阅读说明：本技术 一株促肠蠕动动物双歧杆菌亚种f1-7及应用 (Bifidobacterium subspecies F1-7 for promoting enterokinesia and application thereof ) 是由 张兰威 吕优优 公丕民 刘同杰 易华西 张喆 梁曦 于 2021-05-24 设计创作，主要内容包括：本发明属于食品微生物技术、生物医药领域,公开了一株促肠蠕动动物双岐杆菌亚种F1-7及应用,本发明所述动物双歧杆菌亚种F1-7具有良好的促进肠蠕动功能。本发明还涉及动物双歧杆菌亚种F1-7在制备乳制品、固体饮料、微生态制剂中的应用及含有该菌株的益生菌产品。(The invention belongs to the fields of food microbial technology and biomedicine, and discloses an animal bifidobacterium subspecies F1-7 for promoting intestinal peristalsis and application thereof, wherein the animal bifidobacterium subspecies F1-7 has a good intestinal peristalsis promoting function. The invention also relates to application of the bifidobacterium animalis subspecies F1-7 in preparing dairy products, solid beverages and microecological preparations and probiotic products containing the strain.)

1. Bifidobacterium animalis subspecies F1-7 (A)Bifidobacterium animalis subspF1-7), wherein the Bifidobacterium animalis strain is preserved in China center for type culture Collection, with the preservation date of 2020, 12 months and 2 days, and the preservation number is CCTCC NO: m2020833.

2. The Bifidobacterium animalis F1-7 of claim 1 has good adhesion and excellent tolerance to the simulated gastrointestinal environment.

3. A method for promoting intestinal motility, which comprises using the Bifidobacterium animalis subsp.F 1-7 of claim 1.

4. The bifidobacterium animalis subspecies F1-7 has the function of promoting intestinal peristalsis, and is characterized in that the bifidobacterium animalis subspecies F1-7 can remarkably promote intestinal peristalsis of zebra fish.

5. The Bifidobacterium animalis subspecies F1-7 as claimed in claim 3 is effective in promoting intestinal motility, and is characterized in that the Bifidobacterium animalis subspecies F1-7 is effective in promoting intestinal motility in mice.

6. Use of the bifidobacterium animalis subsp F1-7 of claim 1 in the preparation of dairy products.

Technical Field

The invention belongs to the fields of food microbial technology and biomedicine, and particularly relates to an enterokinesia-promoting animal bifidobacterium subspecies F1-7 and application thereof.

Background

Peristalsis, which refers to the movement of peristalsis like worms, particularly the wavy contractions of the intestine. In recent years, with the increasing pace of life, more and more people suffer from intestinal tract peristalsis dysfunction. The intestinal tract peristalsis is slowed down, so that toxins in the body cannot be discharged out of the body as soon as possible, harmful substances can be accumulated in the body continuously, and long-term peristalsis is slowed down, so that gastrointestinal tract diseases such as intestinal obstruction, constipation and the like can be caused, and cardiovascular and cerebrovascular diseases can be caused. The exact pathogenesis of the peristaltic dysfunction is not clear due to the influence of various factors, and at present, the symptom is improved by mostly relying on purgative, although the effect is quick, the recurrence is easy, and the problem cannot be solved from the root. Therefore, the search for a new way capable of replacing the traditional treatment mode has important practical significance, and the microecological therapy solves various defects of the traditional treatment mode from a brand-new perspective. Research shows that the intestinal flora of patients with intestinal dysfunction is different from healthy people, the abundance of bifidobacterium and lactobacillus is reduced, and the abundance of some potential pathogenic bacteria such as methanogenic archaea and clostridium is increased, so that the changes probably mean disorder of intestinal microecosystem to a certain extent, and the imbalance of the intestinal flora simultaneously aggravates the intestinal dyskinesia. The intestinal flora can produce various metabolites such as short-chain fatty acid and the like through fermenting substrates; or regulate the movement of digestive organs by specific endocrine factors and gastrointestinal hormones. Bifidobacterium and the like as main probiotics planted in intestinal tract at early stage have significance for the development, maturity and establishment of functions of intestinal mucosa immune system and general immune systemThe intended effect. The study of O' Mahony et al found that VSL containing Bifidobacterium^#3The mixed bacteria preparation can promote enterokinesia, and change the composition of intestinal flora, mainly VSL^#3 Promotes the colonization of probiotics in the intestinal tract and inhibits the reproduction of pathogenic bacteria, thereby changing the microenvironment of the intestinal tract and promoting the intestinal peristalsis. Although there are a lot of research reports on the physiological functions of bifidobacteria, there are few reports on the development and detailed mechanism of effective bifidobacteria products promoting intestinal motility. Therefore, the method has extremely strong research significance and application value for screening and developing excellent intestinal peristalsis promoting bifidobacterium products.

