CN102512660B - Protein containing 51st to 89th sites of amino acid basic sequences in protein 3A of coxsackievirus B3 and application for same - Google Patents
Protein containing 51st to 89th sites of amino acid basic sequences in protein 3A of coxsackievirus B3 and application for same Download PDFInfo
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Abstract
The invention discloses a protein containing 51st to 89th sites of amino acid basic sequences (shown in SEQ ID NO. 1) in the protein 3A of coxsackievirus B3 and an application for the same; preferably, the protein is the protein 3A of coxsackievirus B3 and the truncated protein thereof; and more preferably, the protein is the protein shown in SEQ ID NO. 1. The invention further relates to a nucleotide sequence for coding the protein, a carrier containing the nucleotide sequence, and a host cell containing the carrier; and tumour cells can be further induced to generate autophagic apoptosis by transfecting the carrier to the tumour cells (HeLa cells), so that the functions of causing the autophagy of tumour cells and inhibiting the growth of tumour cells of the expressed protein are proved.
Description
Technical field
The present invention relates to a kind of protein and application thereof that causes tumor cell generation autophagy sexual cell apoptosis, be particularly related to the application of cells of coxsackie B 3 virus 3A albumen in preparing antitumor drug or Ancillary drugs in tumor therapy, especially relate to the application of 51-89 amino acids motif in the inhibition tumor cell growth by causing the tumor cell autophagy in the Coxsackie virus nonstructural protein 3A, belong to biomedical sector.
Background technology
Tumor is the important diseases that threatens human health and life, the primary treatment means of tumor are operation, radiotherapy and chemotherapy etc. at present, although these methods obtain certain effect, but also can't thoroughly effect a radical cure tumor, and all there is certain side effect, the bone marrow depression that for example chemotherapy causes, appetite lower, diarrhoea, the caused dysphagia of radiotherapy etc.Moreover, these traditional treatments, except having side effect, also have the blind spot of some treatments, such as drug-fast generation etc.Along with the development of modern molecular biology and technology thereof, the treatment of tumor is more diversified, treatment that some are new or the method for auxiliary treatment have occurred, and this wherein just comprises and utilize virus to prepare antitumor drug.2010, China national Department of Intellectual Property discloses the application (CN101653462A) of a kind of plant virus in the medicine of preparation malignant tumor, it utilizes a kind of plant virus (tobacco mosaic virus (TMV)) transfection to malignant cell (HeLa cell), in tumor cell, copies and inducing tumor cell generation autophagy and apoptosis death.Some virus also can be by tumoricidal growth treatment tumor, and CA group 21 types can be used as the treatment means of potential anti-pleomorphism myeloma.(Au?GG，Lincz?LF，Enno?A，Shafren?DR.Oncolytic?Coxsackievirus?A21?as?a?novel?therapy?for?multiple?myeloma.2007；Br?J?Haematol.137(2)：133-141.)
Research to death of neoplastic cells and anti-dead characteristic, focus mostly in the apoptosis mechanism of tumor cell, yet in tumor cell, the existence of apoptosis resistance has become the major obstacle of oncotherapy, and the key that the research of autophagy and function of tumor addresses this problem just.Autophagy is the biological process that cell utilizes lysosome degraded self macromolecular substances and impaired organelle.Autophagy can promote the catabolism of albumen or increase genome stability to bring into play antitumor action by isolation damaged cell device.Cell autophagy has the effect of the Cell Homeostasis of maintaining, and also affects generation and the development of tumor, becomes the potential target spot of neoplasm targeted therapy, and therefore, effect how in oncotherapy, to bring into play autophagy is the focus of current research.The action pathway of more known antitumor drug is relevant with autophagy at present, can induce MCF-7 Breast Cancer Cell as tamoxifen (tamoxifen) and produce dead (the Bursch W of autophagy, E llinger A, K ienzlH et al.Active cell death induced by the anti-estrogens tamoxifen and ICI 164384in human mammary carcinoma cells (MCF-7) in culture:the role of autophagy.Carcinogenesis, 1996,17 (8): 1595-1607.); Vincristine (vincristine) can be induced the autophagy apoptosis of hepatoma carcinoma cell HepG2, suppresses the medicine of tumor but autophagocytosis is induced in direct performance seldom.
