阅读说明：本技术 环状RNAhsa_circ_0007015在制备预防和治疗慢性肾脏病药物中的应用 (Application of cyclic RNAhsa _ circ _0007015 in preparation of medicine for preventing and treating chronic kidney disease ) 是由 韩飞 聂琬云 林伟强 王毓茜 张颖 于 2021-09-17 设计创作，主要内容包括：本发明公开了环状RNA hsa-circ-0007015在制备预防和治疗慢性肾脏病药物中的应用。hsa-circ-0007015基因的靶向抑制剂可通过RNA干扰诱导hsa-circ-0007015基因沉默,从而减少肾脏纤维化的发生。此种基因治疗法能够显著减少肾小管上皮细胞中纤维化相关蛋白如α-SMA、Collagen I、FN等的表达,同时显著改善成年小鼠缺血再灌注损伤及单侧输尿管梗阻后肾脏纤维化程度。因此,hsa-circ-0007015基因的靶向抑制剂可以起到保护肾脏的作用,其作为新型基因药物能够应用于慢性肾脏疾病的治疗、减轻肾脏纤维化程度、改善肾脏功能等方面。(The invention discloses application of circular RNA hsa _ circ _0007015 in preparation of a medicine for preventing and treating chronic kidney diseases. Targeted inhibitors of the hsa _ circ _0007015 gene can induce silencing of the hsa _ circ _0007015 gene by RNA interference, thereby reducing the occurrence of renal fibrosis. The gene therapy method can obviously reduce the expression of fibrosis-related proteins such as alpha-SMA, Collagen I, FN and the like in renal tubular epithelial cells, and simultaneously obviously improve the degree of renal fibrosis after ischemia-reperfusion injury and unilateral ureteral obstruction of adult mice. Therefore, the targeted inhibitor of the hsa _ circ _0007015 gene can play a role in protecting the kidney, and can be applied to the aspects of treating chronic kidney diseases, reducing the degree of kidney fibrosis, improving the kidney function and the like as a novel gene medicament.)

1. The application of the circular RNA hsa _ circ _0007015 as a molecular marker in preparing a chronic kidney disease diagnostic reagent.

2. Application of circular RNA hsa _ circ _0007015 in preparation of medicines for preventing and treating chronic kidney disease.

Application of hsa _ circ _0007015 gene targeted inhibitor in preparation of drugs for preventing and treating chronic kidney diseases.

4. The use according to claim 3, characterized in that the targeting sequence of the inhibitor is: 5'-TTCTCCGAACGTGTCACGTAA-3' are provided.

5. Use according to claim 3, wherein the inhibitor is an shRNA sequence capable of inhibiting the expression of the hsa _ circ _0007015 gene, the shRNA sequence comprising a sense strand and an antisense strand, the sense and antisense strands being:

sense strand: '-AATTCGCAGGAGGCCACAAGCTGTCTTCAAGAGAGACAGCTTGTGGCCTCCTGTTTTTTG-3';

antisense strand:

5’-GATCCAAAAAACAGGAGGCCACAAGCTGTCTCTCTTGAAGACAGCTTGTGGCCTCCTGCG-3’。

6. the use according to claim 3, wherein the inhibitor is packaged into AAV9 to produce an adeno-associated virus.

7. Use according to any one of claims 1 to 3, wherein the renal disease is an acute renal injury or an incipient stage of chronic renal disease.

(I) technical field

The invention relates to application of circular RNA hsa _ circ _0007015 in preparing a medicament for preventing and treating chronic kidney diseases.

(II) background of the invention

Chronic Kidney Disease (CKD) is defined as a disease in which various causes cause chronic structural or functional impairment of the kidney, which progresses slowly over a period of months to years, eventually leading to chronic renal failure and even death. The incidence of CKD is rising year by year, and is a great public health problem facing all countries. CKD can be secondary to hypertension, diabetes, obesity, and kidney damage from aging. In china, the prevalence of CKD has reached 10.8%. Patients above stage CKD4 need to receive renal replacement therapy including peritoneal dialysis, hemodialysis, renal transplantation, which puts tremendous stress on the patient and society. Therefore, effective prevention and treatment of CKD would bring immeasurable benefits to the patient and society.