In documents and patents or patent applications that have been published so far, for example, CN111466440A discloses a complex strain fermented milk that can be used to improve intestinal motility; CN111493261A discloses a composite bacteria solid beverage for improving intestinal peristalsis; CN111000109A discloses a synbiotic solid beverage for preventing and treating intestinal motility disorder. In view of the prior art, the common points of these patents or patent applications are that a plurality of mixed strains or bacteria are mixed with a plurality of components to prevent or promote the intestinal peristalsis, while the probiotic characteristics, effects and detailed action mechanisms of a single strain which effectively promotes the intestinal peristalsis are still lack of reports, and meanwhile, the preparation forms and product applications of related products are still not wide enough. Through the above analysis, the problems and defects of the prior art are as follows: strains with excellent probiotic properties and peristalsis promoting function have not been effectively developed, and an exhaustive peristalsis promoting mechanism of a single strain is still lack of reports. The difficulty in solving the above problems and defects is: the screening range of the probiotics is wide, the task amount is large, and the potential probiotic characteristics of the existing probiotics are different; meanwhile, the formation reason of the intestinal peristalsis dysfunction is complex, the single animal bifidobacterium capable of effectively promoting the intestinal peristalsis is not effectively developed, and the mechanism is not effectively verified and explored; finally, the preparation forms and applications of the related products are still few. The significance of solving the problems and the defects is as follows: the obtained single strain has outstanding probiotic performance and can effectively promote intestinal peristalsis, the mechanism of the single-strain animal bifidobacterium for promoting the intestinal peristalsis is disclosed, a more efficient product preparation form is developed, the application range is widened, and a reliable theoretical basis and an effective intervention mode are provided for relieving clinical intestinal dysfunction.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a bifidobacterium animalis subspecies F1-7 and application thereof, and particularly relates to a bifidobacterium animalis subspecies F1-7 capable of remarkably promoting intestinal peristalsis and application thereof. The gene sequence of the bifidobacterium animalis subspecies F1-7 is as follows:

GGGTGGGGGGCGTTCTTACACATGCAGTCGAACGGGATCCCTGGCAGCTTGCTGTCGGGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCAACCTGCCCTGTGCACCGGAATAGCTCCTGGAAACGGGTGGTAATACCGGATGCTCCGCTCCATCGCATGGTGGGGTGGGAAATGCTTTTGCGGCATGGGATGGGGTCGCGTCCTATCAGCTTGTTGGCGGGGTGATGGCCCACCAAGGCGTTGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGCGGGATGGAGGCCTTCGGGTTGTAAACCGCTTTTGTTCAAGGGCAAGGCACGGTTTCGGCCGTGTTGAGTGGATTGTTCGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTATCCGGATTTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCCTAACGGTGGATCTGCGCCGGGTACGGGCGGGCTGGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTCACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGCCCTTTCCACGGGTCCCGTGTCGGAGCCAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTGCCGGATCGCCGTGGAGACACGGTTTCCCTTCGGGGCCGGTTCACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCGCATGTTGCCAGCGGGTGATGCCGGGAACTCATGTGGGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAACGCGGTGCGACACGGTGACGTGGGGCGGATCGCTGAAAACCGGTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGGCGGAGTCGCTAGTAATCGCGGATCAGCAACGCCGCGGTGAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGTAGCACCCGAAGCCGGTGGCCCGACCCTTGTGGGGGGAGCCGTCTAAGGGTAAGAACTCG

the invention is realized in such a way that a strain of animal bifidobacterium subspecies F1-7 (with the function of promoting intestinal peristalsis)Bifidobacterium animalis subspF1-7) which is preserved in China center for type culture Collection, wherein the preservation date is 2020, 12 months and 2 days, the preservation number is CCTCC NO: m2020833, status survival.