In some viral disease, also found the generation of autophagocytosis, poliovirus (polio virus) 2BC albumen is expressed the light chain 3 (LC3) of microtubule-associated protein 1 by promotion, thereby promote the autophagy of 293T cell that (Jackson WT occurs, Giddings TH Jr, Taylor MP, Mulinyawe S, Rabinovitch M, Kopito RR, Kirkegaard K.Subversion of cellular autophagosomal machinery by RNA viruses PLoS Biol.2005,3 (5): 861-871.); Human simple herpesvirus (HSV) passes through activated protein kinase R (PKR) thereby formation (the Talloczy Z of promotion autophagosome, Virgin HW 4th, Levine B.PKR-dependent autophagic degradation of herpes simplex virus type 1.Autophagy.2006,2 (1): 24-29.).Coxsackie B virus group 3 types (CVB3) can cause HeLa cell generation autophagy (Wong J, Zhang J, Si X, Gao G, Mao I, McManus BM, Luo H.Autophagosome supports coxsackievirus B3replication in host cells.J Virol.2008; 82 (18): 9143-9153.).But at present also not by the carrier for expression of eukaryon of virus protein, directly promote the cell autophagy effect, thereby suppress the research of tumor.
Summary of the invention
The object of the invention is to by utilizing Coxsackie virus to cause tumor cell generation autophagy, in the 3A albumen of discovery Coxsackie B virus group 3 types, 51-89 amino acids motif can cause tumor cell generation autophagy sexual cell apoptosis and death, and then the application of Coxsackie virus in the medicine of preparation treatment malignant tumor is provided.
Therefore, first technical problem to be solved by this invention has been to provide the application of protein in preparing the medicine for treating tumor thing that contains sequence shown in SEQ ID NO.1;
Preferably, described albumen is cells of coxsackie B 3 virus 3A albumen (meaning with CVB3-3A).
Preferably, described albumen is truncated-type Coxsackie virus 3A recombiant protein, preferred, and the aminoacid sequence of described albumen (is used CVB3-3A as shown in SEQ ID NO.1
51-89mean).
It is the protein shown in SEQ ID NO.1 and the nucleotide sequence of code for said proteins that second technical problem to be solved by this invention has been to provide aminoacid sequence.
Preferably, described nucleotide sequence is as shown in SEQ ID NO.2.
The 3rd technical problem to be solved by this invention has been to provide a kind of expression vector, it is characterized in that containing above-described nucleotide sequence.
Preferably, described expression vector is carrier for expression of eukaryon, and preferred described expression vector is pEGFP-C1.
The 4th technical problem to be solved by this invention has been to provide a kind of host cell, it is characterized in that containing above-described expression vector.
Preferably, described host cell behaviour cervical cancer cell HeLa cell.
The 5th technical problem to be solved by this invention has been to provide nucleotides sequence of the present invention and has been listed in the application prepared in the medicine for treating tumor thing.