In recent years, many studies have found that non-coding RNA has important significance in the development of disease. Circular RNA (circular RNA) in non-coding RNA has important biological functions. Circular RNA is a closed circular structure formed by reverse cleavage of a portion of the exons and/or introns of a linear mRNA, which structure determines its stability. Research has shown that the function of the circular RNA can be achieved through various ways, including competitive inhibition of the molecule sponge microRNA and protein binding so as to regulate the protein, and some circular RNAs can achieve the function through protein translation.

In recent years, many studies have found that non-coding RNA has important significance in the development of disease. Circular RNA (circular RNA) in non-coding RNA has important biological functions. Circular RNA is a closed circular structure formed by reverse cleavage of a portion of the exons and/or introns of a linear mRNA, which structure determines its stability. Research has shown that the function of the circular RNA can be controlled by various ways, including competitive inhibition of the molecule sponge microRNA and protein binding, and some circular RNAs can play the function as translation proteins.

RNA interference (RNAi) technology is a specific gene silencing phenomenon induced by double-stranded RNA (dsRNA) homologous to a target gene sequence. RNAi technology is used to specifically reduce or turn off the expression of a specific gene, and thus has been widely used in the field of gene therapy for exploring gene functions and infectious diseases and malignant tumors. The intracellular transcribed shRNA is processed to incorporate an RNA-induced silencing complex (RISC) that directs nuclease degradation of the target RNA. The shRNA is a hairpin structure which has two short inverted repeats and is composed of a stem loop in the middle, and is commonly used for RNA interference silencing of the expression of target gene mRNA. Viral shRNA vectors can also be used for RNAi interference, and vectors with antibiotic markers can continue to suppress target gene expression in cells for weeks or even longer. Compared with other vectors, the viral shRNA vector can directly infect cells with high efficiency to research gene silencing, has stable transfection effect, and can avoid various inconveniences caused by low plasmid transfection efficiency.

The hsa _ circ _0007015 gene is located in chr1:214625147-214638300, and consists of 2 and 3 exons of the host gene PTPN14, and the sequence length is 498 bp. The complete sequence is: 5'-CTATCTTCCAGAGGGCCACACTGGGCATGGACACCCTTTTCCCTGCCTGGAGGAGCACAGGTGATAGTGTAATTTTCCAGTCACGAAACTGCTAAGGCCATCTCAGGGGCGTGTGCGCCAGGATAGGCGGGCGGCGTCCGAGGACCACATAGCCATGCCTTTTGGTCTGAAGCTCCGCCGGACACGGCGCTACAACGTCCTGAGCAAGAACTGCTTTGTCACACGGATTCGCCTGCTGGACAGCAATGTTATCGAGTGCACGCTGTCGGTGGAAAGCACAGGGCAAGAATGCCTGGAGGCTGTGGCCCAGAGGCTGGAGCTGCGAGAGACGCACTACTTTGGCCTTTGGTTTCTCAGCAAGAGCCAGCAAGCACGATGGGTGGAGCTGGAGAAACCTCTGAAGAAACATCTGGACAAATTCGCTAATGAGCCTTTGCTTTTCTTTGGAGTCATGTTCTATGTGCCAAATGTGTCATGGCTTCAGCAAGAGGCCACAAG-3' at present, the mechanism of action level correlation of hsa _ circ _0007015 gene in the development of kidney fibrosis is not reported, and the application in the gene therapy of kidney fibrosis is not clear, therefore, the research of a hsa _ circ _0007015 gene-related drug for treating kidney fibrosis has the necessity and uniqueness

Disclosure of the invention

Based on the defects in the prior art and the market demand, the invention provides the application of the circular RNA hsa _ circ _0007015 in preparing the medicine for preventing and treating chronic kidney diseases.

