CN101081870B - A kind of anti-tumor biological medicine PTD4-GFP-Apoptin fusion protein and preparation method thereof - Google Patents

Abstract

The biomedicine PTD4-GFP-Apoptin fusion protein for treating tumor has its synthesized PTD4 utilized to introduce Apoptin protein into cell, and its Apoptin further utilized to induce tumor apoptosis, so as to inhibit tumor growth. At the same time, green fluorescent protein (GFP) is utilized for observing the intracellular distribution of the fusion protein and showing the aggregation of the medicine in the target position, realizing the effective monitoring and timely regulation of the treating process. Experiment shows that the fusion protein can penetrate biomembrane to kill tumor cell effectively while generating no damage to health cell. The present invention is applied in treating tumor.

Description

A kind of anti-tumor biological medicine PTD4-GFP-Apoptin fusion protein and preparation method thereof

technical field

The invention relates to biotechnological medicines, especially biotechnological medicines for treating tumors.

Background technique

Cancer is a great threat to human health and life. About 7 million people in the world are newly diagnosed with cancer every year, and more than 5 million people die of cancer every year. At present, about 1.5 million people are newly diagnosed with cancer every year in our country, and about 800,000 people die of cancer every year. Cancer is one of the major diseases that seriously affect human health and threaten human life. Together with cardiovascular and cerebrovascular diseases and accidents, cancer constitutes the top three causes of death in all countries in the world today. Therefore, the World Health Organization and the health departments of governments of various countries have listed conquering cancer as a top priority. Finding new safe and effective drugs and methods for treating tumors is an important topic in the current medical field.

The VP3 protein (also known as apoptin, Apoptin) derived from chicken anemia virus (CAV) has attracted people's attention because of its tumor-specific apoptosis-inducing effect. Both in vivo and in vitro research results show that VP3 (VP3 in this article refers to the VP3 protein of chicken anemia virus, namely CAV VP3, also known as apoptin, namely Apoptin) can induce apoptosis of various tumor cells ^[1] , and its induced The apoptotic effect is independent of p53 ^[2] and not inhibited by anti-apoptotic factors Bcl-2 and Bcl-xL ^[3] . More meaningfully, VP3 only induces the apoptosis of cells with tumorigenic phenotype or transformed phenotype, but has no apoptotic effect and no cytotoxicity on normal cells ^[4] , and VP3 transgenic mice can grow and develop normally ^{[5 ]} . People have actively explored the use of VP3 to treat tumors: at present, direct introduction of exogenous vp3 gene for gene therapy is still the main strategy people adopt; at the same time, different strategies have been tried for different tumors to achieve targeted therapy ^{[6- 7]} , for example, we reported for the first time in the world that the receptor-mediated transfer technology was used to realize the directional transfer and expression of the vp3 gene in vivo in hepatocytes, and specifically induce the apoptosis of liver cancer cells, and achieved a good tumor-suppressing effect ^[8-9] . However, how to give full play to the characteristics of VP3 "tumor cell-specific induction of tumor cell apoptosis", avoid potential risks such as overexpression that may occur after foreign genes are introduced into the body, and further expand the application range of VP3 in the treatment of tumors still needs to be solved urgently The problem.

Protein transduction technology (protein transduction technology) is an emerging macromolecular transfer strategy in recent years: using some proteins or polypeptides with protein transduction domain (PTD) to directly transfer biological macromolecules with therapeutic effects "Send" into cells to exert biological effects ^[10] . This polypeptide with the ability to pass through the cell membrane is called cell-penetrating peptides (Cell-Penetrating Peptides, CPPs), generally no more than 30 amino acids in length and rich in basic amino acids, such as TAT (11 peptide, 13 peptide), Antp (16 peptides), VP22 (34 peptides), Transportan (28 peptides), MAP (18 peptides). CPP can deliver a wide range of substances, such as proteins, DNA, antibodies, imaging reagents, toxins, nano drug particles, liposomes, etc. ^[11] . The CPP delivery system is not only a good tool for studying intracellular biological processes, but also a research object with potential value in biopharmaceutical delivery. This technology is becoming a new idea for tumor biotherapy—it can be used to directly introduce therapeutic substances into cancer cells, making the therapeutic effect more controllable ^[10] . At present, most of the relevant researches are focused on the HIV-TAT (trans-activator of transcription) polypeptide. TAT protein (86 peptide) is a transcriptional activator protein. In 1988, Green ^[11] and Frankel ^[12] independently discovered that HIV-TAT protein has the ability to penetrate biological membranes ^[12-13] ; The ability is mediated by the peptide segment between 47-57 (or 48-60) amino acid residues, and the fusion protein formed by TAT polypeptide and other proteins can also pass through the cell membrane and can exert the biological functions of these proteins ^{[ 14-16]} . Ho et al. ^[17] modified TAT by chemical synthesis, and obtained a series of TAT-PTD polypeptides through amino acid substitutions. The secondary structure of PTD4 is more stable and the transduction efficiency is higher, which can make the target protein transduction successful. The rate is almost 100%, and the amount of protein imported into a single cell is 33 times that of TAT, but whether PTD4 can achieve transmembrane transport of fusion proteins through biosynthesis has not been reported yet.

