CN111424017B - Exosome loading shRNA (short hairpin ribonucleic acid) and construction method and application thereof - Google Patents

Abstract

The invention discloses an exosome for loading shRNA and a construction method and application thereof. The invention clones shRNA sequence capable of effectively silencing target gene into an expression vector to obtain a recombinant expression vector 1; the shRNA sequence comprises a target sequence, a stem-loop structure sequence and a complementary sequence of the target sequence which are connected in sequence; then connecting the gene of the protein A which can be specifically combined with the stem-loop structure with the gene of the exosome membrane protein to obtain a fusion gene sequence, and cloning the obtained fusion gene sequence into an expression vector to obtain a recombinant expression vector 2; and finally, transfecting the cells with the recombinant expression vectors 1 and 2 at the same time, culturing the transfected cells, and collecting exosomes to obtain the exosomes loaded with shRNA. The method is simple and easy to operate, stable in process and good in applicability. The obtained exosome is loaded with a large amount of shRNA and can be used as a high-efficiency drug delivery system.

Description

Exosome loading shRNA (short hairpin ribonucleic acid) and construction method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an exosome for loading shRNA, and a construction method and application thereof.

Background

RNA interference (RNAi) is a process of effectively silencing or inhibiting the expression of a target gene by selective inactivation of the corresponding mRNA of the target gene by double-stranded RNA (dsrna). The silencing mechanism may result in the induction of target mRNA degradation by small interfering rna (sirna) or short hairpin rna (shrna), or the induction of inhibition of translation of specific mrnas by small rna (mirna). Both siRNA and shRNA have been used for gene therapy. Wherein the short hairpin RNA (shRNA) comprises a loop structure, can be processed into siRNA to play a role, and can also specifically realize target mRNA degradation through a complementary binding sequence with the target mRNA. The advantages of shRNA over siRNA include the ability to use viral vectors for transfection, overcoming the difficulty that certain types of cells cannot be transfected, the ability to select inducible promoters to control shRNA expression, and the ability to co-express with reporter genes. In addition, they can reduce off-target effects.

Exosomes (exosomes) are vesicles secreted by cells and having a diameter of 30 to 150nm, and intracellular vesicles formed by the caveolae of the cytoplasmic membrane have the same membrane structure as the cell membrane, can carry a large amount of components such as RNA and protein, and are closely related to the biological functions of cells and the signal transmission between cells. Because of its special structure, compare synthetic drug carrier, the exosome carries out the medicine transportation as drug carrier and has unique advantage, mainly reflects that the membrane structure that is embodied in the exosome is the same with the cell membrane, can improve the efficiency that the medicine got into the cell, can pass through the blood brain barrier even, and the harmful immunoreaction that the exosome arouses simultaneously is extremely low, thereby has fine infiltration detention (EPR) effect to have slow-release effect etc.. At present, research on gene therapy, tumor therapy and the like by carrying siRNA, chemical small molecule drugs and the like by exosome has been tried. The exosome entraps small molecules such as siRNA and the like, and the mode of incubating the exosome and the small molecules is adopted, so that the entrapment rate is generally low.

Therefore, how to realize more efficient loading of the exosome to the shRNA is a key problem to be solved and researched urgently.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a construction method of an exosome for loading shRNA.

Another object of the present invention is to provide an exosome for loading shRNA.

The invention also aims to provide application of the exosome for loading shRNA.

The purpose of the invention is realized by the following technical scheme:

a construction method of an exosome loading shRNA comprises the following steps:

(1) designing and synthesizing shRNA sequence capable of effectively silencing target gene; cloning the shRNA sequence into an expression vector to obtain a recombinant expression vector 1; the shRNA sequence comprises a target sequence, a stem-loop structure (Kt-loop) sequence and a complementary sequence of the target sequence which are connected in sequence;

(2) connecting a gene of a protein capable of being specifically combined with the stem-loop structure with a gene of an exosome membrane protein to obtain a fusion gene sequence, and cloning the obtained fusion gene sequence into an expression vector to obtain a recombinant expression vector 2;

(3) and (3) transfecting the cell with the recombinant expression vector 1 obtained in the step (1) and the recombinant expression vector 2 obtained in the step (2) at the same time, culturing the transfected cell, and collecting an exosome, namely obtaining the exosome loading shRNA.

When the shRNA sequence in the step (1) is designed, a cell is transfected by constructing a recombinant expression vector containing the shRNA sequence, and the function of silencing a target gene is verified.

The sequence of the stem-loop structure in the step (1) is GCTGACCCGAAAGGGCGTGATGC.

The expression vector in the step (1) is preferably pRNAT-U6.1/Neo.

The protein capable of specifically binding to the stem-loop structure described in the step (2) may be at least one selected from the group consisting of RNA-binding protein L7Ae, NHP2 ribonucleoprotein-like protein 1(NH2L1_ HUMAN NHP2-like protein 1), HUMAN nucleoprotein P56(HumanNOP56_ HUMAN Nucleolar protein 56) and HUMAN nucleoprotein P58(HumanNOP58_ HUMAN Nucleolar protein 58); preferably the RNA binding protein L7 Ae.

The amino acid sequence of the RNA binding protein L7Ae is shown in SEQ ID NO. 3.

The nucleotide sequence of the RNA binding protein L7Ae is shown in SEQ ID NO. 4.

The amino acid sequence of the NHP2 ribonucleoprotein-like protein 1 is shown in SEQ ID NO. 5.

The nucleotide sequence of the NHP2 ribonucleoprotein-like protein 1 is shown in SEQ ID NO. 6.

The amino acid sequence of the human nucleoprotein P56 is shown in SEQ ID NO. 7.

The nucleotide sequence of the human nucleoprotein P56 is shown in SEQ ID NO. 8.

The amino acid sequence of the human nucleoprotein P58 is shown in SEQ ID NO. 9.

The nucleotide sequence of the human nucleoprotein P58 is shown in SEQ ID NO. 10.

The exosome membrane protein in step (2) is preferably at least one of CD63, CD81, CD9 and TP 01; more preferably CD 63.

The amino acid sequence of the CD63 is shown in SEQ ID NO. 11.

The nucleotide sequence of the CD63 is shown as SEQ ID NO. 12.

The amino acid sequence of the CD81 is shown in SEQ ID NO. 13.

The nucleotide sequence of the CD81 is shown as SEQ ID NO. 14.

The amino acid sequence of the CD9 is shown in SEQ ID NO. 15.

The nucleotide sequence of the CD9 is shown as SEQ ID NO. 16.

The amino acid sequence of TP01 is shown in SEQ ID NO. 17.

The nucleotide sequence of TP01 is shown in SEQ ID NO. 18.

The connection in the step (2) is preferably through a connector, and the connection sequence is the gene segment of the protein A, the connector and the exosome membrane protein gene segment.

The nucleotide sequence of the linker is:

GGTGGAGGTGGCAGCGGAGGAGGTGGGTCCGGCGGTGGAGGAAGC。

the expression vector in step (2) is preferably pCDNA3.4.

In the step (3), when cells are transfected, the number ratio of the recombinant expression vector 1 to the recombinant expression vector 2 is preferably 1-2: 1-2; more preferably 1: 1.