The technical scheme adopted by the invention is as follows:

the application of the circular RNA hsa _ circ _0007015 as a molecular marker in preparing a chronic kidney disease diagnostic reagent.

The invention also relates to the application of the cyclic RNAhsa _ circ _0007015 in preparing medicines for preventing and treating chronic kidney diseases. Experiments prove that the hsa _ circ _0007015 gene is highly expressed in the kidney fibrosis tissue, and the inhibition of the expression of the hsa _ circ _0007015 gene can reduce renal tubular injury, extracellular matrix deposition and collagen production and reduce kidney fibrosis.

Specifically, the application is the application of the hsa _ circ _0007015 gene targeting inhibitor in preparation of medicines for preventing and treating chronic kidney diseases.

The targeting sequence of the inhibitor is as follows: 5'-TTCTCCGAACGTGTCACGTAA-3' are provided.

The inhibitor is shRNA sequence capable of inhibiting expression of hsa _ circ _0007015 gene, the shRNA template sequence comprises a sense strand and an antisense strand, and the sense strand and the antisense strand are respectively:

sense strand: 5'-AATTCGCAGGAGGCCACAAGCTGTCTTCAAGAGAGACAGCTTGTGGCCTCCTGTTTTTTG-3', respectively;

antisense strand: 5'-GATCCAAAAAACAGGAGGCCACAAGCTGTCTCTCTTGAAGACAGCTTGTGGCCTCCTGCG-3' are provided.

The invention develops an hsa _ circ _0007015 gene targeted inhibitor by utilizing an RNA interference technology, and the inhibitor can be specifically combined with an hsa _ circ _0007015 gene to silence the gene, so that renal tubular injury, extracellular matrix deposition and collagen production are inhibited, and the aim of treating renal fibrosis is fulfilled. The hsa _ circ _0007015 gene targeted inhibitor plays an important role in the field of kidney fibrosis, and provides a novel targeted therapeutic drug for clinical treatment of kidney fibrosis.

The inhibitor can be packaged into AAV9 to prepare adeno-associated virus. Adeno-associated virus (AAV) is a genus dependovirus of the family parvoviridae, and has a virus particle size of about 20-26 nm. AAV has multiple serotypes, and different serotypes have different affinities for different tissues, so that the AAV9 has high affinity for kidney infection and is suitable for various in vivo infection experiments. AAV has been the most promising gene therapy tool due to its advantages of good safety and long expression time.

The kidney disease includes kidney fibrosis caused by poor repair after acute kidney injury. Further, the kidney diseases include acute kidney injury before and after kidney, primary nephropathy and secondary nephropathy, and the kidney fibrosis is in the early stage during the development process.

The drug may be a recombinant nucleic acid construct that inhibits the hsa _ circ _0007015 gene, which is any suitable vector for gene therapy, including viral vectors or non-viral vectors. The viral vector may be an adenoviral vector.

The pharmaceutical dosage form may be an injection. The pharmaceutical dose is any pharmaceutically therapeutically acceptable dose.

The invention has the following beneficial effects:

(1) the hsa _ circ _0007015 gene targeting inhibitor provided by the invention has strong specificity and high silencing efficiency, and can effectively inhibit the expression level of the hsa _ circ _0007015 gene;

(2) according to the invention, the expression of the hsa _ circ _0007015 gene is inhibited by the hsa _ circ _0007015 gene targeted inhibitor, the injury of a fibrosis stimulating factor to renal tubular epithelial cells is protected, and the generation of collagen and extracellular matrix is reduced, so that the aim of preventing and treating renal fibrosis is achieved;

(3) the medicine for inhibiting the hsa _ circ _0007015 gene is the hsa _ circ _0007015 gene which is not clinically researched, and the necessity and uniqueness of researching the hsa _ circ _0007015 gene related medicine for treating kidney fibrosis are realized.

