CN112194717A - Peptide segment Tr for promoting regeneration of cochlear hair cells and application thereof - Google Patents

Abstract

The invention discloses a peptide segment Tr for promoting regeneration of cochlear hair cells and application thereof. According to the invention, by copying a peptide segment Tr and constructing an Anc80L65-Tr virus, one month after injecting the Anc80L65-Tr virus into the inner ear of a P2 young mouse, new hair cells appear near the inner hair cells of the mouse, and SEM data shows that the new hair cells show certain morphological characteristics of the inner hair cells. The surface of the new hair cell is provided with bundled static cilia, the static cilia on the surface of part of the cell is in a ladder-shaped arrangement, and the peptide section Tr has the function of promoting the regeneration of the hair cell. The discovery finds a new treatment direction for sensorineural deafness, realizes the recovery of auditory function in the true sense for replacing the artificial cochlea, and has great clinical potential application value.

Description

Peptide segment Tr for promoting regeneration of cochlear hair cells and application thereof

Technical Field

The invention belongs to the field of biological inner ear research, and particularly relates to a peptide segment Tr for promoting regeneration of cochlear hair cells and application thereof.

Background

Hearing loss is a common sensory deficit affecting more than 6.8% of the world's population. More than half of sensorineural deafness is caused by genetic mutations in hair cells and supporting cells. The main causes of nongenetic sensorineural deafness include noise exposure, ototoxic drug exposure and presbycusis. Sensorineural deafness is mainly manifested by damage to the normal structure of the cochlea, especially the hair cells. It is generally believed that hair cell damage is irreversible.

At present, the deafness treatment method mainly takes instrument assistance as a main treatment method, and the cochlear implant is a main technical tool for restoring the auditory function of a patient at present. For severe SNHL patients, cochlear implants can deliver signals generated by hair cells to residual spiral neurons (SGNs) by electrical stimulation at different frequencies. At present, no other effective means is available for clinically treating hereditary deafness except wearing a hearing aid and implanting a cochlear prosthesis. The signals produced by the cochlear implant are very different from those produced by normal cochlear hair cells, and require long-term rehabilitation to maximize the benefit from the implant. Also, up to 50% of cochlear implants experience progressive hearing loss (i.e., hair cell loss) following cochlear implant. This may occur shortly after implantation as a result of surgical trauma. Furthermore, many patients are reluctant to accept implants because cochlear implants permanently leave the signal receiver device on the surface of the head. Therefore, biological treatment methods for recovery of auditory function are in great demand. It is desirable to recover the number and function of hair cells directly from the inner ear to achieve a hearing recovery effect.

Regenerative medicine, which is centered on stem cells, provides a promising solution for us. The study finds that Lgr5 positive support cells in the inner ear are inner ear stem cells, and the capacity of the inner ear stem cells for regenerating hair cells can be improved after some signal paths in the inner ear stem cells, such as Wnt, Aoth1 and the like, are regulated. Although the prior art can regenerate part of the supporting cells into hair cells, the effect is not ideal, and the main difference is that the stability and the functionality of the regenerated hair cells are different from those of normal hair cells. The molecular mechanisms involved in hair cell regeneration are not well understood, and we cannot precisely intervene from the genetic level to generate functional new hair cells. The regeneration process of inner ear hair cells is extremely complex, is probably the result of the interaction of multiple genes and multiple signal paths, has strong unknown property and higher exploration depth, and needs a longer time to explore the complex process.

Disclosure of Invention

The invention provides a peptide segment Tr for promoting the regeneration of cochlear hair cells and application thereof, aiming at the problem of hair cell regeneration in the auditory system of mammals, and the application fundamentally solves the basic problem of the auditory system, so that animals with hearing impairment can achieve the effect of recovering hearing, thereby replacing artificial cochlea to realize the recovery of auditory function in the true sense.

A peptide stretch Tr, which has an activity of promoting regeneration of cochlear hair cells, and the amino acid sequence of the peptide stretch Tr is as shown in SEQ ID NO: 1 is shown.