For achieving the above object, the present invention has adopted following technical measures:
(1) build the expression vector of Coxsackie virus CVB3-3A albumen and green fluorescent protein fusion gene;
(2) observe Coxsackie virus CVB3-3A albumen and the green fluorescent protein fusion rotein can effectively be expressed in tumor cell;
(3) but observe Coxsackie virus CVB3-3A albumen and green fluorescent protein fusion rotein inducing tumor cell generation autophagy;
(4) build the expression vector of Coxsackie virus CVB3-3A truncated protein and green fluorescent protein fusion gene;
(5) observe Coxsackie virus CVB3-3A truncated protein and the green fluorescent protein fusion rotein can effectively be expressed in tumor cell;
(6) but observe Coxsackie virus CVB3-3A truncated protein and green fluorescent protein fusion rotein inducing tumor cell generation autophagy;
(7) build Coxsackie virus CVB3-3A
51-89expression vector with the green fluorescent protein fusion gene;
(8) observe Coxsackie virus CVB3-3A
51-89with the green fluorescent protein fusion rotein, can effectively in tumor cell, express;
(9) observe Coxsackie virus CVB3-3A
51-89but with green fluorescent protein fusion rotein inducing tumor cell generation autophagy.
Technical scheme provided by the invention is: the application of Coxsackie virus in preparing the medicine for treating tumor thing.
The present invention has successfully built Coxsackie B virus group 3 types (CVB3) albumen 3A and CVB3-3A
51-89expression vector with green fluorescent protein (EGFP) fusion gene, transfection is to tumor cell (HeLa cell), these expression vectors are further expressed virus protein and green fluorescent protein in tumor cell, have finally induced tumor cell generation autophagy sexual cell apoptosis and death.
It is pioneering that the present invention still belongs to, by CVB3 virus protein (CVB3-3A) and green fluorescent protein fusion gene cloning to the eukaryotic expression carrier, structure obtains protokaryon or the carrier for expression of eukaryon of fusion rotein, by its transfection to tumor cell (HeLa cell), further inducing tumor cell generation autophagy sexual cell apoptosis, build the expression vector of many truncated proteins of virus protein 3A and EGFP fusion gene, transfection, to tumor cell (HeLa cell), further determines that the function motif of the protein induced cell autophagy sexual cell of 3A apoptosis is aa51-aa89.Can use it for the treatment of tumor for this characteristic of Coxsackie virus 3A albumen, as shown in specific embodiment result subsequently, by immunofluorescence cell, locate with the protein expression characteristics and assert that CVB3-3A aa51-aa89 motif is the function motif that causes tumor cell autophagy apoptosis.
Except the nucleotide sequence of coding CVB3-3A aa51-aa89 aminoacid sequence, also comprise protokaryon or the carrier for expression of eukaryon that contains above-mentioned nucleotide sequence and contain protokaryon or the host cell of carrier for expression of eukaryon also belongs to the row of protection category of the present invention certainly.
This albumen motif can be applicable to the medicine of preparation treatment tumor, can be made into active component, administration by all means in oncotherapy, including but not limited to the interior administration of tumor and local tumor intravascular administration or packing this virus 3A albumen autophagy function motif by liposome is that a kind of liposomal pharmaceutical preparation directly imports performance killing tumor cells effect in tumor.In addition, this albumen motif also can be used for combining other autophagy functional proteins or its autophagy function motif treatment tumor.Also can be used for the means of associating other treatment malignant tumor as operative treatment means such as X-ray therapy in chemical medicinal treatment, part or tumor, and as a kind of means of adjuvant therapy of tumors.
The Coxsackie virus albumen of take has the following advantages as the active fraction preparation antitumor drug:
(1) as good autophagy derivant, the growth of inhibition tumor cell;
(2) one section aminoacid that this amino acid motif is virus protein, do not contain live virus, no pathogenicity;
(3) this amino acid motif molecular weight is little, is easy to processing, absorbs.
The accompanying drawing explanation
Fig. 1 is the structure ideograph of Coxsackie B virus group 3 type 3A and green fluorescent protein fusion rotein eukaryon expression plasmid;
Fig. 2 is fluorescence microscope CVB3-3A
51-89expression in the HeLa cell;
Fig. 3 is fluorescence microscope CVB3-3A
51-89induce LC3 in the HeLa cell to assemble situation;
Fig. 4 is CVB3-3A
51-89induce the expression of HeLa cell LC3;
Fig. 5 is electron microscopic observation CVB3-3A
51-89cause the HeLa apoptosis.