(IV) description of the drawings

FIG. 1 shows the identification of circular RNA of hsa _ circ _0007015 gene by using sanger DNA sequencing;

FIG. 2 shows that the expression of hsa _ circ _0007015 gene is detected to be increased in 5 days and 14 days of mouse unilateral ureteral obstruction model and 7 days and 14 days of ischemia-reperfusion model by qPCR;

FIG. 3 shows that the expression of hsa _ circ _0007015 gene in kidney fibrotic tissue is significantly reduced after application of hsa _ circ _0007015 gene inhibitor for qPCR and FISH detection.

FIG. 4 is a graph of immunofluorescence assay of kidney tissue using an hsa _ circ _0007015 gene inhibitor to reduce tubular injury, LTL (green) to mark healthy proximal tubular brush border, and KIM-1 (red) to mark injured tubular luminal surface;

FIG. 5 shows that Western Blot and immunohistochemical detection using hsa _ circ _0007015 gene inhibitor resulted in reduced renal Collagen production and reduced extracellular matrix deposition, and α -SMA, Collagen I and Fn are fibrosis marker proteins.

(V) detailed description of the preferred embodiments

The present invention is further illustrated by the following examples, without limiting the scope of the invention thereto.

The model is applied to a plurality of animal models in the research process of kidney fibrosis to simulate the pathological development and development of the kidney fibrosis of a patient, wherein the mouse unilateral ureteral obstruction model is a classical mouse kidney fibrosis model, and the disease is caused by the fibrosis change of renal tubules and interstitium due to renal postrenal obstruction; the ischemia reperfusion model is a pathological model which causes acute injury to the kidney of a mouse and gradually develops into fibrosis in subsequent poor repair and tissue regeneration, and can simulate the initial change of the fibrosis of the kidney in the development process of chronic kidney diseases. The mouse model can be selected to better simulate the development process of the renal fibrosis in clinical diseases. Therefore, the invention carries out disease intervention based on the two models, and the research result can better reflect the clinical relevance and the application potential.

Example 1: sequencing and identifying hsa _ circ _0007015 gene circular RNA

PCR primers are designed on two sides of the reverse connection site of hsa _ circ _0007015, sequences at two wings of the cyclization site of hsa _ circ _0007015 are amplified, and the accurate cyclization site of hsa _ circ _0007015 is obtained through sanger DNA sequencing. The PCR primer sequences were as follows:

hsa_circ_0007015_F:5’-TCGGAGTCATGTTCTATGTGC-3’

hsa_circ_0007015_R:5’-CCTGAGATGGCCTTAGTAGTTTT-3’

the PCR takes mouse kidney cDNA as a template, and the amplification system and conditions are as follows: 2 XPCR MIX (Vazyme company) 20ul, upstream and downstream primers (10mM) 1ul each, cDNA template 2ul, make up with sterilized water 40ul system; pre-denaturation at 94 ℃ for 2min, within 35 cycles: denaturation at 98 deg.C for 30s, annealing at 58 deg.C for 20s, extension at 68 deg.C for 30s, circulation, extension at 68 deg.C for 5min, and storage at 4 deg.C. The PCR product was purified and subjected to sanger DNA sequencing to determine the exact circularization interface of circular RNA hsa _ circ _0007015, as shown in FIG. 1.

The positioning of the hsa _ circ _0007015 gene in chr1:214625147-214638300 is confirmed by an authoritative circular RNA database circBase (http:// www.circbase.org), and the gene consists of exons 2 and 3 of a host gene PTPN 14; PCR primers are designed on both sides of the hsa _ circ _0007015 reverse ligation site, sequences at two wings of the hsa _ circ _0007015 cyclization site are amplified, and the accurate cyclization site (ligation site in figure 1) of hsa _ circ _0007015 is obtained through sanger DNA sequencing.

