctccatcc acgttgctgc aaagacatga 26880

tttcattttt tcatagctgc atagtattcc atggtgtata tgtaccacat ttcctttatc 26940

caatctaccg ttgatggcca cctagattga ttccatggct ttgttattgt gaatagtgct 27000

gtgacggaca tgcaagagca tgtgtctttt tggcagacca attattttcc tttgggtata 27060

tgcccagtaa aggaattgct gggtggaaag gtagttctgt ttttaattat ttcagaaatc 27120

tccaaactgc cttctgcatt aatttttcca taaactaatc gacagtcagg ccaagagtgt 27180

ataagcattc cctttcctct gcagccttga cagcatcttc tatttttttt tttttttttt 27240

tgacgtttta ataatagcca ctctgcagcc aagctctttc aaggccaatg tggggtagag 27300

ccacaaacat cccttttccg gaagagaatt cttgcctgcc cactggtaga attcttgttt 27360

tcatctgtct tttattattt gtttttcaaa gaaaaactgg cctgtaatcc cagcactata 27420

cgtggccgag gcgggcggat cacgaagtca ggagatagag accatcctgg ctaacacagt 27480

gaaatcccgt gtctactaaa aacataaaaa attagccggg tgtggtggca ggtgcctgta 27540

gtcccagcta ctcgggaggc tgaggcggga gaatggcgtg aacctgggag gcaaagcttg 27600

cagtgacctg agatcatgct actgcactcc agcctggaca acagagccag actctgtctc 27660

aaaaaaaaaa aaagaaagaa aagaaaagaa aaaaaagaaa aaccaaaggt aaagtggtaa 27720

gatcttccac acctgaacca gtttgtagcc accagagcct gctgcgaagg ggcccctcaa 27780

gcatgcattc atcttgtcac ctggaatttg agagatcaag aagccccata gcaacctacc 27840

atgcaactgc ccatcagtgc tgatcctttc aagggattat ctcaattctg actctgcaga 27900

cactggcagc cacaggtgac aaaccctgtg tatctgtggg ctttcctcat cacccagggc 27960

cacaatgggg tgcctgccct aggcagagac acagcaacat tctcttaaac tgaaattaag 28020

cataaatcca cttcaccaat aatcatctga gggctcagtc cctgtctcct tcctcgggga 28080

ttttaaaaca cacatttctc tgaccaaaca ggtaggtgag atctgacttt aaagggggga 28140

aattgggttg aattgaggta tcaggaatgg atcacaagtg tttttgtgtg gggacaggca 28200

tccacatccc gaactgtacc tggagatgga gaaaaatgca ggagaggaga agagaaaaaa 28260

aggaggaaaa agtggctgaa agtatttgaa agcttctctc atggttcttt ctggctctgg 28320

ttgcttttcc aaggtcttga gtgcaggcag ggccagctct ggctgggagc tgctgctctg 28380

ggacatggaa gagtcacttc acctccagaa agttcattcg atcatctgta aaatgggcaa 28440

gcccataggt ggttgtgatg ataaaatgaa aaaaaaacat tcttaactta catgagttac 28500

atgctgtata gtaagttatg tgctatatag gaagcacaca gaaatgattc actgttatca 28560

taatagaagg acagtggctt ggtgtgctaa gcactaaata caagccagaa aaaatgtctt 28620

agagaagtag acaaagaaat ctatagttca gaaccaacag taccaagaat gacatgtcct 28680

ccaaattgag cttgggcagg tagatctgaa cctatcctac acatctgagt ctatgatatg 28740

aggtaatagt ctccacctat agctatcttc tctgcctgtg aggtcattcc tttttgaaga 28800

aagctgacct cagactatac tgtgatcatt taaggaagag cacaaaatac tggagaaggc 28860

tagtcttcaa gctctagata cagaaaatcc tcatattctt gggtgttccc tttcagacag 28920

agaatcagag ccacaagcac taatccagaa accccctttc ccaaaaacgt tttccatctc 28980

ctaccaaggt tcacatccct attgagtgct cactagtgtc tatagttgct agagcaagag 29040

tctctcctta ttctctaagg caggagttct gtgaaggccc catgacaagg ggacaaaatg 29100

aaacaacatg tttagggaca acacaggaaa aacccatatt atcaaagctc caaaacctgg 29160

ctacctcagg cttggccagt tattcatgag cagagaggtt tctcatctac atggcagtca 29220

tggcctggat gggtctggaa ctgtggcaag ataaggctct gagacaaggt gccaggagaa 29280

agatgaattc aatcatccat ttctgccagg aagaaaagca ccaggaagga atcagatgta 29340

cagtttgtcc tctggggacc tctgccttga tccgcaataa atctcctttt agatgccacg 29400

tgcagcaatt cccacacttt acaatgctct ccatggcttc tgttggcctt atggtcagct 29460