It is another object of the present invention to provide a bifidobacterium animalis subsp F1-7 with outstanding potential for probiotic properties, characterized by a good adhesive capacity and an outstanding ability to tolerate a simulated gastrointestinal environment.

The invention also aims to provide an application of the bifidobacterium animalis subsp F1-7 in promoting intestinal peristalsis.

The invention also aims to provide a preparation method of the pill for promoting the intestinal peristalsis animal bifidobacterium subsp F1-7.

The invention also aims to provide an application of the bifidobacterium sp F1-7 for promoting the intestinal peristalsis in dairy products, including but not limited to dairy slices and soy milk.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a flow chart of a method for preparing a constipation-improving strain probiotic product according to an embodiment of the present invention; FIG. 2(a) is a schematic diagram of the surface hydrophobicity and self-aggregation capability of probiotic F1-7 provided by the embodiment of the invention; FIG. 2(b) is a schematic diagram of the acid and bile salt resistance of probiotic F1-7 provided by an embodiment of the present invention; fig. 2(c) is a schematic diagram of simulated gastric fluid transport and simulated intestinal fluid transport of probiotics F1-7 provided by the embodiment of the invention; FIG. 3 is a schematic diagram of the colonization effect of probiotics F1-7 in zebra fish according to the embodiment of the invention; FIG. 4 is a schematic diagram of the effect of F1-7 on promoting the intestinal peristalsis of zebra fish provided by the embodiment of the invention; FIG. 5 is a schematic diagram showing the effect of F1-7 on promoting the intestinal peristalsis of mice provided by the embodiment of the invention; FIG. 6 is a schematic diagram of the colonization change of F1-7 in the intestinal tract of a mouse, which is provided by the embodiment of the invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Aiming at the problems in the prior art, the invention provides a bifidobacterium animalis subspecies F1-7 and application thereof, in particular to a bifidobacterium animalis subspecies F1-7 capable of remarkably promoting intestinal peristalsis and application thereof, and the invention is described in detail with reference to the attached drawings.

As shown in fig. 1, the preparation method of the F1-7 for promoting intestinal peristalsis provided by the embodiment of the invention comprises the following steps: s101, F1-7 culture: inoculating bifidobacterium animalis subspecies F1-7 into a bacterium-enriched TPY culture medium of a fermentation tank for culture to obtain fermentation liquor; s102, drying F1-7: sequentially carrying out pre-freezing treatment, main drying treatment and analysis drying treatment on the F1-7 before freezing to finish the preparation of the animal bifidobacterium freeze-dried product; s103, packaging of F1-7: preparing powder F1-7 into pills; s104, application of F1-7: and (3) reasonably proportioning the F1-7 into milk, soybean milk and the like to obtain the milk slice or the soybean milk product.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

Probiotic properties of F1-7: (1) has excellent adhesion capability. And a model strainBifidobacterium animalis 12(BB12), compared with F1-7, the surface hydrophobic capacity reaches 60.74 +/-0.62%, which is close to 67.77 +/-1.39% of BB 12; the self-aggregation ability reaches 13.67 +/-2.57 percent, which is lower than 93.98 +/-0.06 percent of BB12, so that the adhesive property is excellent (FIG. 2 a). (2) Has excellent acid and bile salt resistance. F1-7 can survive well and can realize certain increase after being digested for 3 hours in an acid environment with the pH of 3.00, the increase is up to 101.61 +/-0.04%, which is superior to 101.53 +/-0.03% of BB 12; after digestion of F1-7 in 0.3% bile salt environment for 4 hours, the survival rate was as high as 56.61. + -. 1.07%, approaching 69.03. + -. 0.91% of BB12 (FIG. 2 b). (3) Has excellent tolerance mouldAbility to mimic gastrointestinal fluids. After F1-7 is digested for 3 hours in a simulated gastric juice environment with the pH of 3.00, the survival rate is as high as 100.58 +/-0.03 percent, which is better than 97.52 +/-0.18 percent of BB 12; after subsequent digestion in a simulated intestinal fluid environment at pH 8.00 for 8 hours, good survival was achieved, up to 95.01. + -. 0.22%, close to 95.19. + -. 0.19% for BB12 (FIG. 2 c).