Contents of the invention

The task of the present invention is to provide an anti-tumor biopharmaceutical, which has the characteristics of being able to directly enter tumor cells to induce tumor cell apoptosis, emit fluorescence, and facilitate observation of its distribution in cells.

The anti-tumor biological drug provided by the present invention is a protein transduction domain 4-green fluorescent protein-apoptin fusion protein (PTD4-GFP-Apoptin, or PTD4-GFP-VP3), which is shown in sequence 8 or sequence 9 in the sequence listing The amino acid sequence, its preparation method, comprises the following steps:

a. Construct the prokaryotic expression vector pET28a-PTD4 containing the PTD4 sequence;

b. Construct the prokaryotic expression vector pET28a-PTD4-GFP containing the GFP gene;

c. Construct the prokaryotic expression vector pET28a-PTD4-GFP-Apoptin containing the Apoptin gene;

d. Expression and purification of PTD4-GFP-Apoptin fusion protein.

The method for constructing the prokaryotic expression vector pET28a-PTD4 containing the PTD4 sequence is as follows: design its base sequence according to the amino acid composition of the PTD4 polypeptide, synthesize two oligonucleotide fragments, mix the two oligonucleotide fragments in equimolar ratios, and heat at 95°C for 10 minutes , and then placed at room temperature for 1 hour to renature to form a double-stranded DNA encoding PTD4; the double-stranded DNA was inserted into the Nhe I of pET28a, transformed into DH5α, and identified by restriction enzyme digestion, PCR, and sequencing to prove that the pET28a-PTD4 recombinant plasmid was constructed.

The method for constructing the prokaryotic expression vector pET28a-PTD4-GFP containing the GFP gene is: utilize the PCR method to amplify the GFP gene of the green fluorescent protein, and pass the PCR amplification fragment GFP and the pET28a-PTD4 recombinant plasmid through BamH I and EcoR I double enzymes respectively Cut, recover the target fragment, connect and transform DH5α, amplify and extract the plasmid, and obtain the recombinant pET28a-PTD4-GFP.

The method for constructing the prokaryotic expression vector pET28a-PTD4-GFP-Apoptin containing the Apoptin gene is: utilize the PCR method to amplify the Apoptin gene of the chicken anemia virus, and the PCR amplified fragment Apoptin and the pET28a-PTD4-GFP recombinant plasmid are passed through EcoR I respectively. Digest with Sal I, recover the target fragment, connect and transform DH5α, amplify and extract the plasmid, and obtain recombinant pET28a-PTD4-GFP-Apoptin.

The method of expression and purification of PTD4-GFP-Apoptin fusion protein is: transform the recombinant plasmid pET28a-PTD4-GFP-Apoptin into the expression strain Escherichia coli BL21(DE3) PlysS, and culture it in 5ml LB containing 0.05mg/ml kanamycin Shake culture in medium at 37°C, when A ₆₀₀ = 0.4-0.6, add IPTG to a final concentration of 1.0mM to induce for 8 hours, sonicate bacteria, collect inclusion bodies by centrifugation, use uninduced bacteria as control, and identify by 12.5% SDS-PAGE ; The inclusion body was dissolved in the sample buffer containing 8mol/l urea, and purified by nickel affinity chromatography, and the operation steps were carried out according to the requirements of the kit. SDS-PAGE identified the eluted protein, combined the eluate containing the target protein, dialyzed, concentrated, filtered and sterilized, determined the protein content by BCA method, and stored at -80°C.

The schematic diagram of the construction of the recombinant plasmid of the present invention is shown in FIG. 1 .

The anti-tumor biopharmaceutical PTD4-GFP-Apoptin fusion protein provided by the present invention uses the synthetic PTD4 to directly introduce the Apoptin protein into cells, and then uses Apoptin to specifically induce tumor cell apoptosis, thereby achieving the goal of promoting tumor apoptosis and inhibiting tumor growth. Objective: At the same time, using the characteristics of GFP (Green Fluorescent Protein) to emit green fluorescence, the distribution of the fusion protein in the cell can be observed under a fluorescent microscope, and the aggregation of the drug at the target site can be displayed, so as to achieve an effective treatment process. Monitor and make timely adjustments. It was confirmed by protein transduction experiments that PTD4-GFP and PTD4-GFP-Apoptin fusion protein of the present invention can penetrate biofilm, and it was confirmed by TUNEL method and DAPI staining that PTD4-GFP-Apoptin fusion protein of the present invention can induce tumor cell apoptosis, showing that The PTD4-GFP-Apoptin fusion protein prepared and purified artificially has a good biomembrane penetration effect and can induce tumor cell apoptosis, has a great killing ability on tumor cells, and does not damage normal cells and will not There is a risk of introducing foreign genes, and it can be used for the treatment of tumors.