In the step (3), the method for collecting exosomes is performed by referring to the method and the steps of "exosome extraction" in the step 3 of the step 2 of the chinese patent application CN 201811023079.4.

The construction method of the exosome loading shRNA can be suitable for various target genes. The target gene may be selected as desired, for example, EGFP, bFGF, EGFR, etc.

When the target gene is EGFP, the target sequence is GGCATCAAGGTGAACTTCA.

An exosome loaded with shRNA is obtained by the construction method.

The exosome is applied to a targeted delivery system for gene therapy.

Compared with the prior art, the invention has the following advantages and effects:

1. the invention provides a construction method of an exosome loading shRNA. The invention utilizes a special hairpin structure (Kt-loop) of shRNA and a protein capable of being specifically combined with the Kt-loop to purposefully pull the shRNA into an exosome by a plasmid transfection method, thereby establishing a method for loading a new shRNA into the exosome. The method can obviously improve the loading efficiency of shRNA and the drug-loading rate of exosome, and has the advantages of simple and easy operation, stable process and good applicability.

2. The invention also provides an exosome for loading shRNA, wherein a large amount of shRNA is loaded in the exosome, and the exosome can release the shRNA into a receptor cell in a membrane fusion mode, can be used as an efficient drug delivery system and is applied to researches such as gene therapy and tumor therapy.

Drawings

FIG. 1 is a photograph showing the results of fluorescence from cells in which EGFP was silenced by shRNA in example 1, wherein A is a cell which had not been transfected with a plasmid, B is a cell which had been transfected with pcDNA3.4-EGFP plasmid and pRNAT-U6.1-sh-N plasmid at the same time, and C is a cell which had been transfected with pcDNA3.4-EGFP plasmid and pRNAT-U6.1-Kt-shEGFP plasmid at the same time.

FIG. 2 is an analysis diagram of the result of NTA detection of exosomes.

FIG. 3 is a Western blot identification result of marker proteins in exosomes, wherein a lane 1 is a Kt-shEGFP + pCDNA3.4 group, and a lane 2 is a Kt-shEGFP + CD63-L7Ae group.

FIG. 4 is an analysis chart of the results of detection of shRNA in exosomes in example 6.

FIG. 5 is a Western blot analysis of EGFP expression in example 7, wherein lanes 1-3 are control, lanes 4-6 are Kt-shEGFP + CD63-L7Ae, and lanes 7-9 are Kt-shEGFP + pCDNA3.4.

FIG. 6 is a graph showing analysis results of a gray scale scan of Western blot for EGFP expression in different treatment groups in example 7.

FIG. 7 is a graph showing the results of flow cytometry, wherein the control group, the Kt-shEGFP + CD63-L7Ae group and the Kt-shEGFP + pCDNA3.4 group each had 3 replicates.

FIG. 8 is a histogram of flow cytometry detection.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Example 1 construction and validation of shRNA vectors for silencing EGFP proteins

DNA fragments for chemical synthesis of shRNA

(1) sh-N (non-targeting shRNA as negative control) (SEQ ID NO: 1);

(2) Kt-sh-EGFP (shRNA silencing EGFP with Kt loop) (SEQ ID NO: 2);

the above sequences were chemically synthesized by Ribobio, constructed and ligated between BamHI and HindIII cleavage sites of vector pRNAT-U6.1/Neo (purchased from Ribobio), to obtain recombinant plasmids pRNAT-U6.1-shN and pRNAT-U6.1-Kt-shEGFP, respectively.

Example 2 construction of EGFP-expressing plasmid

Entrusted Suzhou Hongxn Biotechnology Limited company to synthesize the EGFP gene, and the EGFP gene is constructed on an expression vector pcDNA3.4 (purchased from Invitrogen company) to obtain a recombinant plasmid pcDNA3.4-EGFP, which is specifically operated as follows:

1. designing a primer:

EGFP-F:ATAAAAGGTACCATGGTGAGCAAGG；

EGFP-R:ATAAAAGGATCCTTACTTGTACAGCTCG；

EGFP gene amplification:

EGFP-F and EGFP-R primers are used for amplifying the EGFP gene, so that the 5 'end of the amplified target gene is provided with a KpnI enzyme cutting site of a pCDNA3.4 vector, and the 3' end is provided with a BamHI enzyme cutting site, wherein the amplification system is shown in the following table 1, and the amplification conditions are shown in the following table 2.

Table 1 amplification system:

TABLE 2 amplification conditions

3. Enzymatic ligation of the vector

The target gene and the pCDNA3.4 vector are subjected to double enzyme digestion reaction, the target gene double enzyme digestion reaction system is shown in the following table 3, and the pCDNA3.4 vector double enzyme digestion system is shown in the following table 4.

TABLE 3 Dual enzyme digestion reaction System for target genes

TABLE 4 pCDNA3.4 vector double digestion reaction System

The specific conditions are as follows: the digestion was carried out at 37 ℃ for 30 min.

After double digestion, the recombinant pCDNA3.4-EGFP vector was constructed by enzymatic ligation using T4 ligase, the enzyme ligation system is shown in Table 5 below.

Table 5 enzyme linked systems:

the specific conditions are as follows: ligation was performed overnight at 16 ℃.

4. And (3) transformation and identification of the recombinant plasmid:

transferring the enzyme-linked product into E.coli DH5 alpha (bio-engineering (Shanghai) Co., Ltd.) for competent culture for 12 hours, and selecting a monoclonal extracted plasmid to be sent to the bio-engineering (Shanghai) Co., Ltd.) for sequencing identification. The sequencing result shows that the sequence is correct, and the recombinant pCDNA3.4-EGFP vector is successfully constructed.

Example 3 validation of shRNA silencing EGFP

2 shRNA plasmids (pRNAT-U6.1-shNP and pRNAT-U6.1-Kt-shEGFP plasmids) and expression EGFP plasmids (pcDNA3.4-EGFP) are respectively co-transfected into 293T cells (purchased from Biotechnology engineering (Shanghai) GmbH), the untransfected 293T cells are used as blank controls, and the specific operation steps are as follows:

1. one day before transfection, each well in 6-well plates was plated at 1X 10 ⁶ 293T cells were cultured overnight in DMEM medium (Gibco) containing 10% fetal bovine serum (Gibco).

2. The next day, the culture medium in the 6-well plate was aspirated and a suitable amount of serum-free DMEM medium was added immediately before the transfer.