The specific embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment are only exemplary, scope of the present invention are not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can modify or replace details and the form of technical solution of the present invention, but these modifications and replacement all fall within the scope of protection of the present invention.The structure of embodiment 1 Coxsackie B virus group 3 type 3A fusion rotein eukaryon expression plasmids and cellular expression are identified
CVB3-3A in the present embodiment
51-89autophagy function motif aminoacid sequence is (SEQ ID NO.1): Thr-Leu-Gln-Ile-Glu-Lys-His-Val-Ser-Arg-Ala-Phe-Ile-Cys-Leu-Gln-Ala-Ile-Thr-Thr-Phe-Val-Ser-Val-Ala-Gly-Ile-Ile-Tyr-Ile-Ile-Tyr-Lys-Leu-Phe-Ala-Gly-Phe-Gln
CVB3-3A in the present embodiment
51-89autophagy function motif nucleotides sequence is classified (SEQ ID NO.2) as: ACCCTCCAAATTGAAAAACATGTCAGTCGGGCTTTCATCTGCTTGCAGGCAATAAC TACGTTTGTGTCAGTAGCTGGAATCATCTATATAATATACAAGCTCTTTGCAGGCT TTCAA
1.1.CVB3-3A
51-89structure with the expression vector of green fluorescent protein (EGFP) fusion gene
1.1.1CVB3-3A, CVB3-3A
51-89the preparation of nucleic acid fragment
With plasmid pMKS1 (the Mark K.Slifka that contains the full gene of CVB3, Robb Pagarigan, Ignacio Mena, Ralph Feuer, J.Lindsay Whitton.Using recombinant coxsackievirus B3 to evaluate the induction and protective efficacy of CD8
+t cells during picornavirus infection.J Virol.2001; 75 (5): 2377-2387.) be template, primer is:
3A?PF：5‘ATATATAAGCTTGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGGTCCACCAGTATACAGAGAGAT3’；
3A?PR：5‘GCGCGTCTAGATTATTGAAAGCCTGCAAAGAGCTTGTATATTATAT?3’
3A
51-89PF：5‘ATATATAAGCTTGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGACCCTCCAAATTGAAAAACATGT3’；
3A
51-89PR：5‘GCGCGTCTAGATTATTGAAAGCCTGCAAAGAGCTTGTATATTATAT?3’
Primer 3APF, 3APR are for increasing the CVB3-3A full-length gene, primer 3A
51-89pF, 3A
51-89pR is for increasing CVB3-3A
51-89gene.
With above-mentioned primer PCR amplification CVB3-3A gene, CVB3-3A
51-89genetic fragment, archaeal dna polymerase is LA Taq DNA Polymerase (purchased from Japanese TaKaRa).
Reaction condition: 95 ℃ of 5min, 95 ℃ of 45s, 61 ℃ of 45s, 72 ℃ of 1min, totally 30 circulations, then 72 ℃ of abundant 10min that extend.
1.1.2pEGFP-C1 the structure of plasmid platform
Using pcDNA3.1 (+) (purchased from American I nvitrogen) as basic plasmid skeleton, amplify the nucleic acid fragment of EGFP from pEGFP-N1 plasmid (purchased from American I nvitrogen), the EGFP genetic fragment is inserted to pcDNA3.1 (+) plasmid, build pEGFP-C1 plasmid platform, for the structure of the fusion rotein eukaryon expression plasmid of each functional protein of CVB3 and EGFP.