Using the protein transduction domain 4-green fluorescent protein-apoptin fusion protein (PTD4-GFP-Apoptin) provided by the present invention as an active ingredient, plus pharmaceutically acceptable carriers and/or additives, prepared according to conventional methods The antitumor drug preparation can be applied to the tumor site to treat the tumor, and the pharmaceutical carrier can be PBS (phosphate buffer saline), glycerin or urea, etc.

Description of drawings

Figure 1 is a schematic diagram of the construction of the recombinant plasmid of the present invention.

Figure 2 shows the identification results of the recombinant pET28a-PTD4, and the marks in the figure are:

M1: Mraker, molecular weight standards, from large to small are 7000, 5500, 3500, 2000, 1000, 500bp;

A: The result of pET28a-PTD4 digested by Nhe I;

B: PTD4 fragment obtained by step 1;

C: PCR result of pET28a-PTD4;

M2: Mraker, molecular weight standards, from large to small are 600, 500, 400, 300, 200, 100bp.

Figure 3 shows the identification results of the recombinant pET28a-PTD4-GFP, and the marks in the figure are:

M1: Marker, molecular weight standard, from large to small are 7000, 5500, 3500, 2000, 1000, 500bp;

A: the GFP fragment obtained in step 1;

B: pET-28a-PTD4-GFP was digested with BamHI and EcoRI;

C: pET-28a-PTD4-GFP was digested with BamHI.

Figure 4 shows the identification results of the recombinant pET28a-PTD4-GFP-VP3, and the marks in the figure are:

M3: Mraker, molecular weight standards, from large to small are 1500, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100bp;

A: PCR result of pcDNA-vp3;

B: pET28a-PTD4-GFP-VP3 digested by EcoR I and Sal I;

C: pET28a-PTD4-GFP-VP3 digested by EcoR I;

M1: Marker, molecular weight standard, from large to small are 7000, 5500, 3500, 2000, 1000, 500bp.

Figure 5 shows the PAGE results of the PTD4-GFP fusion protein, and the marks in the figure are:

M: protein molecular weight standard, from top to bottom: 116.0kDa, 66.2kDa, 45.0kDa, 35.0kDa, 25.0kDa, 18.4kDa, 14.4kDa;

1: Bacterial total protein not induced to express;

2: Bacterial total protein induced by IPTG;

3: Purified PTD4-GFP fusion protein.

Figure 6 shows the PAGE results of the PTD4-GFP-VP3 fusion protein, and the marks in the figure are:

M: protein molecular weight standard, from top to bottom: 116.0kDa, 66.2kDa, 45.0kDa, 35.0kDa, 25.0kDa, 18.4kDa, 14.4kDa;

4: Bacterial proteins in inclusion bodies;

5: Bacterial total protein not induced to express;

6: Bacterial total protein induced by IPTG;

7: Purified PTD4-GFP-VP3 fusion protein.

Fig. 7 is the result of the fluorescence microscope observation of the transmembrane effect experiment of PTD4-GFP fusion protein;

Figure 8 is the result of optical microscope observation of the membrane penetration effect experiment of PTD4-GFP fusion protein;

Fig. 9 is the result of the fluorescence microscope observation of the transmembrane effect experiment of PTD4-GFP-VP3 fusion protein;

Figure 10 is the result of optical microscope observation of the membrane penetration effect experiment of PTD4-GFP-VP3 fusion protein;

Figure 11 shows the FITC excitation results of PTD4-GFP-VP3 fusion protein-induced apoptosis of HepG ₂ cells;

Fig. 12 is the laser confocal microscope observation DAPI excitation result of PTD4-GFP-VP3 fusion protein-induced apoptosis of HepG ₂ cells;

Figure 13 shows the co-excitation results of FITC and DAPI observed by confocal laser microscopy of PTD4-GFP-VP3 fusion protein-induced apoptosis of HepG ₂ cells;

Fig. 14 shows the results under the confocal laser microscope of PTD4-GFP-VP3 fusion protein-induced apoptosis of HepG ₂ cells.

Detailed ways

Example 1

Construction of prokaryotic expression vector pET28a-PTD4 containing PTD4 sequence

1. Preparation of PTD4 sequence by annealing artificially synthesized oligonucleotide fragments

(1) According to Ho's report ^[17] , the PTD4 polypeptide consists of 11 amino acids, and its base sequence is independently designed according to the characteristics of prokaryotic codons as follows:

S1: 5′-CTAGTTATGCCCGCGCGGCAGCACGACAAGCTCGAGCCC-3′

S2: 5′-CTAGGGGCTCGAGCTTGTCGTGCTGCCGCGCGGGCATAA-3′

Two oligonucleotide fragments were synthesized by Shanghai Bioengineering Company.