SEQ ID NO：1：

PSQRQWVSSATSANDSFEIRPSSKPDMETIPIGDLQARALANLRVNSRNSFVLIPKRKAPGNYPLAGRQFEEPKGEVGWASQSQGLGSQLVSTVDGAPALEKSPLAAEMQWAVRKGACPRPAISDTDKCVRWQRPASPPPFLPATAEAEPAEGLGVPGLAKNGQEPVRPGLPVTFIDEVDSEEEAF

A set of isolated nucleic acid molecules encoding the peptide stretch Tr of claim 1, having a nucleic acid sequence as set forth in SEQ ID NO: 2, respectively.

SEQ ID NO：2：

CCAGGCTTCCCACCCGCTGTGCCGCCCGCTGCGGGCATCCGCGCTGCCGAGGTAGTAGTGTACGAGGCGCCACAGCCCGGCCGTGTCAGCCGCCTGCTGGAAAAGTTCGACTCGCCTGCTGCGCCCTGCCGCCGCGGGAGCCCAGAGCGCTTCCGCCCTGCGCTCCCGCAGCTTCCCGTGGCGTCTGCATCTGCTGCCACGCGCACTCCTACCAATCGGTCGTTGGCTCCTGCCTCACCGGTGCGCCTCAGCCAGCCCGCACCCCCTATTTCGCCTGTCCCCGTTGCCCAGCGCGCGGGTCAGCGCTCTGCCTGTTGCGAGCCCGCGCACCCCGACGGCACTGCCGGCCCCGGGGCCCGGCGCAGCGACTTTTTACAGAAGACCGGCAGCAACTCTTTCACTGTTCACCCCCGGGGCCTGCCCCGCAGTGCGGTCAATCGCTCGCTTTCTAATGGACCCATGACTCAGGAATCCCCTACCGGCCCTGCCAATGGGTTGTCAGGCTCTCCGCCTGTACCGGGAAAGTGGAAGCCAAAGGTGGAGTCAAAGGAACCCTCTCTCCACCCGCCCCCAAGCCCTGGGACTCCAAGTGCCACTTCAGTTGGGCCCCCTGCCTTCCCAGCGCCCAGCCCAGCCAGTGCCACTCCC

A recombinant plasmid incorporating a nucleotide sequence encoding the above-mentioned peptide stretch Tr.

A recombinant vector transformed with the above recombinant plasmid, said recombinant plasmid incorporating a nucleotide sequence encoding the above peptide Tr.

The application of the peptide segment Tr in preparing related products for treating sensorineural deafness.

As an improvement, the product is a pharmaceutical product.

Has the advantages that:

compared with the prior art, the peptide segment Tr for promoting the regeneration of cochlear hair cells and the application thereof have the following advantages:

according to the invention, by copying a peptide segment Tr and constructing an Anc80L65-Tr virus, one month after injecting the Anc80L65-Tr virus into the inner ear of a P2 young mouse, new hair cells appear near the inner hair cells of the mouse, and SEM data shows that the new hair cells show certain morphological characteristics of the inner hair cells. The surface of the new hair cell is provided with bundled static cilia, the static cilia on the surface of part of the cell is in a ladder-shaped arrangement, and the peptide section Tr has the function of promoting the regeneration of the hair cell. The discovery finds a new treatment direction for sensorineural deafness and realizes the recovery of auditory function in the true sense for replacing the artificial cochlea, thereby having great clinical application value.

In addition, the recombinant adeno-associated virus vector derived from non-pathogenic wild AAV has the advantages of low pathogenicity, high safety, weak immunogenicity, stable physical property, stable expression in a host and the like, and has a great application prospect in gene therapy research and development.

Drawings

FIG. 1 is a schematic representation of the promotion of transdifferentiation of inner ear stem cells into hair cells by Tr peptide fragments;

FIG. 2 is a schematic representation of the immunofluorescent staining of HA and Phalloidin following Anc80-Tr-HA infection of cochlear tissue;

FIG. 3 is a schematic scanning electron microscope image of Ant 80-Tr-HA infected cochlear tissue and its control.