1.1.2.1EGFP the preparation of nucleic acid fragment
5 ' end of upstream and downstream primer add respectively restriction enzyme enzymatic nucleic acid site NheI (TAGCTA) and HindIII (AAGCTT) for after carrier be connected.Take pEGFP-N1 as template, utilize primer PF-pEGFP-C1 (5 '-ATATATAGCTAGCATGGTGAGCAAGGGCGAGGAGCT-3 ') and PR-pEGFP-C1 (5 '-GCTTTAAGCTTCTTGTACAGCTCGTCCATGCCG-3 ') pcr amplification 3 ' end containing the EGFP fragment of termination codon.Reaction condition is: 95 ℃ of 5min, and 95 ℃ of 45s, 56 ℃ of 45s, 72 ℃ of 1min, totally 30 circulations, then 72 ℃ of abundant 10min that extend, archaeal dna polymerase is LA Taq DNA Polymerase (purchased from Japanese TaKaRa).Glue reclaims purification EGFP fragment (glue reclaims test kit purchased from Chinese Hua Shun), glue reclaimer operation reference reagent description.
The method reclaimed by glue again after double digestion EGFP fragment is carried out purification.The double digestion reaction condition is 37 ℃ of 4h, restricted enzyme NheI and HindIII (purchased from Japanese TaKaRa), and glue reclaims the purpose fragment after the purification enzyme action.
1.1.2.2 the preparation of cloning vector
Used carrier is reclaimed and is obtained by NheI and the rear glue of HindIII double digestion plasmid pcDNA3.1 (+).The double digestion reaction condition is 37 ℃ of 4h, restricted enzyme NheI and HindIII (purchased from Japanese TaKaRa), and glue reclaims the purpose carrier after the purification enzyme action.
1.1.2.3 the purpose fragment is connected with carrier
By purpose fragment and the carrier mass ratio of 5: the 1 separately system that connects, after mixing, 16 ℃ of connections of spending the night.T4DNA Ligase (purchased from Japanese TaKaRa).
Transform as entered in competent cell 1.1.2.4 will connect product, screen and identify positive colony
1.1.3pEGFP-3A, pEGFP-3A
51-89merge the structure of recombiant plasmid:
Application limitations restriction endonuclease nucleic acid site HindIII (AAGCTT) and XbaI (TCTAGA) carry out the double digestion processing and open loop plasmid after enzyme action are carried out to the glue recovery pEGFP-C1.Again respectively with CVB3-3A, CVB3-3A
51-89nucleic acid fragment connects, and connected in EGFP reading frame downstream and guaranteed relatively independent the flexibly connecting of both sides albumen space structure (5 '-GGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCG-3 '), vector construction as shown in Figure 1, can obtain respectively described two kinds of fusion protein expression vector pEGFP-CVB3-3A
51-89and pEGFP-CVB3-3A.The carrier sequencing result is correct.
1.2. the expression of fusion rotein
1.2.1 eukaryotic cell transfection experiment:
By human cervical carcinoma cell HeLa cell (purchased from U.S. ATCC) with 2 * 10
4the density in individual/hole is laid in 48 orifice plates, is placed in 37 ℃, 5%CO
2cell culture incubator in cultivate.During to the Growth of Cells area 85%-90% that is the orifice plate floor space, by the fusion rotein plasmid pEGFP-CVB3-3A of 0.8 μ g
51-89reach the pEGFP-CVB3-3A transfection to the HeLa cell, transfection reagent used is Lipofectamine 2000 (purchased from American I nvitrogen), transfection operation reference reagent description.
1.2.2 cultivate after 12h the expression at fluorescence microscopy Microscopic observation fusion rotein
Result of the test is shown in Fig. 2, from result of the test, and CVB3-3A, CVB3-3A
51-89all can express green fluorescence with the EGFP fusion rotein.
Embodiment 2, viral protein expression carrier cause HeLa cell generation autophagy sexual cell apoptosis
2.1. merge recombiant plasmid and pmCherry-LC3 plasmid co-transfection HeLa cell observation cell autophagy
2.1.1pmCherry-LC3 plasmid construction
2.1.1.1LC3 obtaining of purpose fragment
Application limitations restriction endonuclease BglII and EcoRI (purchased from Japanese TaKaRa) enzyme action pEGFP-LC3 (Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T.LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.EMBO is J.2000; 19 (21): 5720-5728.) obtain purpose fragment LC3, double digestion nucleic acid fragment reaction condition is 37 ℃ of 4h.Glue reclaims the LC3 purpose fragment after the purification enzyme action.