Example 2: the hsa _ circ _0007015 gene is overexpressed in renal fibrosis

The expression conditions of the hsa _ circ _0007015 gene in normal mouse kidney, two fibrosis model mouse kidneys (mouse ischemia reperfusion and unilateral ureteral obstruction model) and human tubular epithelial cell line HK2 and HK2 after stimulation by TGF beta are respectively detected by a qPCR method. Total RNA of a tissue or a cell line is extracted by using an RNA extraction kit (Yihua corporation), 1ug of RNA is reversely transcribed into cDNA, the cDNA is further used as a template to carry out qPCR detection by a SYBR fluorescent dye method, the PCR primer sequence is used for detecting the amplification cycle number, and GAPDH is used as an internal reference gene to carry out standardization and comparison, and the result is shown in figure 2. The results show that hsa _ circ _0007015 gene expression is elevated in the kidneys of two fibrosis-modeled mice, suggesting a correlation between the hsa _ circ _0007015 gene and renal fibrosis. At the same time, the elevation of the hsa _ circ _0007015 gene in HK2 following TGF β stimulation suggested that the hsa _ circ _0007015 gene was associated with fibrosis and localized to the tubular epithelial cell line.

Example 3: targeted suppression of AAV-9 production for hsa _ circ _0007015 Gene

1. Preparation of AAV9-hsa _ circ _0007015-shRNA

AAV9-hsa _ circ _0007015-shRNA vector was purchased from Hantah Biotechnology (Shanghai) GmbH. The virus type adopted by the adeno-associated virus package is AAV9, and the shRNA sequence of hsa _ circ _0007015 is inserted into the MCS multiple cloning site of pHBAAV-U6-MCS-CMV-EGFP vector (sense strand:

5'-AATTCGCAGGAGGCCACAAGCTGTCTTCAAGAGAGACAGCTTGTGGCCTCCTGTTTTTTG-3', respectively; antisense strand: 5'-GATCCAAAAAACAGGAGGCCACAAGCTGTCTCTCTTGAAGACAGCTTGTGGCCTCCTGCG-3'), obtaining a target vector plasmid, then co-transfecting the target vector plasmid, the pAAV-RC vector plasmid and the pHelper vector plasmid to 293T cells, collecting viruses and purifying to obtain AAV9-hsa _ circ _0007015-shRNA of adeno-associated virus.

2. In vivo injection of AAV9-hsa _ circ _0007015-shRNA

An SPF-grade C57BL/6J male mouse with age of 8-10 weeks is selected for virus injection, a 1% sodium pentobarbital is used for intraperitoneal injection to anaesthetize the mouse, the left kidney is exposed by a back incision, and a Hamilton microsyringe is used for in-situ injection to the kidney, wherein 60ul is obtained at each injection point by 10 ul. The onset of the virus was 3 weeks. After 3 weeks, the kidney RNA was extracted and subjected to the above qPCR assay to detect the expression level of hsa _ circ _0007015 gene, and the results are shown in FIG. 3. The results show that after the AAV9-hsa _ circ _0007015-shRNA, namely hsa _ circ _0007015 gene targeted inhibitor is injected, the expression of the hsa _ circ _0007015 gene is remarkably reduced, which indicates that the hsa _ circ _0007015 gene targeted inhibitor can be successfully prepared and can specifically inhibit the expression of the hsa _ circ _0007015 gene.

3. Fluorescence in situ hybridization technique (FISH)

The mouse injected with the AAV9-hsa _ circ _0007015-shRNA and the control is subjected to fluorescence in situ hybridization to detect the expression level of hsa _ circ _0007015, and the method comprises the following specific steps: after 3 weeks, mice were anesthetized with isoflurane, perfused with sterile PBS, the side kidney of the model was fixed to 4% PFA, transferred to 30% sucrose solution after 24h, embedded with OCT after the kidney had settled, and sectioned into 6um tissue sections on a cryomicrotome.