3. Adding plasmids with the total amount of 2 mug (the quantitative ratio of the two plasmids is 1:1) into each hole, and calculating the amount of the plasmids;

4. calculating the amount of PEI (Polysciences, MW25000) which is a required transfection reagent according to the amount of the plasmid, and preheating the PEI in hot water at 60 ℃; (DNA: PEI concentration ratio of 1: 2);

5. taking a sterilized 1.5mL EP tube, diluting needed PEI and plasmid DNA respectively with serum-free DMEM, adding the PEI and the plasmid DNA into the EP tube, mixing, and standing the mixed solution for about 5 min;

6. adding the DNA/PEI mixed solution into the cell suspension to ensure that the cells are fully contacted with the DNA/PEI complex and are uniformly mixed;

7. at 37 ℃ 5% CO ₂ Culturing under culture condition, and supplementing new culture after 3 hrFresh DMEM culture medium containing 10% fetal calf serum is added to 5mL and placed on an incubator to be cultured continuously;

8. the fluorescence of the cells was measured the next day after the transient, and the results are shown in FIG. 1, in which the left side of each graph is the observation under visible light conditions and the right side is the observation under fluorescent conditions. As can be seen from the figure, the cells transfected with the expression plasmids pcDNA3.4-EGFP and pRNAT-U6.1-sh-N plasmids had a significantly larger amount of fluorescence (FIG. 1B) compared to the cells transfected with no plasmids (FIG. 1A), indicating that the expression plasmids could well express EGFP and shN could not silence EGFP; meanwhile, the cell transfected with pRNAT-U6.1-Kt-shEGFP + pcDNA3.4-EGFP has greatly reduced fluorescence (figure 1C), which shows that the shRNA of the targeted silent EGFP can inhibit the expression of the EGFP.

Example 4 construction of expression vector for CD63-L7Ae

1. Proteins that can bind to K-turn (Kt sequence)

Searching for proteins that can bind to the Kt sequence, and thenhttps://www.ncbi.nlm.nih.govThe gene library in (1) obtains the corresponding gene sequence:

l7Ae [ Archaeoglobubusfulgidus ]: the amino acid sequence is shown as SEQ ID NO.3, and the nucleotide sequence is shown as SEQ ID NO. 4.

NH2L1_ HUMAN NHP2-like protein 1: the amino acid sequence is shown as SEQ ID NO.5, and the nucleotide sequence is shown as SEQ ID NO. 6.

Humanop 56_ HUMAN Nucleolar protein 56: the amino acid sequence is shown as SEQ ID NO.7, and the nucleotide sequence is shown as SEQ ID NO. 8.

NOP58_ HUMAN Nucleolar protein 58: the amino acid sequence is shown as SEQ ID NO.9, and the nucleotide sequence is shown as SEQ ID NO. 10.

Reference documents:

[1]ROZHDESTVENSKY,S.T.Binding of L7Ae protein to the K-turn of archaeal snoRNAs:a shared RNA binding motif for C/D and H/ACA box snoRNAs in Archaea[J].Nucleic Acids Research,31(3):869-77.

[2]KUHN J F,TRAN E J,STUART M E.Archaeal ribosomal protein L7 is a functional homolog of the eukaryotic 15.5kD/Snu13p snoRNP core protein[J].Nucleic Acids Research,2002,4):4.

[3]OMER A D,ZIESCHE S,EBHARDT H,et al.In vitro reconstitution and activity of a C/D box methylation guide ribonucleoprotein complex[J].Proceedings of the National Academy of Sciences of the United States of America,99(8):5289-94.

[4]ARNAUD,PAUL,DECEBAL,et al.Bcd1p controls RNA loading of the core protein Nop58 during C/D box snoRNP biogenesis[J].

2. sequence of exosome membrane protein

Exosome membrane proteins such as CD63, CD81, CD9, TP01 (see chinese patent application CN201811023079.4) and the like are all proved to be capable of entering exosomes in a targeted manner, and are expressed in a fusion manner with other proteins, so that target proteins can be brought into exosomes to play a role in carrying proteins.

CD 63: the amino acid sequence is shown as SEQ ID NO.11, and the nucleotide sequence is shown as SEQ ID NO. 12.

CD 81: the amino acid sequence is shown as SEQ ID NO.13, and the nucleotide sequence is shown as SEQ ID NO. 14.

CD9 sequence: the amino acid sequence is shown as SEQ ID NO.15, and the nucleotide sequence is shown as SEQ ID NO. 16.

TP01 sequence: the amino acid sequence is shown as SEQ ID NO.17, and the nucleotide sequence is shown as SEQ ID NO. 18.

And carrying out fusion protein expression on the protein of the target exosome and the Kt binding protein, bringing the Kt binding protein into the exosome, combining the Kt binding protein with the KT ring on the Kt-shRNA, and further bringing the shRNA with the Kt ring into the exosome so as to realize the exosome entrapment of the shRNA.

3. The following experimental verification is preferably performed, taking L7Ae and CD63 as examples.

(1) Designing primers F-CD63-L7Ae and R-CD63-L7Ae, and introducing enzyme cutting sites of Nhe1 and BamH1 into the tail ends of the primers; the CD63-L7Ae gene template was synthesized by the company, Biotechnology engineering (Shanghai) Ltd. The two sequences were linked by linker. The specific construction method refers to the embodiment 1 of the Chinese patent application CN 201811023079.4.

Related sequence information:

F-CD63-L7Ae：5’-ATAAAAGCTAGCATGGCGGTGGAAGGAGGAATGAAATGTGTG-3’

R-CD63-L7Ae：5’-ATAAAAGGATCCTTAGTGGTGGTGGTGGTGGTGCTTCTGAAGG-3’

Linker：5’-GGTGGAGGTGGCAGCGGAGGAGGTGGGTCCGGCGGTGGAGGAAGC-3’。

example 5 preparation and identification of exosomes containing shRNA

1. Plasmid cotransfection 293T cell

The recombinant plasmid pRNAT-U6.1-Kt-shEGFP was co-transfected into 293T cells (purchased from Biotechnology engineering (Shanghai) Co., Ltd.) with pCDNA3.4-CD63-L7Ae and a blank pCDNA3.4 plasmid, respectively, as described in example 3.

2. Collection and purification of exosomes

Refer to the method and steps of "exosome extraction" in step 3 of step 2 of Chinese patent application CN 201811023079.4.

3. NTA detection of exosomes: refer to the method and steps of "particle size detection of exosome" in step 3 of step 2 of Chinese patent application CN 201811023079.4. The NTA detection results are shown in FIG. 2, which shows that the particle sizes extracted by us are all around 100nm, which is in accordance with the particle size range of exosomes.

4. Detection of exosome Marker protein and recombinant L7Ae protein

Western blot is used for detecting the marker protein and recombinant CD63-L7Ae protein amount of the exosome. Because the L7Ae protein is connected with a His tag, an anti-His antibody (purchased from proteintech, cat # 66005-1-lg) is used for detection, and the specific method refers to the method and steps of step 4 'detection of recombinant protein carried in exosome' in example 2 of Chinese patent application CN 201811023079.4.

As shown in FIG. 3, the content of recombinant proteins CD63 and L7Ae in the exosomes (lane 2) collected from cells co-transfected with pRNAT-U6.1-Kt-shEGFP + pCDNA3.4CD63-L7Ae was high, compared with the exosomes (lane 1) collected from cells co-transfected with pRNAT-U6.1-Kt-shEGFP and blank pCDNA3.4 plasmids, indicating that many CD63-L7Ae were loaded in the exosomes.

Example 6 detection of shRNA in exosomes

1. Extraction of exosome RNA:

collecting the exosomes extracted in the step 2 of the example 5, and extracting total RNA of the exosomes, wherein the specific operation is as follows:

(1) exosomes were mixed with 1ml of trizol (takara) and mixed thoroughly overnight.