F1-7 functionality: (1) f1-7 has good peristalsis promoting effect on zebra fish. 5dpf zebra fish fries are randomly selected and divided into a Model + LGG group, a Model + F1-7 group, and 48 fries in each group. Each concentration was 10⁸And (5) soaking the zebra fish for 24 hours by using CFU/mL bacterial liquid. At four time points of 0,8,16,24h, 12 seedlings were randomly picked at each time point, washed twice with E3 medium, and placed on agar plates and washed with 5. mu.L gentamicin 100. mu.g/mL for 5 minutes to remove surface bacteria. Washed larvae were broken and plated on MRS plates and incubated overnight at 37 deg.C, colonies were quantified and the average number of cells per tail was calculated. Each set of experiments was performed in triplicate. The in vivo retention capacity of F1-7 was further evaluated using a live zebrafish as a model. After the zebra fish is bathed by F1-7, the zebra fish can reach 2.70 +/-0.00 Log/CFU/Larvae after 8 hours, and is close to a model strain LGG; after 16h, the strain can reach 3.69 +/-0.01 Log/CFU/Larvae, which is obviously higher than that of a model strain LGG; after 24h, 3.09. + -. 0.02 Log/CFU/Larvae can be reached, close to the model strain LGG. Therefore, F1-7 can be effectively colonized in zebrafish bodies (see figure 3). 5dpf of zebra fish fries are selected as experimental objects. Constructing a peristaltic zebra fish Model (Model) by using a loperamide hydrochloride method (the concentration of the loperamide hydrochloride is 10 mu g/mL), and performing microscopic examination on the zebra fish juvenile fish after bathing for 24 h. Selecting successfully constructed zebra fish models and randomly grouping, wherein 12 fish models are selected in each group: the test pieces were divided into 1 Model group, 1 Model + positive drug control group (Phenolphthalain Tablets, 50. mu.g/mL), 1 Model + LGG group, and 1 Model + F1-7 group. At the same time, 1 Normal control group (Normal) was set. Each concentration was 10⁸After 24 hours of soaking and bathing the constipation zebra fish with CFU/mL bacterial solution, observing the times of intestinal peristalsis of each group of zebra fish in unit time (1 min) under an inverted fluorescence microscope. In the observation of intestinal peristalsis at 1min, the first peristalsis peak of the intestinal peristalsis of the zebra fish in the normal group appears at about 28s, while the model group does not appear within 1minThe peristalsis phenomenon appears, which indicates that the peristalsis model is good. After the positive drugs and the strain dry prognosis are applied, the positive drug group has first peristalsis about 22s and second peristalsis about 42 s; the phenomenon of peristalsis is not seen in LGG within 1min, F1-7 shows first peristalsis in about 22s and second peristalsis in about 42s, and the effect is close to that of a positive drug (see figure 4). Therefore, F1-7 has good function of promoting the intestinal peristalsis of zebra fish. (2) F1-7 has good function of promoting intestinal peristalsis for mice. Probiotic intervention for a period of 20 days was performed after successful induction of mouse intestinal motility disorder by Day7 using loperamide hydrochloride method. After the intervention is finished, compared with a model group, F1-7 obviously promotes the intestinal transit rate of mice to 88.19 +/-4.10% from 1.04 +/-1.81% ((the ratio is increased by 1.04 +/-1.81%) (p<0.01), near positive drug levels, superior to the model strain LGG (fig. 5). Using CK as a control, and distributing a large amount of fluorescent dye from the stomach to the colon part within 1 hour; f1-7 was also uniformly distributed throughout the stomach to colon, although not as intense as the fluorochrome. At 3h, the fluorescence intensity of F1-7 in the ileocecal part is increased, which indicates that F1-7 gradually moves from the stomach and duodenum to the jejunum and ileocecum. When the fluorescence is disappeared in 6h, F1-7 duodenum has a large amount of fluorescence, jejunum fluorescence remains, cecum fluorescence intensity is obviously enhanced, and colorectal fluorescence is increased. When the time is 12 hours, the small intestine part of F1-7 still has a little fluorescence, which indicates that a little bacterial strain is still fixedly planted, the fluorescence intensity of the caecum and the colon is more consistent with that of 3 hours and 6 hours, and the fluorescence is still strong, which indicates that F1-7 can be effectively fixedly planted in the large intestine part. In general, probiotic F1-7 was less colonized in the small intestine, including duodenum, and jejunum, and was able to colonize the large intestine, including ileocecum and colon rectum (see FIG. 6).