The two oligonucleotide fragments were mixed equimolarly, 95°C for 10 min, and then placed at room temperature for 1 h to anneal to form a double-stranded DNA encoding PTD4.

2. Construction of recombinant pET28a-PTD4

(1) The prokaryotic expression vector pET-28a(+) was purchased from Novagen.

(2) The construction method adopts the conventional gene cloning method.

The double strand obtained by the above method was inserted into Nhe I of pET28a, transformed into DH5α, identified by enzyme digestion, PCR, and sequencing, and it was proved that the pET28a-PTD4 recombinant plasmid was constructed. DNA sequence determination was completed by Shanghai Boya. PCR identification primers are:

P1: GGCAGCACGACAAGCTCGAG

P2: AACCCCTCAAGACCCGTTTAGAG, fragment size: 298bp

PCR amplification reaction conditions

The cycle parameters were denaturation at 95°C for 5 min, 1 min at 94°C, 1 min at 50°C, and 1 min at 72°C, 30 cycles of amplification, and finally 10 min at 72°C.

The identification results of the recombinant pET28a-PTD4 are shown in Figure 2. From the results in Figure 2, it can be judged that the recombinant pET28a-PTD4 was successfully constructed.

Example 2

Construction of prokaryotic expression vector pET28a-PTD4-GFP containing GFP gene

1. Using the PCR method to amplify the GFP gene of green fluorescent protein

(1) pEGFP-C1 was purchased from CLONETECH.

(2) PCR primer sequences are as follows:

P3: 5'-ACGGATCCATGGTGAGCAAGGGCG-3';

P4: 5′-GCGAATTCCTTGTACAGCTCGTCCATGC-3′

The 5′ ends of the two primers contain recognition sites for restriction endonucleases BamH I and EcoR I, respectively.

(3) PCR amplification reaction conditions

The cycle parameters are 94°C for 5min, 94°C for 55sec, 62°C for 55sec, and 72°C for 1min, and the cycle amplification is 30 times, and finally, keep at 72°C for 10min. The PCR product was identified by 1.5% agarose gel electrophoresis, and the band size was correct.

2. Construction of prokaryotic expression vector pET28a-PTD4-GFP

(1) For the construction of the prokaryotic expression vector pET28a-PTD4, see step (1).

(2) The construction method adopts the conventional gene cloning method.

The PCR amplified fragment GFP and pET28a-PTD4 recombinant plasmid were digested by BamH I and EcoR I respectively, the target fragment was recovered, ligated and transformed into DH5α, and the extracted plasmid was amplified.

The recombinant identification results are shown in Figure 3, from the results in Figure 3 it can be judged that the recombinant pET28a-PTD4-GFP was successfully constructed.

Example 3

Construction of prokaryotic expression vector pET28a-PTD4-GFP-VP3 containing vp3 gene

1. Utilize the PCR method to amplify the vp3 gene of chicken anemia virus

(1) Construct the eukaryotic expression vector pcDNA-vp3 containing the vp3 gene. For specific methods, see: Wang Yuzhe, Tian Jun, Qu Shen, etc., Construction of chicken anemia virus vp3 gene and research on its apoptosis-inducing effect in vitro, Journal of Tongji Medical University, 2001 , 30(4):300-4. ^[18]

(2) PCR primer sequences are as follows:

P5: 5′-AGGAATTCATGAACGCTCTCCAAG-3′

P6: 5′-GCGTCGACTTACAGTCTTATACGCC-3′

The 5' ends of the two primers contain recognition sites for restriction endonucleases EcoR I and Sal I, respectively.

(3) PCR amplification reaction conditions

The cycle parameters are 94°C for 5min, 94°C for 55sec, 60°C for 50sec, and 72°C for 55sec, cycle amplification 30 times, and keep warm at 72°C for 10min. The PCR products were identified correctly by 1.5% agarose gel electrophoresis.

2. Construction of prokaryotic expression vector pET28a-PTD4-VP3 containing vp3 gene

(1) For the construction of the prokaryotic expression vector pET28a-PTD4-GFP, see step (2).

(2) The construction method adopts the conventional gene cloning method.

The PCR amplified fragments vp3 and pET28a-PTD4-GFP recombinant plasmids were digested with EcoR I and Sal I respectively, the target fragments were recovered, ligated and transformed into DH5α, and the extracted plasmids were amplified.

The results of recombinant identification are shown in Figure 4, from the results in Figure 4 it can be judged that the recombinant pET28a-PTD4-GFP-VP3 was successfully constructed.

Example 4

Expression and purification of PTD4-GFP and PTD4-GFP-VP3 fusion proteins

1. The prokaryotic expression strain E.coli BL21(DE3)PlysS was purchased from Novagen. The nickel affinity chromatography column was purchased from Promega. IPTG acquires BBI Corporation. BCA protein assay kit was purchased from Pierce Company.