(2) After the exosomes were fully lysed and thoroughly mixed, 5. mu.l of 200nM exosome (MiDETECTTM) (Ribobio) was added, thoroughly mixed and left to stand for 5-10 minutes.

(3) Mu.l of chloroform (purchased from Tianjin Baishi chemical Co., Ltd.) was added to an EP tube, shaken vigorously up and down for 15 seconds, allowed to stand for 15 minutes, and centrifuged at 12000g at 4 ℃ for 15 minutes.

(4) Aspirate 360. mu.l of supernatant into a new RNase-free EP tube.

(5) Adding equal volume of isopropanol (purchased from Tianjin Baishi chemical Co., Ltd.), mixing well, standing for 10 min, 12000g, centrifuging at 4 deg.C for 15 min. The white precipitate was collected, added with 75% ethanol (purchased from Tianjin Baishi chemical Co., Ltd.), blown carefully, 12000g, and centrifuged at 4 ℃ for 10 minutes.

(6) Air-dried at room temperature, and 30. mu.l of DEPC water (obtained from Biotechnology (Shanghai) Co., Ltd.) was added

2. Detection of shRNA-EGFP in exosomes

RT-PCR detection of the amount of shRNA in exosomes: using a detection kit (Ribobio, product code: C10211), detection was carried out according to the procedures provided in the kit.

As shown in FIG. 4, compared with exosomes collected from cells co-transfected with pRNAT-U6.1-Kt-shEGFP and blank pCDNA3.4 plasmid, the amount of Kt-shEGFP in exosomes collected from cells co-transfected with pRNAT-U6.1-Kt-shEGFP + pCDNA3.4CD63-L7Ae was significantly increased, indicating that L7Ae on the membrane of exosomes can bind to Kt-shEGFP and carry it into exosomes.

Example 7 exosomes can introduce an entrained shRNA into other cells

The exosomes extracted in step 2 of example 5 were transfected into a blank 293T cell (purchased from Biotechnology engineering (Shanghai) Co., Ltd.), and simultaneously transfected with an expression plasmid pCDNA3.4-EGFP of EGFP, and the silencing of EGFP was observed and detected using a Western blot and a flow cytometer using 293T cells transfected with only pCDNA3.4-EGFP as a control.

Western blot detection

The specific procedure was followed as in example 5, step 4, using EGFP antibody (purchased from proteintech, cat # 66002-1-lg) and an antibody to the internal reference protein GAPDH (purchased from proteintech, cat # 60004-1-lg). The results are shown in FIGS. 5 and 6.

2. Flow cytometry detection in cells

The specific method refers to the method and steps of 'flow cytometry detection' in step 2 of the embodiment 3 of Chinese patent application CN 201811023079.4. The results are shown in FIGS. 7 and 8.

Western blot results (FIGS. 5 and 6) and flow cytometry results (FIGS. 7 and 8) show that EGFP expression in the group of exosomes containing L7Ae is significantly reduced compared to exosomes collected from the control group and cells co-transfected with pRNAT-U6.1-Kt-shEGFP and the blank pCDNA3.4 plasmid, indicating that shEGFP in Kt + CD63-L7Ae exosomes can inhibit EGFP expression.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

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Thr Tyr Cys Arg Leu Ala Gln Phe Ile Gly Asn Arg Arg Glu Leu Asn

210 215 220

Glu Asp Lys Leu Glu Lys Leu Glu Glu Leu Thr Met Asp Gly Ala Lys

225 230 235 240

Ala Lys Ala Ile Leu Asp Ala Ser Arg Ser Ser Met Gly Met Asp Ile

245 250 255

Ser Ala Ile Asp Leu Ile Asn Ile Glu Ser Phe Ser Ser Arg Val Val

260 265 270

Ser Leu Ser Glu Tyr Arg Gln Ser Leu His Thr Tyr Leu Arg Ser Lys

275 280 285

Met Ser Gln Val Ala Pro Ser Leu Ser Ala Leu Ile Gly Glu Ala Val

290 295 300

Gly Ala Arg Leu Ile Ala His Ala Gly Ser Leu Thr Asn Leu Ala Lys

305 310 315 320

Tyr Pro Ala Ser Thr Val Gln Ile Leu Gly Ala Glu Lys Ala Leu Phe

325 330 335

Arg Ala Leu Lys Thr Arg Gly Asn Thr Pro Lys Tyr Gly Leu Ile Phe

340 345 350

His Ser Thr Phe Ile Gly Arg Ala Ala Ala Lys Asn Lys Gly Arg Ile

355 360 365

Ser Arg Tyr Leu Ala Asn Lys Cys Ser Ile Ala Ser Arg Ile Asp Cys

370 375 380

Phe Ser Glu Val Pro Thr Ser Val Phe Gly Glu Lys Leu Arg Glu Gln

385 390 395 400

Val Glu Glu Arg Leu Ser Phe Tyr Glu Thr Gly Glu Ile Pro Arg Lys

405 410 415

Asn Leu Asp Val Met Lys Glu Ala Met Val Gln Ala Glu Glu Ala Ala

420 425 430

Ala Glu Ile Thr Arg Lys Leu Glu Lys Gln Glu Lys Lys Arg Leu Lys

435 440 445

Lys Glu Lys Lys Arg Leu Ala Ala Leu Ala Leu Ala Ser Ser Glu Asn

450 455 460

Ser Ser Ser Thr Pro Glu Glu Cys Glu Glu Met Ser Glu Lys Pro Lys

465 470 475 480

Lys Lys Lys Lys Gln Lys Pro Gln Glu Val Pro Gln Glu Asn Gly Met

485 490 495

Glu Asp Pro Ser Ile Ser Phe Ser Lys Pro Lys Lys Lys Lys Ser Phe

500 505 510

Ser Lys Glu Glu Leu Met Ser Ser Asp Leu Glu Glu Thr Ala Gly Ser

515 520 525

Thr Ser Ile Pro Lys Arg Lys Lys Ser Thr Pro Lys Glu Glu Thr Val

530 535 540

Asn Asp Pro Glu Glu Ala Gly His Arg Ser Gly Ser Lys Lys Lys Arg

545 550 555 560

Lys Phe Ser Lys Glu Glu Pro Val Ser Ser Gly Pro Glu Glu Ala Val

565 570 575

Gly Lys Ser Ser Ser Lys Lys Lys Lys Lys Phe His Lys Ala Ser Gln

580 585 590

Glu Asp

<210> 8

<211> 1785

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> HumanNOP56_ HUMAN Nucleolar protein 56 nucleotide sequence