Dyeing: FISH experiments are carried out by using a Basescope kit (ACD company) and an hsa _ circ _0007015 specific fluorescent probe according to the kit specification, renal tubules are stained after RNA in situ hybridization is finished, the luminal and the basal surfaces of the renal tubules are stained by using renal tubular affinity agglutinin LTL and PNA respectively, sealing pieces containing DAPI are used after the renal tubular affinity agglutinin LTL and PNA are finished, pictures are taken by a Zeiss confocal microscope, and the data are analyzed, wherein the result is shown in figure 3. Confocal microscopy results show that, compared with an injected control virus group, the expression of hsa _ circ _0007015 is obviously reduced after AAV9-hsa _ circ _0007015-shRNA is applied, which indicates that the hsa _ circ _0007015 gene targeting inhibitor can specifically inhibit the expression of the hsa _ circ _0007015 gene.

Example 4: inhibition of hsa _ circ _0007015 gene in mouse ischemia reperfusion injury and unilateral ureteral ligation model reduces kidney injury

1. Ischemia reperfusion model

Selecting an SPF (specific pathogen free) grade C57BL/6J male mouse with the age of 8-10 weeks for molding, placing the mouse on a heating pad with the temperature of 37 ℃ in the molding process, keeping the body temperature, carrying out intraperitoneal injection anesthesia on the mouse by using 1% sodium pentobarbital, exposing a left kidney from a back incision, separating and exposing a left kidney blood vessel, clamping an artery and a vein by using a mouse artery clamp for 30 minutes, confirming the color recovery of the kidney after loosening the clamp, and suturing and disinfecting layer by layer.

2. Unilateral ureter ligation model

Selecting an SPF (specific pathogen free) grade C57BL/6J male mouse with the age of 8-10 weeks for molding, placing the mouse on a heating pad at 37 ℃ in the molding process, keeping the body temperature, carrying out intraperitoneal injection anesthesia on the mouse by using 1% sodium pentobarbital, exposing a left kidney from a back incision, separating and exposing a left ureter, ligating the ureter by using 4-0 silk thread, and suturing and disinfecting layer by layer.

3. Tissue immunofluorescence

And (3) after the model mouse reaches a corresponding time point, carrying out anesthesia by using isoflurane, perfusing by using sterile PBS, fixing the kidney at the model side to 4% PFA, transferring to 30% sucrose solution after 24h, embedding by using OCT after the kidney sinks, and slicing into a tissue section of 6um by using a freezing microtome. Dyeing: OCT on sections was washed off using PBS, blocked and ruptured using 5% donkey serum and 0.5% triton x100, at room temperature for 1 hour. The mixture was diluted with 0.5% donkey serum at 1: KIM-1 primary antibody was diluted at a rate of 500 and incubated overnight at four degrees. After three washes with PBS, anti-coat secondary antibody and LTL dye were incubated at room temperature for 1 hour and finally mounted with mounting medium containing DAPI dye. The images were photographed on a Zeiss confocal microscope and the data were analyzed, the results are shown in FIG. 4. Confocal microscopy results show that compared with a sham operation control group and an AAV 9-no-load control group, the KIM-1 expression is obviously reduced after the hsa _ circ _0007015 gene targeting inhibitor is applied, which indicates that the hsa _ circ _0007015 gene targeting inhibitor can reduce renal tubular injury.

Example 5: inhibition of hsa _ circ _0007015 gene in mouse ischemia reperfusion injury and unilateral ureteral ligation model reduces renal fibrosis

1、Western blot

Extracting total mouse kidney protein by RIPA protein lysate, and quantifying the extracted protein by a BCA protein quantification method; 5% SDS-PAGE concentrated gel and 10% SDS-PAGE separating gel are prepared, and the total loading protein is 20 micrograms; running protein electrophoresis for 80V 40 min and 120V 1 h; rotating the film for 120 minutes by adopting 300 mA; sealing 5% skimmed milk for 30 min; alpha-SMA, Collagen I, Fn and internal reference beta-actin are used as primary antibodies, and the antibody is incubated overnight at 4 ℃; the following day, incubation with anti-Rabbit IgG secondary antibody at normal temperature for 1h, TBST washing 3 times for 10min each time, then subsequent ECL chemiluminescence, data analysis, and results are shown in FIG. 5. WB results show that the expression levels of alpha-SMA, Collagen I and Fn are reduced after the hsa _ circ _0007015 gene targeting inhibitor is used, which indicates that the kidney Collagen production is reduced, the extracellular matrix deposition is reduced, and the kidney fibrosis degree is reduced.