Detailed Description

Materials and sources:

young mice: the Nanjing university model animal institute; AAV vector: the Nanjing Kinshire company is synthesized and constructed; donkey serum: shanghai assist, Sheng Biotech Co., Ltd;

dilution ratio of each antibody to staining:

a first antibody: myosin 7A (Myo7A, #25-6790Proteus Biosciences, 1:1000), HA (HA, #11867423001, Roche, 1: 200);

secondary antibody: secondary antibody labeled anti-rat secondary antibody carrying non-Alexa 555 fluorescent molecule, from thermo fisher;

actin staining reagent: the actin dye phallodin carrying Alexa 488;

cell culture reagents: DMEM (Hyclone), fetal bovine serum (Lensa), diabody (ThermoFisher);

cell and tissue culture consumables: including various culture dishes, centrifuging tube, pipette, general consumptive materials such as disposable filter, purchase in Corning company.

EXAMPLE 1 construction of AAV packaging plasmid pAAV-CAG-Tr-eGFP-WPRE-SV40 comprising peptide fragment

Packaging of AAV requires three plasmids: genome plasmid containing target gene, Rep-Cap plasmid and Helper plasmid

And (4) granulating. Therefore, it is necessary to clone the Tr peptide fragment into the plasmid of the target gene first.

Step 1, firstly, a cDNA sequence corresponding to the Tr sequence is amplified by Polymerase Chain Reaction (PCR). The template was derived from mouse cochlear cDNA,

the primers were designed as follows:

the upstream primer is shown as SEQ ID NO: 3, showing:

5’---TTGGCAAAGAATTGGATCGAATTCGCCACCATGCCAGGCTTCCCACCCGCTGT---3’；

the downstream primer is shown as SEQ ID NO: 4, and (2) is as follows:

5’---AGGAACATCGTATGGGTAGGATCCGGGAGTGGCACTGGCTGGGCT---3’；

wherein, the bold font is the restriction enzyme cutting site, the straight line sequence is drawn as the homologous arm of the carrier, and the dotted line sequence is the Tr PCR primer sequence really combined with the cDNA template; the Tr sequence was designed to have the same restriction sites at both ends as the linear vector plasmid.

The PCR reaction used the Phanta Max Super-Fidelity DNA Polymerase kit (# P505) from Vazyme, which contained all the required reagents except primers and template. PCR systems and procedures were used according to kit instructions; the whole PCR reaction system is set to be 30 mu L; the primers were synthesized by Nanjing Kinshire corporation (www. genscript. com. cn).

Step 2, the vector plasmid was linearized using restriction enzymes (ThermoFisher Corp.), the two ends of the linearized plasmid carrying EcoR1 and BamH1 cleavage sites.

And 3, performing agarose gel electrophoresis on the PCR product and the linear plasmid, performing gel tapping recovery, recovering DNA by using a gel recovery kit (Axygen: AP-GX-250G), and detecting the concentration of the recovered fragment by using Nanodrop 2000.

And 4, carrying out recombination and connection (Novoprotein: NR001A) on the recovered PCR product and a linear plasmid vector to obtain circular plasmid DNA. The linking system is as follows: 2 mu L of buffer, 30ng of linear framework vector, 20ng of DNA fragment of Tr peptide fragment, 0.5 mu L of recombinant ligase and ddH₂The amount of O was adjusted to 10. mu.L, and the reaction was carried out at 50 ℃ for 20 minutes.

Step 5, transformation of the recombinant product

The conversion steps are as follows: 100 μ L of competent Stbl3 cells (TransGen: CD201) were thawed on ice; 10 μ L of the ligation product of step 4 was gently mixed with competent cells and left on ice for 30 min; heat shock at 42 ℃ for 60 seconds; placing on ice for 2 minutes, adding 900 μ L resuscitation non-resistant LB medium (MDBio: L001-1kg), and shaking for 45 minutes; after centrifugation (4000 g, 2 min), 900uL of the supernatant was discarded, and the remaining supernatant was used to resuspend the cells, applied to an ampicillin plate (50. mu.g/ml), and cultured in a 37 ℃ incubator for 14-16 hours.

Step 6, monoclonal colonies on the plate were picked the next day, expanded in 4mL LB medium for ampicillin resistance and extracted for small plasmid quantities according to the kit instructions (Axygene: APMN-P-250G). The obtained plasmid is subjected to concentration detection, 10 mu L of the obtained plasmid is taken for sequencing, and the positive plasmid is pAAV-CAG-Tr-eGFP-WPRE-SV40 and then stored at the temperature of-20 ℃ for later use.