<400> 8

atggtgctgt tgcacgtgct gtttgagcac gcggtcggct acgcgctgct ggcgctgaag 60

gaagtggagg agatcagtct gctgcagccg caggtggagg agtctgtgct caacctgggc 120

aaattccaca gcatcgttcg tctggtggcc ttttgtccct ttgcctcatc ccaggttgcc 180

ttggaaaatg ccaacgccgt gtctgaaggg gttgttcatg aggacctccg cctgctcttg 240

gagacccacc tgccgtccaa aaagaagaaa gtactcttgg gagttgggga tcccaagatt 300

ggtgccgcaa tacaggagga gttagggtac aactgccaga ctggaggagt catagctgag 360

atcctgcgag gagttcgtct gcacttccac aatctggtga agggtctgac cgatctgtca 420

gcttgtaaag cacagctggg gctgggacac agctattccc gtgccaaagt taagtttaat 480

gtgaaccggg tggacaatat gatcatccag tccattagcc tcctggacca gctggataag 540

gacatcaata ccttctctat gcgtgtcagg gagtggtacg ggtatcactt tccggagctg 600

gtgaagatca tcaacgacaa tgccacatac tgccgtcttg cccagtttat tggaaaccga 660

agggaactga atgaggacaa gctggagaag ctggaggagc tgacaatgga tggggccaag 720

gctaaggcta ttctggatgc ctcacggtcc tccatgggca tggacatatc tgccattgac 780

ttgataaaca tcgagagctt ctccagtcgt gtggtgtctt tatctgaata ccgccagagc 840

ctacacactt acctgcgctc caagatgagc caagtagccc ccagcctgtc agccctaatt 900

ggggaagcgg taggtgcacg tctcatcgca catgctggca gcctcaccaa cctggccaag 960

tatccagcat ccacagtgca gatccttggg gctgaaaagg ccctgttcag agccctgaag 1020

acaaggggta acactccaaa atatggactc attttccact ccaccttcat tggccgagca 1080

gctgccaaga acaaaggccg catctcccga tacctggcaa acaaatgcag tattgcctca 1140

cgaatcgatt gcttctctga ggtgcccacg agtgtattcg gggagaagct tcgagaacaa 1200

gttgaagagc gactgtcctt ctatgagact ggagagatac cacgaaagaa tctggatgtc 1260

atgaaggaag caatggttca ggcagaggaa gcggctgctg agattactag gaagctggag 1320

aaacaggaga agaaacgctt aaagaaggaa aagaaacggc tggctgcact tgccctcgcg 1380

tcttcagaaa acagcagtag tactccagag gagtgtgagg agatgagtga aaaacccaaa 1440

aagaagaaaa agcaaaagcc ccaggaggtt cctcaggaga atggaatgga agacccatct 1500

atctctttct ccaaacccaa gaaaaagaaa tctttttcca aggaggagtt gatgagtagc 1560

gatcttgaag agaccgctgg cagcaccagt attcccaaga ggaagaagtc tacacccaag 1620

gaggaaacag ttaatgaccc tgaggaggca ggccacagaa gtggctccaa gaaaaagagg 1680

aaattctcca aagaggagcc ggtcagcagt gggcctgaag aggcggttgg caagagcagc 1740

tccaagaaga agaaaaagtt ccataaagca tcccaggaag attag 1785

<210> 9

<211> 529

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> HumanNOP58_ HUMAN Nucleolar protein 58 amino acid sequence

<400> 9

Met Leu Val Leu Phe Glu Thr Ser Val Gly Tyr Ala Ile Phe Lys Val

1 5 10 15

Leu Asn Glu Lys Lys Leu Gln Glu Val Asp Ser Leu Trp Lys Glu Phe

20 25 30

Glu Thr Pro Glu Lys Ala Asn Lys Ile Val Lys Leu Lys His Phe Glu

35 40 45

Lys Phe Gln Asp Thr Ala Glu Ala Leu Ala Ala Phe Thr Ala Leu Met

50 55 60

Glu Gly Lys Ile Asn Lys Gln Leu Lys Lys Val Leu Lys Lys Ile Val

65 70 75 80

Lys Glu Ala His Glu Pro Leu Ala Val Ala Asp Ala Lys Leu Gly Gly

85 90 95

Val Ile Lys Glu Lys Leu Asn Leu Ser Cys Ile His Ser Pro Val Val

100 105 110

Asn Glu Leu Met Arg Gly Ile Arg Ser Gln Met Asp Gly Leu Ile Pro

115 120 125

Gly Val Glu Pro Arg Glu Met Ala Ala Met Cys Leu Gly Leu Ala His

130 135 140

Ser Leu Ser Arg Tyr Arg Leu Lys Phe Ser Ala Asp Lys Val Asp Thr

145 150 155 160

Met Ile Val Gln Ala Ile Ser Leu Leu Asp Asp Leu Asp Lys Glu Leu

165 170 175

Asn Asn Tyr Ile Met Arg Cys Arg Glu Trp Tyr Gly Trp His Phe Pro

180 185 190

Glu Leu Gly Lys Ile Ile Ser Asp Asn Leu Thr Tyr Cys Lys Cys Leu

195 200 205

Gln Lys Val Gly Asp Arg Lys Asn Tyr Ala Ser Ala Lys Leu Ser Glu

210 215 220

Leu Leu Pro Glu Glu Val Glu Ala Glu Val Lys Ala Ala Ala Glu Ile

225 230 235 240

Ser Met Gly Thr Glu Val Ser Glu Glu Asp Ile Cys Asn Ile Leu His

245 250 255

Leu Cys Thr Gln Val Ile Glu Ile Ser Glu Tyr Arg Thr Gln Leu Tyr

260 265 270

Glu Tyr Leu Gln Asn Arg Met Met Ala Ile Ala Pro Asn Val Thr Val

275 280 285

Met Val Gly Glu Leu Val Gly Ala Arg Leu Ile Ala His Ala Gly Ser

290 295 300

Leu Leu Asn Leu Ala Lys His Ala Ala Ser Thr Val Gln Ile Leu Gly

305 310 315 320

Ala Glu Lys Ala Leu Phe Arg Ala Leu Lys Ser Arg Arg Asp Thr Pro

325 330 335

Lys Tyr Gly Leu Ile Tyr His Ala Ser Leu Val Gly Gln Thr Ser Pro

340 345 350

Lys His Lys Gly Lys Ile Ser Arg Met Leu Ala Ala Lys Thr Val Leu

355 360 365

Ala Ile Arg Tyr Asp Ala Phe Gly Glu Asp Ser Ser Ser Ala Met Gly

370 375 380

Val Glu Asn Arg Ala Lys Leu Glu Ala Arg Leu Arg Thr Leu Glu Asp

385 390 395 400

Arg Gly Ile Arg Lys Ile Ser Gly Thr Gly Lys Ala Leu Ala Lys Thr

405 410 415

Glu Lys Tyr Glu His Lys Ser Glu Val Lys Thr Tyr Asp Pro Ser Gly

420 425 430

Asp Ser Thr Leu Pro Thr Cys Ser Lys Lys Arg Lys Ile Glu Gln Val

435 440 445

Asp Lys Glu Asp Glu Ile Thr Glu Lys Lys Ala Lys Lys Ala Lys Ile

450 455 460

Lys Val Lys Val Glu Glu Glu Glu Glu Glu Lys Val Ala Glu Glu Glu

465 470 475 480

Glu Thr Ser Val Lys Lys Lys Lys Lys Arg Gly Lys Lys Lys His Ile

485 490 495

Lys Glu Glu Pro Leu Ser Glu Glu Glu Pro Cys Thr Ser Thr Ala Ile

500 505 510

Ala Ser Pro Glu Lys Lys Lys Lys Lys Lys Lys Lys Arg Glu Asn Glu

515 520 525

Asp

<210> 10

<211> 1590

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> HumanNOP58_ HUMAN Nucleolar protein 58 nucleotide sequence