2. Tissue immunohistochemistry

And (3) after the model mouse reaches a corresponding time point, carrying out anesthesia by using isoflurane, perfusing by using sterile PBS, fixing the kidney at the model side to 4% PFA, transferring to gradient ethanol for dehydration after 24 hours, carrying out paraffin embedding, slicing, and cutting into 3um paraffin sections. Dewaxing by using dimethylbenzene, removing benzene by using gradient ethanol, blocking endogenous peroxide by using 1.5% hydrogen peroxide, boiling by using sodium citrate for 20min for antigen repair, blocking for 1 hour by using 5% BSA, diluting alpha-SMA and Fn primary antibody by using 1% BSA, and incubating overnight at 4 ℃; and (3) incubating the mixture for 20min at normal temperature by using anti-Rabbit IgG secondary antibody on the next day, performing DAB color development, performing hematoxylin nucleus staining, performing 1% hydrochloric acid differentiation, performing neutral resin sealing after gradient ethanol and xylene are transparent, photographing by using a Leica upright microscope, and analyzing data, wherein the result is shown in figure 5. Leica upright microscope results show that after the hsa _ circ _0007015 gene targeted inhibitor is used, the expression level of Collagen I and Fn is reduced, and the results indicate that the kidney Collagen production is reduced, the extracellular matrix deposition is reduced, and the kidney fibrosis degree is reduced.

Sequence listing

<110> Zhejiang university medical college affiliated to the first hospital

<120> application of cyclic RNAhsa _ circ _0007015 in preparation of drugs for preventing and treating chronic kidney disease

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 498

<212> DNA

<213> Unknown (Unknown)

<400> 1

ctatcttcca gagggccaca ctgggcatgg acaccctttt ccctgcctgg aggagcacag 60

gtgatagtgt aattttccag tcacgaaact gctaaggcca tctcaggggc gtgtgcgcca 120

ggataggcgg gcggcgtccg aggaccacat agccatgcct tttggtctga agctccgccg 180

gacacggcgc tacaacgtcc tgagcaagaa ctgctttgtc acacggattc gcctgctgga 240

cagcaatgtt atcgagtgca cgctgtcggt ggaaagcaca gggcaagaat gcctggaggc 300

tgtggcccag aggctggagc tgcgagagac gcactacttt ggcctttggt ttctcagcaa 360

gagccagcaa gcacgatggg tggagctgga gaaacctctg aagaaacatc tggacaaatt 420

cgctaatgag cctttgcttt tctttggagt catgttctat gtgccaaatg tgtcatggct 480

tcagcaagag gccacaag 498

<210> 2

<211> 21

<212> DNA

<213> Unknown (Unknown)

<400> 2

ttctccgaac gtgtcacgta a 21

<210> 3

<211> 60

<212> DNA

<213> Unknown (Unknown)

<400> 3

aattcgcagg aggccacaag ctgtcttcaa gagagacagc ttgtggcctc ctgttttttg 60

<210> 4

<211> 60

<212> DNA

<213> Unknown (Unknown)

<400> 4

gatccaaaaa acaggaggcc acaagctgtc tctcttgaag acagcttgtg gcctcctgcg 60

<210> 5

<211> 21

<212> DNA

<213> Unknown (Unknown)

<400> 5

tcggagtcat gttctatgtg c 21

<210> 6

<211> 23

<212> DNA

<213> Unknown (Unknown)

<400> 6

cctgagatgg ccttagtagt ttt 23