2.1.1.2 the preparation of cloning vector pmCherry-C1
Application limitations restriction endonuclease BglII and EcoRI enzyme action carrier pmCherry-C1 (purchased from Japanese Clontech company), double digestion nucleic acid fragment reaction condition is 37 ℃ of 4h.Glue reclaims the carrier after the purification enzyme action.
2.1.1.3 the purpose fragment is connected with carrier
By purpose fragment and the carrier mass ratio of 5: 1 system that connects, after mixing, 16 ℃ of connections of spending the night.T4DNA Ligase (purchased from Japanese TaKaRa).
Be transformed in competent cell 2.1.1.4 will connect product, screen and identify positive colony
2.1.2 merge recombiant plasmid and pmCherry-LC3 plasmid co-transfection HeLa
By human cervical carcinoma cell HeLa cell (purchased from U.S. ATCC) with 2 * 10
4the density in individual/hole is laid in 48 orifice plates, is placed in 37 ℃, the cell culture incubator of 5%CO2 and cultivates.During to the Growth of Cells area 85%-90% that is the orifice plate floor space, by fusion rotein plasmid pEGFP-CVB3-3A, the pEGFP-CVB3-3A of 0.8 μ g
51-89, with the pmCherry-LC3 plasmid of 0.8 μ g, cotransfection is to the HeLa cell respectively, and transfection reagent used is Lipofectamine 2000 (purchased from American I nvitrogen), transfection operation reference reagent description.Cultivate after 12h in the location of fluorescence microscopy Microscopic observation fusion rotein and LC3 situation.
Result of the test is shown in Fig. 3, from result of the test, with the LC3 generation point-like of red fluorescence, assembles, and fusion rotein EGFP-CVB3-3A, EGFP-CVB3-3A are described
51-89cause cell autophagy.
2.2. integrative gene expression vector is induced the expression of autophagy associated protein LC3-II and LC3-I in the HeLa cell
2.2.1 merge Transfected Recombinant Plasmid HeLa cell:
By human cervical carcinoma cell HeLa cell (purchased from U.S. ATCC) with 1 * 10
5the density in individual/hole is laid in 6 orifice plates, is placed in 37 ℃, 5%CO
2cell culture incubator in cultivate.During to the Growth of Cells area 85%-90% that is the orifice plate floor space, by fusion rotein plasmid pEGFP-CVB3-3A, the pEGFP-CVB3-3A of 4 μ g
51-89and negative control pEGFP-C1 (NC), transfection is to the HeLa cell respectively, and transfection reagent used is Lipofectamine2000 (purchased from American I nvitrogen), transfection operation reference reagent description.
2.2.2 the results of expression product:
After transfection 12h, every hole adds 1 * PBS of 200 μ L, with cell scraper, cell is scraped off, and puts into the 2ml centrifuge tube, and 1500 leave heart 5min.Carefully discard supernatant, in centrifuge tube, add 300 μ L to put the protein lysate of protease inhibitor, cracking 10min on ice well.Centrifugal 14,000 turn, 4 ℃ of 10min, collect supernatant with the 1.5mL centrifuge tube ,-20 ℃ of refrigerator storage are put in packing.