<400> 10

atgttggtgc tgtttgaaac gtctgtgggt tacgccatct ttaaggttct aaatgagaag 60

aaacttcaag aggttgatag tttatggaaa gaatttgaaa ctccagagaa agcaaacaaa 120

atagtaaagc taaaacattt tgagaaattt caggatacag cagaagcatt agcagcattc 180

acagctctga tggagggcaa aatcaataag cagctgaaaa aagttctgaa gaaaatagta 240

aaagaagccc atgaaccgct ggcagtagct gatgctaaac taggaggggt cataaaggaa 300

aagctgaatc tcagttgtat ccatagtcct gttgttaatg aacttatgag aggaattcgt 360

tcacaaatgg atggattaat ccctggggta gaaccacgtg aaatggcagc tatgtgtctt 420

ggattggctc acagcctgtc tcgatataga ttgaagttta gcgctgataa agtagacaca 480

atgattgttc aggcaatttc cttgttagat gacttggata aagaactaaa caactacatt 540

atgcgatgta gagaatggta tggctggcat ttccctgaat taggaaaaat tatttcagat 600

aatttaacat actgcaagtg tttacagaaa gttggcgata ggaagaacta tgcctctgcc 660

aagctttctg agttgctgcc agaagaagtt gaagcagaag tgaaagcagc tgcagagata 720

tcaatgggaa cagaggtttc agaagaagat atttgcaata ttctgcatct ttgcacccag 780

gtgattgaaa tctctgaata tcgaacccag ctctatgaat atctacaaaa tcgaatgatg 840

gccattgcac ccaatgttac agtcatggtt ggggaattag ttggagcacg gcttattgct 900

catgcaggtt ctcttttaaa tttggccaag catgcagctt ctaccgttca gattcttgga 960

gctgaaaagg cacttttcag agccctcaaa tctagacggg atacccctaa gtatggtctc 1020

atttatcatg cttcactcgt gggccagaca agtcccaaac acaaaggaaa gatttctcga 1080

atgctggcag ccaaaaccgt tttggctatc cgttatgatg cttttggtga ggattcaagt 1140

tctgcaatgg gagttgagaa cagagccaaa ttagaggcca ggttgagaac tttggaagac 1200

agagggataa gaaaaataag tggaacagga aaagcattag caaaaacaga aaaatatgaa 1260

cacaaaagtg aagtgaagac ttacgatcct tctggtgact ccacacttcc aacctgttct 1320

aaaaaacgca aaatagaaca ggtagataaa gaggatgaaa ttactgaaaa gaaagccaaa 1380

aaagccaaga ttaaagttaa agttgaagaa gaggaagaag aaaaagtggc agaagaagaa 1440

gaaacatctg tgaagaagaa gaagaaaagg ggtaaaaaga aacacattaa ggaagaacca 1500

ctttctgagg aagaaccatg taccagcaca gcaattgcta gtccagagaa aaagaagaaa 1560

aagaaaaaaa agagagagaa cgaggattaa 1590

<210> 11

<211> 238

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CD63 amino acid sequence

<400> 11

Met Ala Val Glu Gly Gly Met Lys Cys Val Lys Phe Leu Leu Tyr Val

1 5 10 15

Leu Leu Leu Ala Phe Cys Ala Cys Ala Val Gly Leu Ile Ala Val Gly

20 25 30

Val Gly Ala Gln Leu Val Leu Ser Gln Thr Ile Ile Gln Gly Ala Thr

35 40 45

Pro Gly Ser Leu Leu Pro Val Val Ile Ile Ala Val Gly Val Phe Leu

50 55 60

Phe Leu Val Ala Phe Val Gly Cys Cys Gly Ala Cys Lys Glu Asn Tyr

65 70 75 80

Cys Leu Met Ile Thr Phe Ala Ile Phe Leu Ser Leu Ile Met Leu Val

85 90 95

Glu Val Ala Ala Ala Ile Ala Gly Tyr Val Phe Arg Asp Lys Val Met

100 105 110

Ser Glu Phe Asn Asn Asn Phe Arg Gln Gln Met Glu Asn Tyr Pro Lys

115 120 125

Asn Asn His Thr Ala Ser Ile Leu Asp Arg Met Gln Ala Asp Phe Lys

130 135 140

Cys Cys Gly Ala Ala Asn Tyr Thr Asp Trp Glu Lys Ile Pro Ser Met

145 150 155 160

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

165 170 175

Cys Gly Ile Asn Phe Asn Glu Lys Ala Ile His Lys Glu Gly Cys Val

180 185 190

Glu Lys Ile Gly Gly Trp Leu Arg Lys Asn Val Leu Val Val Ala Ala

195 200 205

Ala Ala Leu Gly Ile Ala Phe Val Glu Val Leu Gly Ile Val Phe Ala

210 215 220

Cys Cys Leu Val Lys Ser Ile Arg Ser Gly Tyr Glu Val Met

225 230 235

<210> 12

<211> 717

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CD63 nucleotide sequence

<400> 12

atggcggtgg aaggaggaat gaaatgtgtg aagttcttgc tctacgtcct cctgctggcc 60

ttttgcgcct gtgcagtggg actgattgcc gtgggtgtcg gggcacagct tgtcctgagt 120

cagaccataa tccagggggc tacccctggc tctctgttgc cagtggtcat catcgcagtg 180

ggtgtcttcc tcttcctggt ggcttttgtg ggctgctgcg gggcctgcaa ggagaactat 240

tgtcttatga tcacgtttgc catctttctg tctcttatca tgttggtgga ggtggccgca 300

gccattgctg gctatgtgtt tagagataag gtgatgtcag agtttaataa caacttccgg 360

cagcagatgg agaattaccc gaaaaacaac cacactgctt cgatcctgga caggatgcag 420

gcagatttta agtgctgtgg ggctgctaac tacacagatt gggagaaaat cccttccatg 480

tcgaagaacc gagtccccga ctcctgctgc attaatgtta ctgtgggctg tgggattaat 540

ttcaacgaga aggcgatcca taaggagggc tgtgtggaga agattggggg ctggctgagg 600

aaaaatgtgc tggtggtagc tgcagcagcc cttggaattg cttttgtcga ggttttggga 660

attgtctttg cctgctgcct cgtgaagagt atcagaagtg gctacgaggt gatgtag 717

<210> 13

<211> 236

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CD81 amino acid sequence

<400> 13

Met Gly Val Glu Gly Cys Thr Lys Cys Ile Lys Tyr Leu Leu Phe Val

1 5 10 15

Phe Asn Phe Val Phe Trp Leu Ala Gly Gly Val Ile Leu Gly Val Ala

20 25 30

Leu Trp Leu Arg His Asp Pro Gln Thr Thr Asn Leu Leu Tyr Leu Glu

35 40 45

Leu Gly Asp Lys Pro Ala Pro Asn Thr Phe Tyr Val Gly Ile Tyr Ile

50 55 60

Leu Ile Ala Val Gly Ala Val Met Met Phe Val Gly Phe Leu Gly Cys

65 70 75 80

Tyr Gly Ala Ile Gln Glu Ser Gln Cys Leu Leu Gly Thr Phe Phe Thr

85 90 95

Cys Leu Val Ile Leu Phe Ala Cys Glu Val Ala Ala Gly Ile Trp Gly

100 105 110

Phe Val Asn Lys Asp Gln Ile Ala Lys Asp Val Lys Gln Phe Tyr Asp

115 120 125

Gln Ala Leu Gln Gln Ala Val Val Asp Asp Asp Ala Asn Asn Ala Lys

130 135 140

Ala Val Val Lys Thr Phe His Glu Thr Leu Asp Cys Cys Gly Ser Ser

145 150 155 160

Thr Leu Thr Ala Leu Thr Thr Ser Val Leu Lys Asn Asn Leu Cys Pro

165 170 175