Example 2 packaging of Anc80L65-Tr Virus in HEK293 cells

Step 1, passage of HEK 293T cells

10cm dishes (90% -100% confluency) were passaged to 15cm dishes at night and after 24h 25kD PEI (1 mg/mL, Polyscience, # 23996) transfection was performed. The medium comprises DMEM High Glucose (Biological Industries, #01-052-1 ACS) +10% FBS (Biological Industries, # 04-007-1A) +1% P/S (penillin-Streptomyces, # 15140122)

Step 2, PEI transfection

Respectively mixing PEI or plasmid DNA with DME (vortex, 3000rpm, 5 s), standing at room temperature for 5min, then suspending PEI, dropwise adding the PEI into the DNA, gently mixing uniformly, and standing at room temperature for 20-25 min;

step 3, dropwise adding the PEI/DNA compound into a 15cm culture dish to avoid violent shaking of culture solution;

and 4, sucking out the culture solution after 12-16h, washing once by using preheated PBS, replacing the culture medium with preheated 1% FBS to continuously culture for 72-96h, and finally collecting the supernatant and the cells.

Example 3 purification of Anc80L65-Tr-eGFP using gradient iodixanol ultracentrifugation

Step 1, cell lysis and AAV release

Collecting supernatant and cells, lysing the cells by using a circulating cold-hot alternating method, alternately placing the cells in ethanol precooled by dry ice and water bath at 37 ℃ for three times, and then centrifuging at 4 ℃ for 1167g for 15 min;

step 2, removal of genomic and plasmid DNA

Transferring the supernatant to a 50mL centrifuge tube, adding DNase and RNase to the final concentrations of 10U/mL and 10mg/mL respectively, and incubating at 37 ℃ for 30 min;

step 3, centrifuging for 20min at 4 ℃ and at the speed of 13490 rpm;

step 4, filtering

Filtering the supernatant from the last centrifugation step using a sterile 50mL syringe and a 0.22um filter;

step 5, preparing gradient ioxol

8mL of 15% (v/v) iodixanol, 5.5mL of 25% (v/v) iodixanol, 5mL of 40% (v/v) iodixanol and 4.5mL of 60% (v/v) iodixanol; in the step, iodixanol stimulates eyes, skin and respiratory tract, and needs to be protected and carried out in a fume hood;

step 6, carefully dripping the supernatant obtained in the step 4 on the surface of 15% iodixanol;

step 7, ultracentrifugation

Centrifuging for 1 h 40 min at 12 deg.C at 301580 g speed by using fixed angle titanium rotor, and using maximum acceleration and deceleration during centrifugal acceleration and deceleration;

step 8, AAV acquisition

Purified AAV is obtained between 40% and 60% iodixanol interface, and is carefully inserted and sucked by stainless steel blunt needle;

step 9, AAV titer determination

The titer of AAV was measured by QPCR, and the content was expressed as GC/mL, GC was a oligonucleotide concentration, and it was confirmed that the titer of the purified virus was 10e +12GC/mL or more. The primer for QPCR was designed in WPRE elements.

The primer sequence is as follows:

the upstream primer is shown as SEQ ID NO: and 5, as follows: 5'-GTCAGGCAACGTGGCGTGGTGTG-3', respectively;

the downstream primer is shown as SEQ ID NO: 6, showing: 5'-GGCGATGAGTTCCGCCGTGGC-3' are provided.

Example 4 injection of Anc80L65-Tr into the cochlear canal of a young mouse (P2-P3) by round window injection

The newborn mice are anesthetized by adopting a low-temperature induction anesthesia method. P2 (2 days after birth) mice were placed in an ice bath for 2-3min, and removed on an ice pad for subsequent surgical procedures. Surgery was performed only on the left ear of each mouse, and the right ear was a negative control. During surgery, the round window is exposed at the posterior auricular incision of the left ear. Care was taken to avoid damage to the facial nerve during surgery. Then, Anc80L65-Tr was injected from the round window into the cochlea through a capillary glass electrode (10 mm diameter) using a microinjection system (Nanoliter 2000, WPI). The cochlea of the young mouse can contain 2 mu L of the Anc80L65-Tr virus solution, and the injection virus volume is 1-2 mu L. After surgery, the wound was adhered and coated with analgesic and anti-inflammatory agents, and the rats were allowed to recover on a hot plate at 37 ℃.