ctgccaagat ctgtattctt gggcaagaca gaatccttct gaatctcagt ctcttcctct 29520

ataaatggaa acaatttggc tctacacaaa tcacaattag catgtttgtg ggaagttaaa 29580

ccgtgaaact cagcatcaat gaaaagcaat gtaccactgt tgcttatgaa aactcttgta 29640

aagttgtcag gcaccatgca ccatctaagt acagcagtgt taggtataag cccatcagtg 29700

attagagggg gtatggggtg gagagggggt ctctgagcag tgggtctccg ggctctgggg 29760

aagctggctg ggtggccata ctaggagctg aacccgaagg tactctgtgt gtcctctcaa 29820

ctccacgggc cccactttgg cattttctcc aattccctct cgtttccatt tgcttcctcc 29880

ctctagcccc ctggctgtgt ccaatttcaa caccaagttt ctgagctttt ctcattttgt 29940

tcactgtctc cagaacatcc ctgcttcttt tacctttcaa agaattcaaa cttgaactct 30000

gctcactcct gcccatcacc aacctgagct cagagttcct gagaagggaa agcaccctca 30060

taaggaaact cctccttttc ttacatcatt agacttgagc acagacagca tttccatctc 30120

cacactgctc tggccaggtt gtgcctcagt caattacaca actgtgtctc agtagccttg 30180

gtagggccca aatcaggata ttctttggga agtttcactt tgtactgagc caagcataat 30240

ctcccgggat ctaaaataga aaattctaga cccacccctc cacatcttcg ttattgaacc 30300

caatatgtca cccatttacc atgtgctcaa atctctacct actgctgatt ctcagatcaa 30360

atcgtaaccc aggtttacca tggtccaaac tctgacatct gattggggcc atgtcatcct 30420

ggttcaggct gtcagcccca agcaggtggg tcagtttgcc tcaccctgag agcaggttgc 30480

cagggcaacc atgaggctgc ataaaaagaa cctatgacag gatgcatatg agagagacaa 30540

atgtcttcac attgaagaag gggaggagtg caccattggt tttccatcct ccagatgcac 30600

taagctccta cctaaactgt gccccctcct tctttgaaac acatcccatc cttaagcctt 30660

ggtaggaagg agagccatct ggaagccctg aatcctatgg aatgctgagt ctcccttctc 30720

cttacccctt agtgttggaa ttctcattcc ccaaagtctt ggatttgacc ttgcccgaca 30780

ctgactggag agaagcatct tcatattcac aggaatgagc cttagatcag actttgctcc 30840

tctcctggga gctccaggac actgaaaagc caagcttgtt accaatactt gcccttcctt 30900

aagtacatta aatacatccc aaatagttaa acagattcct aaacagccag gccattctcc 30960

ctccttctaa ccctcatctc ctcggcctac gaaagcccag atgtgtgatc cagggcttgg 31020

cttcccctgt cttccccatc ccagcatcct tgcgggaaag ggctgctctc tctctctgct 31080

ttcagaaggc aaatttcctt atcacctatg agtcacaaac ccagagagtg gccaaacata 31140

gccaagctga tgcaagaccc tgggaagggg aaagctgcag gtgtgtttgt gctgggagga 31200

gtgctgaccc tcacctcaga gacacctcct ctccagatcc tcaggaggta taaagacagg 31260

tcctccacca ccagtcaggc acactctacc accatgaatc cactcctgat ccttgccttt 31320

gtgggagctg ctggcgagtt tcatgccatg cctcaggccc caaccacccc ctttcctggc 31380

agacacttgc cctgccattc ttgccacctt tcccattttg actgtgttct gatattctat 31440

ttcctccatc tctccttccc atcctccttc ggctctcttt aagcctcacc tgtttcagct 31500

tctccatgat ttcactccca ccactgtcat tcatccatat ctgagctgtg gctggagaag 31560

ctgggaaggg agaccaggtg gggcgggccc acaaaatgaa gcagcagcct tcaggcttgg 31620

ctccatagca ccagtatagc accattatat ctgctcttaa cctcaaatgc acctggggag 31680

gttgaaaaat tactcatatc agagactcag ctttagaagt tctaacttca ttctctgggg 31740

tgcaacctgt gtactgggct tttaacattc ccgggtgatt tttaacattt ccatgcatgc 31800

agccaaggtt aagaattgct gttctattgg ccaataacaa tgtctacctt tgttctgcca 31860

aagtgagcct gggggctgcc ctcaactctg ccctgactac acaaatctga gctatggggg 31920

aagctggtca tggccaggtc tacgcagcca gggggttttc ctagcttggc caaagtatac 31980

tgacaatcca gggctcaaat cgccagggga agtacacagg tgatgaataa aagagagaag 32040