Pass through 2.2.3 utilize specific antibody the expression that western blotting method detects LC3-II and LC3-I
2.2.3.1 polyacrylamide gel (PAGE) electrophoresis:
Add 25 μ L6 * loading buffer (0.35M Tris-HCl pH6.8 in the cell conditioned medium of 125 μ l results, 30% glycerol, 10%SDS, 0.012% bromophenol blue, 6% beta-mercaptoethanol) or 6 * non-reduced loading buffer (0.35M Tris-HCl pH6.8,30% glycerol, 10%SDS, 0.012% bromophenol blue), fully mix, 100 ℃ are boiled 2min, centrifugal 8000 turn, 4 ℃ of 10min, and protein sample is loaded in the 8%SDS-PAGE gel, 80V electrophoresis 1h, 120V electrophoresis 2h.。
2.2.3.2Western?blotting：
Electrophoresis to pvdf membrane, spends the night with 5% defatted milk powder the protein transfer printing after finishing in 4 ℃ of sealings.The anti-human LC3 monoclonal antibody of pvdf membrane after sealing and rabbit (1: 500, purchased from U.S. MBL) is hatched 1h in 37 ℃, then hatches 1h with the goat anti-rabbit igg (purchased from China fir Golden Bridge in China) of horseradish peroxidase (HRP) labelling in 37 ℃.Add peroxidase substrate, show protein band.
Result of the test is shown in Fig. 4, and from result of the test, CVB3-2B, CVB3-2C, CVB3-3A group can promote the ratio of LC3II/LC3I to increase, but CVB3-3B acts on without this.CVB3-2B
36-83, CVB3-2C
1-79, CVB3-3A
51-89group also can promote the ratio of LC3II/LC3I to increase.
Induce the HeLa apoptosis 2.3. utilize the electron microscopic observation integrative gene expression vector
By human cervical carcinoma cell HeLa cell (purchased from U.S. ATCC) with 1 * 10
5the density in individual/hole is laid in 6 orifice plates, is placed in 37 ℃, 5%CO
2cell culture incubator in cultivate.During to the Growth of Cells area 85%-90% that is the orifice plate floor space, fusion rotein plasmid by 4 μ g, 5 μ L liposome transfections are to the HeLa cell, and transfection reagent used is Lipofectamine 2000 (purchased from American I nvitrogen), transfection operation reference reagent description.Cultivate after 12h the expression at fluorescence microscopy Microscopic observation fusion rotein.Discard culture fluid in plate, add 1mL1 * PBS (pH7.4), standing 5min, discard repetition 3 times, with Digestive system digestion, with 500 μ L1 * PBS (pH7.4), cell blown to the even 500 μ L centrifuge tubes of putting into, and 1500 leave heart 10min abandons supernatant.Add 500 μ L glutaraldehyde fixatives in each centrifuge tube, fixedly after 48h, electron microscopic observation.
Result of the test is shown in Fig. 5, from result of the test, and pEGFP-CVB3-3A, pEGFP-CVB3-3A
51-89karyopycnosis, apoptosis all appear in the transfection group cell.
Claims (7)
1. the application of cells of coxsackie B 3 virus 3A albumen in preparing the medicine for treating tumor thing.
2. the application of truncated-type Coxsackie virus 3A recombiant protein in preparing the medicine for treating tumor thing, the aminoacid sequence of described albumen is as shown in SEQ ID NO.1.
3. aminoacid sequence is the protein shown in SEQ ID NO.1.
4. the encode nucleotide sequence of protein claimed in claim 3.
5. nucleotide sequence as claimed in claim 4, is characterized in that described nucleotide sequence is as shown in SEQ IDNO.2.
6. an expression vector, is characterized in that containing just like the described nucleotide sequence of claim 4 or 5, and described expression vector is pEGFP-C1.
7. nucleotides sequence claimed in claim 4 is listed in the application prepared in the medicine for treating tumor thing.
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Non-Patent Citations (2)
| Title |
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| Complete nucleotide sequence of the genome of coxsackievirus B1;Narushi izuka et al.;《Viology》;19870131;第158卷(第1期);第64-73页 * |
| Narushi izuka et al..Complete nucleotide sequence of the genome of coxsackievirus B1.《Viology》.1987,第158卷(第1期),第64-73页. |
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