Example 5 Observation of changes in mouse Hair cells following infection

In mouse P30, cochlea infected with the Anc80L65-Tr virus was subjected to immunofluorescence imaging and SEM imaging, and the regeneration of cochlear auditory hair cells was observed.

Injecting 1.5 μ L of Ant 80L65-Tr virus (the virus titer is 4E +12GC/mL) carrying Tr gene into perilymph fluid of cochlear bone order by using the round window injection technology, and referring to the fourth step in the concrete method. The mice used were suckling mice of the C57BL/6 strain 2 days after birth. After 22 days of injection, virus-infected cochlea was stripped for immunostaining identification. Cochlear samples were fixed in 4% (v/v) PFA and then incubated for 2 hours at room temperature using 1XPBS containing 10% (v/v) donkey serum and 0.5% (v/v) Triton X-100, followed by the addition of HA antibodies and corresponding secondary antibodies. While secondary antibody staining was performed, the actin-labeling dye Phalloidin-488 was added to represent the emerging hair structure on the hair cell surface. The cochlea staining sample adopts an anti-fluorescence quenching agent as a medium sealing sheet, and then is observed by adopting confocal observation.

The results are shown in FIG. 2, the left panel shows HA immunostaining, representing the signal of the Tr peptide. The right panel shows immunostaining of actin filaments. The cells above the white dotted line represent normal in situ hair cells (IHCs), the cells below the white dotted line represent ectopically regenerated hair cells (new hair cells, new HCs), and the scale represents the actual distance of 5 μm.

Meanwhile, after injecting 1.5 uL of Anc80L65-Tr virus (the virus titer is 4E +12GC/mL) by the same method, the cochlea infected with the virus is stripped off for the identification of a scanning electron microscope experiment after 28 days. Cochlear samples were subjected to fixation treatment in 2.5% (v/v) fresh glutaraldehyde, and then the samples were post-fixed using 1% (v/v) osmic acid solution to preserve the complete surface topography of the samples. Then dehydrated by gradient ethanol. The volume concentration of the gradient ethanol is respectively 30%, 50%, 70%, 80%, 90%, 100% and 100% in sequence, and the next ethanol concentration is carried out after each treatment is placed at 4 ℃ for reaction for 10 min. The gradient ethanol treatment was followed by critical point drying for 1 hour 10 minutes and sputtering of metallic Pt to a thickness of about 10nm onto the sample surface. And finally, adhering the sample on the conductive copper paste to perform environmental scanning electron microscope imaging under the condition of 10KV voltage.

The results are shown in fig. 3, and the scanning electron microscope imaging clearly reflects the morphology of the cochlea surface and the morphology of the emerging hair. Three groups are set in the experiment, namely a blank control group (Ctrl), a Tc peptide injection group (Tc peptide and Tr peptide are from different position sequences of the same gene) and a Tr peptide injection group. The cells above the white dotted line represent normal in situ inner hair cells, and the cells below the white dotted line represent ectopically regenerated hair cells. The ≧ represents the regenerative ectopic hair cells, and the surfaces of the oriented ectopic regenerative hair cells have high-low arrangement of static cilia. The result shows that the Tr peptide segment can obviously promote the inner ear stem cells near the inner hair cells to regenerate new hair cells, and the new hair cells have a cilium structure. The scale represents an actual distance of 5 μm.

The DNA sequence of the Tc peptide fragment is shown as SEQ ID NO: 7, and:

AGTCAGCGCCAGTGGGTCTCCTCTGCCACCAGCGCCAATGATTCCTTTGAAATAAGGCCATCCTCCAAGCCAGACATGGAAACTATCCCAATCGGGGACCTTCAGGCCAGGGCCCTGGCCAACCTCCGAGTGAACTCCCGCAACTCCTTCGTGTTGATCCCCAAGCGCAAAGCCCCTGGGAACTATCCTTTGGCGGGGAGGCAATTTGAGGAGCCAAAGGGGGAGGTGGGCTGGGCCTCTCAGAGCCAGGGGCTCGGATCcCAGCTGGTGTCTACAGTGGATGGTGCACCTGCCCTAGAGAAGAGTCCCTTGGCTGCTGAGATGCAGTGGGCAGTTAGGAAGGGGGCCTGCCCCAGGCCAGCAATTTCTGACACAGACAAGTGTGTTAGGTGGCAGAGACCAGCCTCGCCACCTCCATTTCTACCAGCCACAGCTGAAGCTGAGCCGGCTGAAGGCTTGGGGGTTCCTGGCTTGGCCAAGAATGGCCAGGAGCCTGTGAGGCCAGGACTTCCAGTCACCTTCATTGATGAAGTAGACTCAGAGGAGGAGGCC

the amino acid sequence is shown as SEQ ID NO: 8, showing:

SQRQWVSSATSANDSFEIRPSSKPDMETIPIGDLQARALANLRVNSRNSFVLIPKRKAPGNYPLAGRQFEEPKGEVGWASQSQGLGSQLVSTVDGAPALEKSPLAAEMQWAVRKGACPRPAISDTDKCVRWQRPASPPPFLPATAEAEPAEGLGVPGLAKNGQEPVRPGLPVTFIDEVDSEEEA

the experiment shows that the exogenous Tr peptide segment can obviously promote the differentiation of inner ear stem cells to hair cells, and the newly-generated hair cells formed by differentiation have high-low arranged static cilia-like structures.

The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.

Sequence listing

<110> university of southeast

<120> peptide segment Tr for promoting regeneration of cochlear hair cells and application thereof

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 186

<212> PRT

<213> Amino Acid (Amino Acid)

<400> 1

Pro Ser Gln Arg Gln Trp Val Ser Ser Ala Thr Ser Ala Asn Asp Ser

1 5 10 15

Phe Glu Ile Arg Pro Ser Ser Lys Pro Asp Met Glu Thr Ile Pro Ile

20 25 30

Gly Asp Leu Gln Ala Arg Ala Leu Ala Asn Leu Arg Val Asn Ser Arg

35 40 45

Asn Ser Phe Val Leu Ile Pro Lys Arg Lys Ala Pro Gly Asn Tyr Pro

50 55 60

Leu Ala Gly Arg Gln Phe Glu Glu Pro Lys Gly Glu Val Gly Trp Ala

65 70 75 80

Ser Gln Ser Gln Gly Leu Gly Ser Gln Leu Val Ser Thr Val Asp Gly

85 90 95

Ala Pro Ala Leu Glu Lys Ser Pro Leu Ala Ala Glu Met Gln Trp Ala

100 105 110

Val Arg Lys Gly Ala Cys Pro Arg Pro Ala Ile Ser Asp Thr Asp Lys

115 120 125

Cys Val Arg Trp Gln Arg Pro Ala Ser Pro Pro Pro Phe Leu Pro Ala

130 135 140

Thr Ala Glu Ala Glu Pro Ala Glu Gly Leu Gly Val Pro Gly Leu Ala

145 150 155 160

Lys Asn Gly Gln Glu Pro Val Arg Pro Gly Leu Pro Val Thr Phe Ile

165 170 175

Asp Glu Val Asp Ser Glu Glu Glu Ala Phe

180 185

<210> 3

<211> 648

<212> DNA

<213> Gene (Gene)

<400> 3

ccaggcttcc cacccgctgt gccgcccgct gcgggcatcc gcgctgccga ggtagtagtg 60

tacgaggcgc cacagcccgg ccgtgtcagc cgcctgctgg aaaagttcga ctcgcctgct 120

gcgccctgcc gccgcgggag cccagagcgc ttccgccctg cgctcccgca gcttcccgtg 180

gcgtctgcat ctgctgccac gcgcactcct accaatcggt cgttggctcc tgcctcaccg 240

gtgcgcctca gccagcccgc accccctatt tcgcctgtcc ccgttgccca gcgcgcgggt 300

cagcgctctg cctgttgcga gcccgcgcac cccgacggca ctgccggccc cggggcccgg 360

cgcagcgact ttttacagaa gaccggcagc aactctttca ctgttcaccc ccggggcctg 420

ccccgcagtg cggtcaatcg ctcgctttct aatggaccca tgactcagga atcccctacc 480

ggccctgcca atgggttgtc aggctctccg cctgtaccgg gaaagtggaa gccaaaggtg 540

gagtcaaagg aaccctctct ccacccgccc ccaagccctg ggactccaag tgccacttca 600

gttgggcccc ctgccttccc agcgcccagc ccagccagtg ccactccc 648

<210> 3

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ttggcaaaga attggatcga attcgccacc atgccaggct tcccacccgc tgt 53