Probiotic products containing the bifidobacterium animalis subspecies F1-7 of the invention: (1) preparation of pill of Bifidobacterium animalis subspecies F1-7. The preparation method of the bifidobacterium animalis subspecies F1-7 pill comprises the following steps: selecting 5% of jasmine flower honey as a wetting agent, 3-5% of oligosaccharide as a protective agent, dispersing the jasmine flower honey into melted edible antarctic krill oil to form a grease mixture, and adding vitamin E as an antioxidant; then adding the Bifidobacterium animalis subspecies F1-7 jelly into the oil and fat mixtureThe ratio of the dry fungus powder to the mixture of the oil is 1:1-6(m/V), and the mixture is stirred and mixed evenly. Adding gelatin as matrix, stirring in cooled oil phase, dripping into flowing cooled corn oil at 50-55 deg.C through a concentric double-layer nozzle to obtain viable bacteria gelatin dripping pill. Filtering the oil phase, adding CaCl₂(0.1-1mol/L) solution to recover viable bacteria gelatin dripping pill coated with gelatin, washing with 0.9% NaCl solution, filtering, and recovering to obtain viable bacteria gelatin dripping pill. Finally, drying by air blast for 3 hours at 30 ℃ and under the condition that the relative humidity of air is 10 percent, reducing the water content of the product to 8 to 10 percent to obtain a finished product, wherein the viable count in the wet dropping pill is more than or equal to 10⁹CFU/g. (2) Application of Bifidobacterium animalis subsp.F 1-7 in dairy products is provided.

The bifidobacterium animalis subsp.f 1-7 of the invention can be used in the preparation of dairy products, including milk slices, soymilk and the like. However, the dairy product of the present invention is not limited thereto, and includes other forms of dairy products. The various dairy products of the invention can be prepared by taking the bifidobacterium animalis subspecies F1-7 bacterial powder as a raw material and adding the powder in a conventional ratio according to a conventional method in the field.

Example 1: bifidobacterium animalis subspecies F1-7 milk tablet. A probiotic milk tablet for promoting intestinal peristalsis is prepared from the following raw materials in parts by weight: the preparation method of the probiotic milk tablet for promoting intestinal peristalsis comprises the following steps of: inoculating probiotics into a tomato-cabbage juice (80% of tomato juice and 20% of cabbage juice) culture medium containing 3% of peptone, 2.5% of yeast extract powder and 1% of calcium carbonate, culturing for 15h at 37 ℃, centrifuging the bacterium solution subjected to proliferation culture at 4 ℃ at 6000r/min for 5min, removing supernatant, and washing with an equal volume of PBS (phosphate buffer solution) until the thalli are colorless; re-suspending the collected thallus with a freeze-drying protective agent containing 10% of skimmed milk powder, 1.5% of xylitol, 2% of sodium acetate and 6% of sodium glutamate, pre-freezing at-25 ℃ for 1h, then freezing at-80 ℃ for 12h, and placing the thallus in a freeze dryer for freeze-drying at-50 ℃ under 5Pa for 12h in vacuum; collecting lyophilized powder, and placing in a refrigerator at-80 deg.C; crushing 50 parts of full-fat milk powder, 35 parts of defatted milk powder, 5 parts of probiotic powder and 4 parts of xylitol, and then sieving the crushed materials through a 60-mesh sieve to obtain a milk tablet crude mixture; and (3) uniformly mixing 3 parts of sodium carboxymethylcellulose and 2 parts of magnesium stearate with the crude mixture of the milk tablets, tabletting, and packaging by adopting plastic and aluminum foil plates to obtain the probiotic milk tablets.