2. Fusion protein expression process

The purified two recombinant plasmids pET28a-PTD4-GFP and pET28a-PTD4-GFP-VP3 were respectively transformed into the expression strain Escherichia coli BL21(DE3)PlysS. Shake culture in 5ml LB medium containing 0.05mg/ml kanamycin at 37°C. When A ₆₀₀ = 0.4~0.6, add IPTG to a final concentration of 1.0mM for induction for 8 hours, sonicate bacteria, and collect inclusion bodies by centrifugation. Using uninduced bacteria as a control, it was identified by 12.5% SDS-PAGE.

3. Fusion protein purification process

The inclusion body was dissolved in the loading buffer solution containing 8mol/l urea, and purified by nickel affinity chromatography column, and the operation steps were carried out according to the requirements of the kit. SDS-PAGE identified the eluted protein, combined the eluate containing the target protein, dialyzed, concentrated, filtered and sterilized, determined the protein content by BCA method, and stored at -80°C.

4. The identification results of PTD4-GFP fusion protein are shown in Figure 5 and Figure 6. The electrophoresis result in Figure 5 shows that a pure PTD4-GFP fusion protein is obtained; the electrophoresis result in Figure 6 shows that a pure PTD4-GFP-VP3 fusion protein is obtained.

Example 5

In vitro membrane permeation effect experiment of the fusion protein of the present invention

1. Experimental materials:

(1) PBS (phosphate buffer: 0.8% NaCl, 0.02% KCl, 0.144% Na ₂ HPO ₄ , 0.024% KH ₂ PO ₄ ) solution of PTD4-GFP and PTD4-GFP-VP3.

(2) The experimental cells were the human liver cancer cell line HepG ₂ purchased from CCTCC.

2. Experimental method

Human liver cancer cells HepG ₂ were inoculated on the culture plate. After the cells adhered to the wall, the cells were incubated with 1 μmol/L fusion protein PTD4-GFP and PTD4-GFP-VP3. After 2 hours, the culture medium was sucked off, washed three times with PBS, and then placed in Observed under a fluorescence microscope.

3. Experimental results

PTD4-GFP and PTD4-GFP-VP3 fusion proteins were added to HepG ₂ cells cultured in vitro, and obvious green fluorescence could be seen in the cells after 2 hours, as shown in Figure 7, Figure 8, Figure 9 and Figure 10.

Combined with the results of Fig. 7, Fig. 8, Fig. 9 and Fig. 10, it is shown that PTD4-GFP and PTD4-GFP-VP3 fusion proteins all successfully penetrate the cell membrane and enter HepG ₂ cells, indicating that the synthesized PTD4 of the present invention has the ability to mediate fusion protein penetration. function of the membrane.

Example 6

Experiment of _HepG2 Cell Apoptosis Induced by Fusion Protein PTD4-GFP-VP3 of the Present Invention

1. Experimental materials:

(1) PBS (phosphate buffer: 0.8% NaCl, 0.02% KCl, 0.144% Na ₂ HPO ₄ , 0.024% KH ₂ PO ₄ ) solution of PTD4-GFP-VP3.

(2) The experimental cells were the human liver cancer cell line HepG ₂ purchased from CCTCC. TUNEL detection kit was purchased from Promega Company.

2. Experimental method

The treated coverslips were placed in a six-well plate, and human liver cancer cells HepG ₂ were seeded in a six-well plate at an appropriate density. After the cells adhered to the wall, the cells were incubated with 1 μmol/L fusion protein PTD4-GFP-VP3 4 -5 days, washed three times with PBS, fixed with 4% paraformaldehyde for 30 min, detected cell apoptosis by TUNEL (FITC staining) and counterstained with DAPI, the operation process was carried out according to the instructions. Observed under a confocal laser microscope.

3. Experimental results

The fusion protein PTD4-GFP-VP3 was incubated with HepG ₂ cells for 4-5 days. TUNEL was used to detect cell apoptosis and counterstained with DAPI. Under the laser confocal microscope, it was obvious that the nucleus shrunk and bordered, indicating that the fusion protein could induce cell apoptosis. , see Figure 11, Figure 12, Figure 13 and Figure 14.

Combining the results of Fig. 11, Fig. 12, Fig. 13 and Fig. 14, it shows that the PTD4-GFP-VP3 fusion protein induces the apoptosis effect of HepG ₂ cells.

Example 7

With the PTD4-GFP-VP3 fusion protein provided by the present invention as the active ingredient, add PBS (phosphate buffer: 0.8% NaCl, 0.02% KCl, 0.144% Na ₂ HPO ₄ , 0.024% KH ₂ PO ₄ ) solution, press Prepare antitumor drug preparations by conventional methods.

Example 8

The PTD4-GFP-VP3 fusion protein provided by the invention is used as an active ingredient, plus PBS solution and 10% glycerin, and prepared into an antitumor drug preparation according to a conventional method.