<210> 4

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

aggaacatcg tatgggtagg atccgggagt ggcactggct gggct 45

<210> 5

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gtcaggcaac gtggcgtggt gtg 23

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggcgatgagt tccgccgtgg c 21

<210> 7

<211> 552

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

agtcagcgcc agtgggtctc ctctgccacc agcgccaatg attcctttga aataaggcca 60

tcctccaagc cagacatgga aactatccca atcggggacc ttcaggccag ggccctggcc 120

aacctccgag tgaactcccg caactccttc gtgttgatcc ccaagcgcaa agcccctggg 180

aactatcctt tggcggggag gcaatttgag gagccaaagg gggaggtggg ctgggcctct 240

cagagccagg ggctcggatc ccagctggtg tctacagtgg atggtgcacc tgccctagag 300

aagagtccct tggctgctga gatgcagtgg gcagttagga agggggcctg ccccaggcca 360

gcaatttctg acacagacaa gtgtgttagg tggcagagac cagcctcgcc acctccattt 420

ctaccagcca cagctgaagc tgagccggct gaaggcttgg gggttcctgg cttggccaag 480

aatggccagg agcctgtgag gccaggactt ccagtcacct tcattgatga agtagactca 540

gaggaggagg cc 552

<210> 8

<211> 184

<212> PRT

<213> Amino Acid (Amino Acid)

<400> 8

Ser Gln Arg Gln Trp Val Ser Ser Ala Thr Ser Ala Asn Asp Ser Phe

1 5 10 15

Glu Ile Arg Pro Ser Ser Lys Pro Asp Met Glu Thr Ile Pro Ile Gly

20 25 30

Asp Leu Gln Ala Arg Ala Leu Ala Asn Leu Arg Val Asn Ser Arg Asn

35 40 45

Ser Phe Val Leu Ile Pro Lys Arg Lys Ala Pro Gly Asn Tyr Pro Leu

50 55 60

Ala Gly Arg Gln Phe Glu Glu Pro Lys Gly Glu Val Gly Trp Ala Ser

65 70 75 80

Gln Ser Gln Gly Leu Gly Ser Gln Leu Val Ser Thr Val Asp Gly Ala

85 90 95

Pro Ala Leu Glu Lys Ser Pro Leu Ala Ala Glu Met Gln Trp Ala Val

100 105 110

Arg Lys Gly Ala Cys Pro Arg Pro Ala Ile Ser Asp Thr Asp Lys Cys

115 120 125

Val Arg Trp Gln Arg Pro Ala Ser Pro Pro Pro Phe Leu Pro Ala Thr

130 135 140

Ala Glu Ala Glu Pro Ala Glu Gly Leu Gly Val Pro Gly Leu Ala Lys

145 150 155 160

Asn Gly Gln Glu Pro Val Arg Pro Gly Leu Pro Val Thr Phe Ile Asp

165 170 175

Glu Val Asp Ser Glu Glu Glu Ala

180

Claims (6)

1. Peptide stretch Tr, wherein the peptide stretch Tr has an activity of promoting regeneration of cochlear hair cells, and the amino acid sequence of the peptide stretch Tr is as shown in SEQ ID NO: 1 is shown.

2. An isolated nucleic acid molecule encoding the peptide stretch Tr of claim 1, having the nucleic acid sequence of SEQ ID NO: 2, respectively.

3. A recombinant plasmid incorporating a nucleotide sequence encoding the peptide Tr of claim 1.

4. A recombinant vector transformed with the recombinant plasmid of claim 3.

5. Use of the peptide stretch Tr according to claim 1 for the preparation of a product related to the treatment of sensorineural deafness.

6. Use according to claim 5, wherein the product is a pharmaceutical product.

Images