Example 2: bifidobacterium animalis subspecies F1-7 soybean milk beverage. A bifidobacterium animalis subspecies F1-7 soybean milk beverage for promoting intestinal peristalsis comprises the following steps: fresh camel milk and soybean milk are mixed according to the proportion of 2: 1 proportion, heating to more than 50 ℃, adding 7-9 percent of white granulated sugar, stirring until the white granulated sugar is completely dissolved, preheating at 55-65 ℃, homogenizing under the pressure of 20Mpa, sterilizing at 95 ℃ for 3-5 minutes, cooling to 45 ℃, inoculating 0.005-0.008 percent of leavening agent and 0.004-0.006 percent of bifidobacterium animalis F1-7. Fermenting at 39-44 deg.C to pH of 4.5-4.8, cooling and refrigerating to obtain Bifidobacterium animalis subspecies F1-7 soybean milk beverage.

Finally, it should be noted that: the above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.

Sequence listing

<110> China oceanic university

<120> one strain of Bifidobacterium bifidum subspecies F1-7 for promoting enterokinesia and application thereof

<130> 2021

<150> 2020115856034

<151> 2020-12-29

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1444

<212> DNA

<213> Bifidobacterium animalis subsp. lactis strain

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gggtgggggg cgttcttaca catgcagtcg aacgggatcc ctggcagctt gctgtcgggg 60

tgagagtggc gaacgggtga gtaatgcgtg accaacctgc cctgtgcacc ggaatagctc 120

ctggaaacgg gtggtaatac cggatgctcc gctccatcgc atggtggggt gggaaatgct 180

tttgcggcat gggatggggt cgcgtcctat cagcttgttg gcggggtgat ggcccaccaa 240

ggcgttgacg ggtagccggc ctgagagggt gaccggccac attgggactg agatacggcc 300

cagactccta cgggaggcag cagtggggaa tattgcacaa tgggcgcaag cctgatgcag 360

cgacgccgcg tgcgggatgg aggccttcgg gttgtaaacc gcttttgttc aagggcaagg 420

cacggtttcg gccgtgttga gtggattgtt cgaataagca ccggctaact acgtgccagc 480

agccgcggta atacgtaggg tgcgagcgtt atccggattt attgggcgta aagggctcgt 540

aggcggttcg tcgcgtccgg tgtgaaagtc catcgcctaa cggtggatct gcgccgggta 600

cgggcgggct ggagtgcggt aggggagact ggaattcccg gtgtaacggt ggaatgtgta 660

gatatcggga agaacaccaa tggcgaaggc aggtctctgg gccgtcactg acgctgagga 720

gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg taaacggtgg 780

atgctggatg tggggccctt tccacgggtc ccgtgtcgga gccaacgcgt taagcatccc 840

gcctggggag tacggccgca aggctaaaac tcaaagaaat tgacgggggc ccgcacaagc 900

ggcggagcat gcggattaat tcgatgcaac gcgaagaacc ttacctgggc ttgacatgtg 960

ccggatcgcc gtggagacac ggtttccctt cggggccggt tcacaggtgg tgcatggtcg 1020

tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa ccctcgccgc 1080

atgttgccag cgggtgatgc cgggaactca tgtgggaccg ccggggtcaa ctcggaggaa 1140

ggtggggatg acgtcagatc atcatgcccc ttacgtccag ggcttcacgc atgctacaat 1200

ggccggtaca acgcggtgcg acacggtgac gtggggcgga tcgctgaaaa ccggtctcag 1260

ttcggatcgc agtctgcaac tcgactgcgt gaaggcggag tcgctagtaa tcgcggatca 1320

gcaacgccgc ggtgaatgcg ttcccgggcc ttgtacacac cgcccgtcaa gtcatgaaag 1380

tgggtagcac ccgaagccgg tggcccgacc cttgtggggg gagccgtcta agggtaagaa 1440

ctcg 1444