Example 9

The PTD4-GFP-VP3 fusion protein provided by the present invention is used as an active ingredient, and a certain amount of urea (such as 4M) is added to prepare an antitumor drug preparation according to a conventional method.

references

1. Noteborn MHM. Chicken anemia virus induced apoptosis: underlying molecular mechanisms. VetMicorbiol, 2004, 98: 89-94.

2. Zhuang SM, shvarts A, van Ormondt H, Jochemsen AG, van der Eb AJ, and Noteborn MHM. Apoptin, a protein derived from chicken anemia virus, induces p53-independent apoptosis in humanosteosarcoma cells. Cancer Res, 1995, 5:5 486-489.

3. Schoop RA, Kooistra K, Baatenburg De Jong RJ, and Noteborn MHM. Bcl-xL inhibits p53-butnot apoptin-induced apoptosis in head and neck squamous cell carcinoma cell line. Int JCancer, 2004, 109: 38-42.

4. Zhang YH, Leliveld SR, Kooistra K, Molenaar C, Rohn JL, Tanke HJ, Abrahams JP, and NotebornMHM. Recombinant Apoptin multimers kill tumor cells but are nontoxic and epitope-shielded in a normal-cell-specific C fashion Res. , 2003, 289: 36-46.

5. Pietersen AM. Preclinical studies with Apoptin. PhD thesis, Erasmus University, Rotterdam, The Netherlands, 2003.

6. Van der Eb MM, Pietersen AM, Speetjens FM, Kuppen PJ, van de Velde CJ, Notetorn MHM, and Hoeben RC. Gene therapy with apoptin induces regression of xenografted human hepatomas. Cancer Gene Ther, 2002, 9: 53-61.

7. Song JS. Enhanced expression of apoptin by the myc-max binding motif and SV40 enhancer for SCLC gene therapy. Biosci Biotechnol Biochem, 2005, 69: 51-55.

8. Sun Jun, Wang Yuzhe, Peng Dongjun, Zong Yiqiang, Qu Shen, Experimental study of asialoglycoprotein receptor-mediated vp3 gene-targeted therapy for liver cancer, Journal of Medical Molecular Biology, 2004, 1(1): 5- 9.

9. WANG YZ, Peng DJ, SUN J, TIAN J, Zhang YH, Noteborn MHM, Qu S. Inhibition of hepatocarcinomaby systemic delivery of Apoptin gene to the hepatic asiaglycorprotein receptor. Cancer GeneTherapy, 2006, submitted.

10. KG Ford, BE Souberbielle, D Darling and F Farzaneh, Protein Transduction: an alternative to genetic intervention? Gene Therapy, 2001, 8:1-4.

11. Gupta B, Levchenko TS, Torchilin VP, Intracellular delivery of large molecules and small particles by cell-penetrating protein and peptides. Advanced Drug Delivery Reviews. 2005, 57: 637-651.

12. Green, M., and Loewenstein, P.M. Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein. Cell, 1988, 55: 1179-1188.

13. Frankel, A.D., and Pabo, C.O. Cellular uptake of the tat protein from human immunodeficiency virus. Cell, 1988, 55: 1189-1193.

14. Nagahara, H., Vocero-Akbani, A.M., Snyder, E.L., Ho, A., Latham, D.G., Lissy, N.A., Becker-Hapak, M., Ezhevsky, S.A., and Dowdy, S.F.Transduction of full length tat Fusion proteins into mammalian cells: p27Kipl mediates cell migration. Nat. Med., 1998, 4: 1449-1452.

15. Schwarze, S., Ho, A., Vocero-Akbani, A., and Dowdy, S.F. In vivo protein transduction: delivery of biologically active protein into the mouse. Science (Washington DC), 1999, 285: 1569-1572 .

16. Schwarze, S.R., Hruska, K.A., and Dowdy, S.F. Protein transduction: unrestricted delivery into all cells? Trends Cell Biol. 2000, 10: 290-295.

17. Ho A., Schwarze SR., Ermelstein SJ., Waksman G. and Dowdy SF., Synthetic Protein Transduction Domains: Enhanced Transduction Poteintial in Vitro and in Vivo., Cancer Research, 61, 474-477, Jan.15, 2001.

18. Wang Yuzhe, Tian Jun, Qu Shen, etc., Construction of Chicken Anemia Virus vp3 Gene and Study on Induction of Apoptosis in Vitro, Journal of Tongji Medical University, 2001, 30(4): 300-4.