cactcagtgg gtgagacaac cacatcccaa ttcctatccc actggaagca ttgtgaggac 32100

attccttgaa tcctcagcct ggtgaccccg gggagaactg agcccctgca gggtacctag 32160

ctacatgccc tgcagacaca gagacttggg agccacatgc agtgatgctt accaggggtg 32220

acagcgctcc ctcccttacc tagcctcact gtgctcgtta aggatttcta attagcagga 32280

agcaaccgaa ggctgggagc gctaccccta acatgctact gacttgcctt ctcctttccc 32340

atctccactc cagttgcttc ccctttcgac gacgatgaca acatcattta gtggtatacc 32400

tgtgaggaga attctgtccc ctaccaggtg tccctgaatt ctgggtacca cttctgcagt 32460

ggctccctcc tcagcgaaca gtgggtggtg tcagcagctc actgctacaa gtagtaagtg 32520

tggggcccct gactgcaaag ctcccagcca ggctgcctgg gagagcttgg cttcagccca 32580

gggaactact gaggttgggt aagatggatg ggagaggtgg tggagaagaa aacttgttgg 32640

cagcagcgga ctctccagag cagagagcga acacaagaca ggaagctctc acacccaggc 32700

aaatccatga aacagcaagg gttgtggtca taaaagcagg cagggatgat cttggtgttt 32760

ggcagagcta gtaagaacag caggcaagta ccctttgctg gttagctaca catcaaagtc 32820

acttaagaaa gagtttttaa aagtactgat gcctgtgtcc tatcccaggg caattatcag 32880

gaattttcag gaagagggtg tgaatatcag tgagtattaa acattctacc tctggtaact 32940

gtagattgta tagacagagc tgagaactgc tccctacacc aagaactctc aaacctgagt 33000

ttgcctcaga accacctgca gggttgttaa agcacaaatc actgggaccc atccccaagg 33060

ttctgagcag taggtggggg taaggaccaa gaatttacat ttctaacaag ttcccaggag 33120

atgctaatgc tatggctacc cttaggttag attacacaga agggtggtgc tcaccaggcc 33180

aagaaaggag ggaggaacag gcactgtgca cagttagcaa aggcctgggg tgaagaatgc 33240

tgggaaaact tcaaagagct ccttgtgccc acagtgctag tgactgtgga gattgtggga 33300

aagaggctgg gaaggagggt taaggagcat cctccggtgg aatccttttg actcttccca 33360

accccattac caccaacctc tgaaacagaa aggtcctggg tctcacaact tcactgaccc 33420

ccatccctct cctgcccatg tgatatggcc acacacccca cccgatgcct ccagagctgc 33480

ccatgagcag ggagcttgag gaccctgggg aaagtaagat gggtgtccca gctgtgggag 33540

aaggtcttca ccatgcctgc cctgcccatc agccgcatcc aggtgagact gggagagcac 33600

aacgtcgaag ttctggaggg gaatgagcag ttcatcaatg cagccaagat catccgccac 33660

cccaaataca acagttggac tctggacaat gacatcctgc tgatcaagct ctccatgcct 33720

gctgtcatca atgcccacat gtccaccatc tctctaccca ccgcccctcc agctgctggc 33780

accaagttcc tcatctctgg ctagggcaac actctgagct ctggtggtga gtgggaccct 33840

ttgtccttct ccttccctcc atcctcacaa tttccagaac aaaccatgcc ccttaacttg 33900

aatcctctca cctccaggct taagacacat ttctagtgcc cattacacac aggctgtaca 33960

gtggatggac atcagagaga tgcaaagtct caaggacttg tcgacccatc cagctgcagc 34020

tgtcaaacat gagaattaca acttatatcg tatggggctg acttcaggtg ctacatttga 34080

agagataaat tgcactgaaa tctagaaata ttttatctga ttaataagat gatcttcttg 34140

agatcgtttt ggtctgcgcg taatctcttg ctctgaaaac gaaaaaaccg ccttgcaggg 34200

cggtttttcg aaggttctct gagctaccaa ctctttgaac cgaggtaact ggcttggagg 34260

agcgcagtca ccaaaacttg tcctttcagt ttagccttaa ccggcgcatg acttcaagac 34320

taactcctct aaatcaatta ccagtggctg ctgccagtgg tgcttttgca tgtctttccg 34380

ggttggactc aagacgatag ttaccggata aggcgcagcg gtcggactga acggggggtt 34440

cgtgcataca gtccagcttg gagcgaactg cctacccgga actgagtgtc aggcgtggaa 34500

tgagacaaac gcggccataa cagcggaatg acaccggtaa accgaaaggc aggaacagga 34560

gagcgcacga gggagccgcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 34620