Ser Gly Ser Asn Ile Ile Ser Asn Leu Phe Lys Glu Asp Cys His Gln

180 185 190

Lys Ile Asp Asp Leu Phe Ser Gly Lys Leu Tyr Leu Ile Gly Ile Ala

195 200 205

Ala Ile Val Val Ala Val Ile Met Ile Phe Glu Met Ile Leu Ser Met

210 215 220

Val Leu Cys Cys Gly Ile Arg Asn Ser Ser Val Tyr

225 230 235

<210> 14

<211> 711

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CD81 nucleotide sequence

<400> 14

atgggagtgg agggctgcac caagtgcatc aagtacctgc tcttcgtctt caatttcgtc 60

ttctggctgg ctggaggcgt gatcctgggt gtggccctgt ggctccgcca tgacccgcag 120

accaccaacc tcctgtatct ggagctggga gacaagcccg cgcccaacac cttctatgta 180

ggcatctaca tcctcatcgc tgtgggcgct gtcatgatgt tcgttggctt cctgggctgc 240

tacggggcca tccaggaatc ccagtgcctg ctggggacgt tcttcacctg cctggtcatc 300

ctgtttgcct gtgaggtggc cgccggcatc tggggctttg tcaacaagga ccagatcgcc 360

aaggatgtga agcagttcta tgaccaggcc ctacagcagg ccgtggtgga tgatgacgcc 420

aacaacgcca aggctgtggt gaagaccttc cacgagacgc ttgactgctg tggctccagc 480

acactgactg ctttgaccac ctcagtgctc aagaacaatt tgtgtccctc gggcagcaac 540

atcatcagca acctcttcaa ggaggactgc caccagaaga tcgatgacct cttctccggg 600

aagctgtacc tcatcggcat tgctgccatc gtggtcgctg tgatcatgat cttcgagatg 660

atcctgagca tggtgctgtg ctgtggcatc cggaacagct ccgtgtactg a 711

<210> 15

<211> 227

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CD9 amino acid sequence

<400> 15

Met Pro Val Lys Gly Gly Thr Lys Cys Ile Lys Tyr Leu Leu Phe Gly

1 5 10 15

Phe Asn Phe Ile Phe Trp Leu Ala Gly Ile Ala Val Leu Ala Ile Gly

20 25 30

Leu Trp Leu Arg Phe Asp Ser Gln Thr Lys Ser Ile Phe Glu Gln Glu

35 40 45

Thr Asn Asn Asn Asn Ser Ser Phe Tyr Thr Gly Val Tyr Ile Leu Ile

50 55 60

Gly Ala Gly Ala Leu Met Met Leu Val Gly Phe Leu Gly Cys Cys Gly

65 70 75 80

Ala Val Gln Glu Ser Gln Cys Met Leu Gly Leu Phe Phe Gly Phe Leu

85 90 95

Leu Val Ile Phe Ala Ile Glu Ile Ala Ala Ala Ile Trp Gly Tyr Ser

100 105 110

His Lys Asp Glu Val Ile Lys Glu Val Gln Glu Phe Tyr Lys Asp Thr

115 120 125

Tyr Asn Lys Leu Lys Thr Lys Asp Glu Pro Gln Arg Glu Thr Leu Lys

130 135 140

Ala Ile His Tyr Ala Leu Asn Cys Cys Gly Leu Ala Gly Gly Val Glu

145 150 155 160

Gln Phe Ile Ser Asp Ile Cys Pro Lys Lys Asp Val Leu Glu Thr Phe

165 170 175

Thr Val Lys Ser Cys Pro Asp Ala Ile Lys Glu Val Phe Asp Asn Lys

180 185 190

Phe His Ile Ile Gly Ala Val Gly Ile Gly Ile Ala Val Val Met Ile

195 200 205

Phe Gly Met Ile Phe Ser Met Ile Leu Cys Cys Ala Ile Arg Arg Asn

210 215 220

Arg Glu Met

225

<210> 16

<211> 687

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CD9 nucleotide sequence

<400> 16

atgccggtca aaggaggcac caagtgcatc aaatacctgc tgttcggatt taacttcatc 60

ttctggcttg ccgggattgc tgtccttgcc attggactat ggctccgatt cgactctcag 120

accaagagca tcttcgagca agaaactaat aataataatt ccagcttcta cacaggagtc 180

tatattctga tcggagccgg cgccctcatg atgctggtgg gcttcctggg ctgctgcggg 240

gctgtgcagg agtcccagtg catgctggga ctgttcttcg gcttcctctt ggtgatattc 300

gccattgaaa tagctgcggc catctgggga tattcccaca aggatgaggt gattaaggaa 360

gtccaggagt tttacaagga cacctacaac aagctgaaaa ccaaggatga gccccagcgg 420

gaaacgctga aagccatcca ctatgcgttg aactgctgtg gtttggctgg gggcgtggaa 480

cagtttatct cagacatctg ccccaagaag gacgtactcg aaaccttcac cgtgaagtcc 540

tgtcctgatg ccatcaaaga ggtcttcgac aataaattcc acatcatcgg cgcagtgggc 600

atcggcattg ccgtggtcat gatatttggc atgatcttca gtatgatctt gtgctgtgct 660

atccgcagga accgcgagat ggtctag 687

<210> 17

<211> 243

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> TP01 amino acid sequence

<400> 17

Met Pro Ala Pro Ala Leu Leu Ala Ala Leu Cys Gly Ala Leu Leu Cys

1 5 10 15

Ala Pro Ser Leu Leu Val Ala Leu Ala Ile Cys Ser Leu Ala Pro Cys

20 25 30

His Ala Gly Gly Leu Cys Gly Gly Ile Ser Gly Gly Val Ala Gly Ala

35 40 45

Val Pro Pro Ser Thr Thr Cys Thr Cys Leu Leu Gly Thr Ala Gly Ala

50 55 60

His Cys Gly Thr Leu Cys Val Gly Pro Leu Gly Leu Gly Ala Gly Ala

65 70 75 80

Ile Ala Ala Ser Gly Ile Ala Ala Ser Ser Val Ala Val Thr Pro Leu

85 90 95

Gly Leu Gly His Thr Val Pro Gly Leu Ala Ala Leu Ala Ala Ala Gly

100 105 110

Met Val Ala Ala Thr Thr Pro Ser Ser Ala Ala Ala Ala Pro Thr Ile

115 120 125

Gly Val Ala Leu Leu Ala Ala Met Thr Val Thr Gly Val Val Thr Gly

130 135 140

Gly Ala Ser Ala Leu Ala Ser His Gly Thr Leu Leu Ala Pro Leu Val

145 150 155 160

Ala Thr Ser Leu Ala Gly His Gly Pro Ala Pro Ile His Ala Val Ala

165 170 175

Leu Leu His Leu Gly Pro Val Gly Ala Thr Ala Leu Ala Ala Val His

180 185 190

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

195 200 205

Pro Thr Ser Cys His Thr Ala Cys Thr Leu Ala Pro Gly Leu Leu Gly

210 215 220

Cys Gly Leu Ala Ala Ala Leu Ala Ala Leu Ala Ala Gly Ala Ala Ala

225 230 235 240

Ala Gly Gly

<210> 18

<211> 732

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> TP01 nucleotide sequence

<400> 18

atgccgcgcc cccgcctgct ggccgcgctg tgcggcgcgc tgctctgcgc ccccagcctc 60

ctcgtcgccc tggatatctg ttccaaaaac ccctgccaca acggtggttt atgcgaggag 120

atttcccaag aagtgcgagg agatgtcttc ccctcgtaca cctgcacgtg ccttaagggc 180

tacgcgggca accactgtga gacgaaatgt gtcgagccac tgggcctgga gaatgggaac 240

attgccaact cacagatcgc cgcctcgtct gtgcgtgtga ccttcttggg tttgcagcat 300