The following are the nucleotide sequences and amino acid sequences involved in the present invention, and the sequences in the table are respectively:

Sequence 1 is the DNA sequence of PTD4 (only the sequence of the coding strand is shown, the same below);

Sequence 2 is the amino acid sequence of PTD4;

Sequence 3 is the DNA sequence of Apoptin;

Sequence 4 is the amino acid sequence of Apoptin;

Sequence 5 is the amino acid sequence of Apoptin;

Sequence 6 is the DNA sequence of GFP;

Sequence 7 is a DNA sequence expressing PTD4-GFP-Apoptin fusion protein;

Sequence 8 is the amino acid sequence of PTD4-GFP--Apoptin fusion protein;

Sequence 9 is the amino acid sequence of PTD4-GFP--Apoptin fusion protein digested by thrombin.

sequence listing

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ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaag 717

<210>6

<211>239

<212>PRT

<213>Aequorea victoria

<400>6

Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu

1 5 10 15

Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly

20 25 30

Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile

35 40 45

Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr

50 55 60

Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys

65 70 75 80

Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu

85 90 95

Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu

100 105 110

Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly

115 120 125

Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr

130 135 140

Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn

145 150 155 160

Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser

165 170 175

Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly

180 185 190

Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu

195 200 205

Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe

210 215 220

Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys

225 230 235

<210>7

<211>1230

<212>DNA

<213> Artificial sequence

<400>7

atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60

atggctagtt atgcccgcgc ggcagcacga caagctcgag cccctagcat gactggtgga 120

cagcaaatgg gtcgcggatc catggtgagc aagggcgagg agctgttcac cggggtggtg 180

cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag 240

ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag 300

ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc 360

cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac 420

gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg 480

aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag 540

gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc 600

atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag 660

gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc 720

gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac 780

gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc 840

atggacgagc tgtacaagga attcatgaac gctctccaag aagatactcc acccggacca 900

tcaacggtgt tcaggccacc aacaagttca cggccgttgg aaacccctca ctgcagagag 960

atccggattg gtatcgctgg aattacaatc actctatcgc tgtgtggctg cgcgaatgct 1020

cgcgctccca cgctaagatc tgcaactgcg gacaattcag aaagcactgg tttcaagaat 1080

gtgccggact tgaggaccga tcaacccaag cctccctcga agaagcgatc ctgcgacccc 1140

tccgagtaca gggtaagcga gctaaaagaa agcttgatta ccactactcc cagccgaccc 1200

cgaaccgcaa aaaggcgtat aagactgtaa 1230

<210>8

<211>409

<212>PRT

<213> Artificial sequence

<400>8

Met Gly Ser Ser His His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Ala Ser Tyr Ala Arg Ala Ala Ala Arg Gln Ala

20 25 30

Arg Ala Pro Ser Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Met

35 40 45

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

50 55 60

Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu

65 70 75 80

Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys

85 90 95

Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu

100 105 110

Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln

115 120 125

His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg

130 135 140

Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val

145 150 155 160

Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile

165 170 175

Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn

180 185 190

Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly

195 200 205

Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val

210 215 220

Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro

225 230 235 240

Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser

245 250 255

Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val

260 265 270

Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Glu Phe

275 280 285

Met Asn Ala Leu Gln Glu Asp Thr Pro Pro Gly Pro Ser Thr Val Phe

290 295 300

Arg Pro Pro Thr Ser Ser Arg Pro Leu Glu Thr Pro His Cys Arg Glu

305 310 315 320

Ile Arg Ile Gly Ile Ala Gly Ile Thr Ile Thr Leu Ser Leu Cys Gly

325 330 335

Cys Ala Asn Ala Arg Ala Pro Thr Leu Arg Ser Ala Thr Ala Asp Asn

340 345 350

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

355 360 365

Pro Lys Pro Pro Ser Lys Lys Arg Ser Cys Asp Pro Ser Glu Tyr Arg

370 375 380

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

385 390 395 400

Arg Thr Ala Lys Arg Arg Ile Arg Leu

405

<210>9

<211>392

<212>PRT

<213> Artificial sequence

<400>9

Gly Ser His Met Ala Ser Tyr Ala Arg Ala Ala Ala Arg Gln Ala Arg

1 5 10 15

Ala Pro Ser Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Met Val

20 25 30

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

35 40 45

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

50 55 60

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr

65 70 75 80

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr

85 90 95

Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His

100 105 110

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

115 120 125

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

130 135 140

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

145 150 155 160

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr

165 170 175

Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile

180 185 190

Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln

195 200 205

Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val

210 215 220

Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys

225 230 235 240

Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr

245 250 255

Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Glu Phe Met

260 265 270

Asn Ala Leu Gln Glu Asp Thr Pro Pro Gly Pro Ser Thr Val Phe Arg

275 280 285

Pro Pro Thr Ser Ser Arg Pro Leu Glu Thr Pro His Cys Arg Glu Ile

290 295 300

Arg Ile Gly Ile Ala Gly Ile Thr Ile Thr Leu Ser Leu Cys Gly Cys

305 310 315 320

Ala Asn Ala Arg Ala Pro Thr Leu Arg Ser Ala Thr Ala Asp Asn Ser

325 330 335

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

340 345 350

Lys Pro Pro Ser Lys Lys Arg Ser Cys Asp Pro Ser Glu Tyr Arg Val

355 360 365

Ser Glu Leu Lys Glu Ser Leu Ile Thr Thr Thr Pro Ser Arg Pro Arg

370 375 380

Thr Ala Lys Arg Arg Ile Arg Leu

385 390

Claims (9)

1. protein transduction domain 4-green fluorescent protein-apoptosis plain fusion protein PTD4-GFP-Apoptin, its aminoacid sequence is shown in sequence in the sequence table 8 or sequence 9.