cgccaccact gatttgagcg tcagatttcg tgatgcttgt caggggggcg gagcctatgg 34680

aaaaacggct ttgccgcggc cctctcactt ccctgttaag tatcttcctg gcatcttcca 34740

ggaaatctcc gccccgttcg taagccattt ccgctcgccg cagtcgaacg accgagcgta 34800

gcgagtcagt gagcgaggaa gcggaatata tcctgtatca catattctgc tgacgcaccg 34860

gtgcagcctt ttttctcctg ccacatgaag cacttcactg acaccctcat cagtgccaac 34920

atagtaagcc agtatacact ccgctagcgc tgaggtctgc ctcgtgaaga aggtgttgct 34980

gactcatacc aggcctgaat cgccccatca tccagccaga aagtgaggga gccacggttg 35040

atgagagctt tgttgtaggt ggaccagttg gtgattttga acttttgctt tgccacggaa 35100

cggtctgcgt tgtcgggaag atgcgtgatc tgatccttca actcagcaaa agttcgattt 35160

attcaacaaa gccacgttgt gtctcaaaat ctctgatgtt acattgcaca agataaaaat 35220

atatcatcat gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg 35280

agccatattc aacgggaaac gtcttgctcg aggccgcgat taaattccaa catggatgct 35340

gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat 35400

cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt 35460

gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt 35520

ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc 35580

cccgggaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt 35640

gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt 35700

aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt 35760

gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa 35820

atgcataagc ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt 35880

gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga 35940

atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct 36000

tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg 36060

cagtttcatt tgatgctcga tgagtttttc taatcagaca tgttctttcc tgcgttatcc 36120

cctgattctg tggataaccg tattaccgct agcatggatc tcggggacgt ctaactacta 36180

agcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg gaagactggg 36240

cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg 36300

gagcggattt gaacgttgtg aagcaacggc ccggagggtg gcgggcagga cgcccgccat 36360

aaactgccag gcatcaaact aagcagaagg ccatcctgac ggatggcctt tttgcgtttc 36420

tacaaactct tcctgttagt tagttactta agctcgggcc 36460

Claims (1)

1. A method for constructing a mouse model of human acute pancreatitis is characterized by comprising the following steps:

constructing a targeting vector plasmid containing PRSS1 gene, and electrically transferring the targeting vector plasmid containing PRSS1 gene into mouse embryonic stem cells to obtain the mouse embryonic stem cells carrying recombinant PRSS1 gene; microinjecting the mouse embryonic stem cells carrying the recombinant PRSS1 gene into albino mouse blastocyst, transferring the injected blastocyst into a pseudopregnant female mouse uterus for future birth, and obtaining a chimeric mouse after transplantation; combining a male chimeric mouse with a chimeric rate of more than 50% with a wild C57BL/6J female mouse, breeding to generate a next generation mouse, and screening a mouse carrying a PRSS1 gene, namely a mouse model simulating human acute pancreatitis; the nucleotide sequence of the targeting vector plasmid containing the PRSS1 gene is shown as SEQ ID NO. 1.

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