tgggtcccgg agctggcccg cctgaaccgc gcaggcatgg tcaatgcctg gacacccagc 360

agcaatgacg ataacccctg gatccaggtg aacctgctgc ggaggatgtg ggtaacaggt 420

gtggtgacgc agggtgccag ccgcttggcc agtcatgagt acctgaaggc cttcaaggtg 480

gcctacagcc ttaatggaca cgaattcgat ttcatccatg atgttaataa aaaacacaag 540

gagtttgtgg gtaactggaa caaaaacgcg gtgcatgtca acctgtttga gacccctgtg 600

gaggctcagt acgtgagatt gtaccccacg agctgccaca cggcctgcac tctgcgcttt 660

gagctactgg gctgtgagct gaacgcaagg aaggcagact tgaggcgagg agcagatgac 720

agagagcagt aa 732

<210> 19

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> EGFP-F

<400> 19

ataaaaggta ccatggtgag caagg 25

<210> 20

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> EGFP-R

<400> 20

ataaaaggat ccttacttgt acagctcg 28

<210> 21

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> F-CD63-L7Ae

<400> 21

ataaaagcta gcatggcggt ggaaggagga atgaaatgtg tg 42

<210> 22

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> R-CD63-L7Ae

<400> 22

ataaaaggat ccttagtggt ggtggtggtg gtgcttctga agg 43

<210> 23

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Linker

<400> 23

ggtggaggtg gcagcggagg aggtgggtcc ggcggtggag gaagc 45

Claims (7)

1. A construction method of an exosome loading shRNA is characterized by comprising the following steps: the method comprises the following steps:

(1) designing and synthesizing shRNA sequence capable of effectively silencing target gene; cloning the shRNA sequence into an expression vector to obtain a recombinant expression vector 1; the shRNA sequence comprises a target sequence, a stem-loop structure sequence and a complementary sequence of the target sequence which are connected in sequence;

(2) connecting a gene of a protein capable of being specifically combined with the stem-loop structure with a gene of an exosome membrane protein to obtain a fusion gene sequence, and cloning the obtained fusion gene sequence into an expression vector to obtain a recombinant expression vector 2;

(3) transfecting the cell with the recombinant expression vector 1 obtained in the step (1) and the recombinant expression vector 2 obtained in the step (2) at the same time, culturing the transfected cell, and collecting an exosome to obtain the exosome loaded with shRNA;

the sequence of the stem-loop structure in the step (1) is GCTGACCCGAAAGGGCGTGATGC;

the protein capable of specifically binding to the stem-loop structure in the step (2) is selected from at least one of RNA binding protein L7Ae, NHP2 ribonucleoprotein-like protein 1, human nucleoprotein P56 and human nucleoprotein P58;

the exosome membrane protein in the step (2) is at least one of CD63, CD81, CD9 and TP 01.

2. The method for constructing an shRNA-loading exosome according to claim 1, characterized in that:

the amino acid sequence of the RNA binding protein L7Ae is shown in SEQ ID NO. 3;

the amino acid sequence of the NHP2 ribonucleoprotein-like protein 1 is shown in SEQ ID NO. 5;

the amino acid sequence of the human nucleoprotein P56 is shown in SEQ ID NO. 7;

the amino acid sequence of the human nucleoprotein P58 is shown in SEQ ID NO. 9;

the amino acid sequence of the CD63 is shown as SEQ ID NO. 11;

the amino acid sequence of the CD81 is shown as SEQ ID NO. 13;

the amino acid sequence of the CD9 is shown as SEQ ID NO. 15;

the amino acid sequence of TP01 is shown in SEQ ID NO. 17.

3. The method for constructing an shRNA-loading exosome according to claim 1, characterized in that:

the nucleotide sequence of the RNA binding protein L7Ae is shown as SEQ ID NO. 4;

the nucleotide sequence of the NHP2 ribonucleoprotein-like protein 1 is shown in SEQ ID NO. 6;

the nucleotide sequence of the human nucleoprotein P56 is shown in SEQ ID NO. 8;

the nucleotide sequence of the human nucleoprotein P58 is shown in SEQ ID NO. 10;

the nucleotide sequence of the CD63 is shown as SEQ ID NO. 12;

the nucleotide sequence of the CD81 is shown as SEQ ID NO. 14;

the nucleotide sequence of the CD9 is shown as SEQ ID NO 16;

the nucleotide sequence of TP01 is shown in SEQ ID NO. 18.

4. The method for constructing an shRNA-loading exosome according to claim 1, characterized in that:

the connection in the step (2) is through a joint, and the connection sequence is a gene fragment of the protein A, a joint and an exosome membrane protein gene fragment;

the nucleotide sequence of the linker is:

GGTGGAGGTGGCAGCGGAGGAGGTGGGTCCGGCGGTGGAGGAAGC。

5. the method for constructing an shRNA-loading exosome according to claim 1, characterized in that:

the expression vector in the step (1) is pRNAT-U6.1/Neo;

the expression vector in the step (2) is pCDNA3.4.

6. The method for constructing an shRNA-loading exosome according to claim 1, characterized in that:

in the step (3), the number ratio of the recombinant expression vector 1 to the recombinant expression vector 2 is 1-2: 1-2 when cells are transfected.

7. An exosome for loading shRNA, characterized in that: obtained by the construction method according to any one of claims 1 to 6.

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