2. contain protein transduction domain 4-green fluorescent protein-apoptosis plain fusion protein PTD4-GFP-Apoptin Prokaryotic Expression carrier, the nucleotide sequence of described protein transduction domain 4-green fluorescent protein-apoptosis plain fusion protein PTD4-GFP-Apoptin gene is shown in sequence in the sequence table 7.

3. the application of the described fusion rotein of claim 1 in the preparation medicines resistant to liver cancer.

4. the preparation method of the described protein transduction domain 4-green fluorescent protein of claim 1-apoptosis plain fusion protein PTD4-GFP-Apoptin may further comprise the steps:

A. make up the prokaryotic expression carrier pET28a-PTD4 that contains the PTD4 sequence;

B. make up and contain PTD4-GFP Prokaryotic Expression carrier pET28a-PTD4-GFP;

C. structure contains PTD4-GFP-Apoptin Prokaryotic Expression carrier pET28a-PTD4-GFP-Apoptin, and the nucleotide sequence of described PTD4-GFP-Apoptin gene is shown in sequence in the sequence table 7;

D.PTD4-GFP-Apoptin Expression of Fusion Protein and purifying.

5. the preparation method of protein transduction domain 4-green fluorescent protein according to claim 4-apoptosis plain fusion protein PTD4-GFP-Apoptin; It is characterized in that the method that said structure contains the prokaryotic expression carrier pET28a-PTD4 of PTD4 sequence is: form its base sequence of design according to the PTD4 polypeptide amino acid; Synthetic two oligonucleotide fragments mix moles such as two oligonucleotide fragments 95 ℃ of 10min; Room temperature is placed the 1h renaturation then, forms the double-stranded DNA of coding PTD4; This two strands is inserted into the Nhe I place of pET28a, transforms DH5 α, cut evaluation, PCR, order-checking, prove to be built into the pET28a-PTD4 recombinant plasmid through enzyme.

6. the preparation method of protein transduction domain 4-green fluorescent protein according to claim 4-apoptosis plain fusion protein PTD4-GFP-Apoptin; It is characterized in that the method that said structure contains GFP Prokaryotic Expression carrier pET28a-PTD4-GFP is: the GFP gene that utilizes PCR method amplification green fluorescent protein; With pcr amplified fragment GFP and pET28a-PTD4 recombinant plasmid respectively through BamH I and EcoR I double digestion; Reclaim the purpose segment; Connect, transform DH5 α, plasmid is extracted in amplification, obtains recombinant chou pET28a-PTD4-GFP.

7. the preparation method of protein transduction domain 4-green fluorescent protein according to claim 4-apoptosis plain fusion protein PTD4-GFP-Apoptin; It is characterized in that the method that said structure contains PTD4-GFP-Apoptin Prokaryotic Expression carrier pET28a-PTD4-GFP-Apoptin is: the Apoptin gene that utilizes PCR method amplification chicken anaemia virus; With pcr amplified fragment Apoptin and pET28a-PTD4-GFP recombinant plasmid respectively through through EcoR I and Sal I double digestion; Reclaim the purpose fragment; Connect, transform DH5 α, plasmid is extracted in amplification, obtains recombinant chou pET28a-PTD4-GFP-Apoptin.

8. the preparation method of protein transduction domain 4-green fluorescent protein according to claim 4-apoptosis plain fusion protein PTD4-GFP-Apoptin; The method that it is characterized in that said PTD4-GFP-Apoptin Expression of Fusion Protein and purifying is: recombinant plasmid pET28a-PTD4-GFP-Apoptin is transformed expression strain e. coli bl21 PlysS; Contain in the LB substratum of 0.05mg/ml kantlex 37 ℃ of concussions at 5ml and cultivate, to A ₆₀₀=0.4～0.6 o'clock, add IPTG and induced 8 hours to final concentration 1.0mM, carrying out ultrasonic bacteria breaking, centrifugal collection inclusion body does not compare to induce bacterium, identifies through 12.5%SDS-PAGE; Inclusion body is dissolved in the sample-loading buffer that contains 8mol/l urea, and through the nickel affinity chromatography column purification, operation steps is undertaken by the test kit requirement; SDS-PAGE identifies eluted protein, merges the elutriant that contains target protein, dialysis, concentrated, filtration sterilization; The BCA method is measured protein content ,-80 ℃ of preservations.

9. the gene of protein transduction domain 4-green fluorescent protein-apoptosis plain fusion protein PTD4-GFP-Apoptin, its nucleotide sequence is shown in sequence in the sequence table 7.

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