WO2015078237A1 - 增强trail抗肿瘤活性的双靶点融合蛋白及其体外表达方法和应用 - Google Patents

增强trail抗肿瘤活性的双靶点融合蛋白及其体外表达方法和应用 Download PDF

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WO2015078237A1
WO2015078237A1 PCT/CN2014/088301 CN2014088301W WO2015078237A1 WO 2015078237 A1 WO2015078237 A1 WO 2015078237A1 CN 2014088301 W CN2014088301 W CN 2014088301W WO 2015078237 A1 WO2015078237 A1 WO 2015078237A1
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sequence
trail
fusion protein
target fusion
protein
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French (fr)
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陈守春
潘凤
闫娟
徐琦
胡海洋
李昭君
朱文彦
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成都华创生物技术有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • the invention relates to the field of genetic engineering medicine technology, in particular to a double target fusion protein for enhancing TRAIL antitumor activity and an in vitro expression method and application of the fusion protein.
  • Tumor necrosis factor-related apoptosis-inducing ligand is a member of the tumor necrosis factor (TNF) superfamily.
  • TNF tumor necrosis factor
  • the gene sequences were respectively in 1995 by Wiley et al. (Wiley et al. Al. 1995) and 1996 were independently cloned by Pitti et al. (Pitti et al. 1996). The latter named it an apoptin 2 ligand (Apo2ligand, Apo2L). Later studies confirmed that Apo2L is the same protein as TRAIL, so it is customary to call it Apo2L/TRAIL.
  • the coding gene for human Apo2L/TRAIL is located on chromosome 3q26 (Miriani and Krammer. 1998).
  • the complete Apo2L/TRAIL cDNA sequence encodes a 281 amino acid full length precursor protein with a relative molecular mass of 32.5 kDa.
  • the natural Apo2LTRAIL protein is expressed as a type II transmembrane protein, which is divided into three parts: the extracellular C-terminal region, the transmembrane region, and the intracellular N-terminal region.
  • the N-terminal 1-14aa of Apo2LTRAIL is a short intracellular segment with no signal peptide sequence; the 15th to 40th aa is a hydrophobic region, forming a transmembrane structure; the C-terminal 41-281 aa is an extracellular region, which is highly conserved .
  • the C-terminus can form several ⁇ -sheets, which form a typical ⁇ -sandwich structure, which in turn forms a subunit of homotrimers, which is the main structural region in which the protein functions.
  • the Apo2L/TRAIL precursor protein is hydrolyzed by a specific protease to form soluble TRAIL, and its 114-281aa is a soluble fragment of the protein (Miriani and Krammer. 1998).
  • the C-terminal (extracellular region) sequence of human Apo2L/TRAIL is as highly conserved as TNF and Fas ligands, and the extracellular domain of human TRAIL molecule is associated with Fas ligand, tumor necrosis factor alpha, lymphotoxin-alpha and lymphotoxin-beta. The homology was 28%, 23%, and 22%, respectively (Wiley et al. 1995). The most unique difference between TRAIL and other members of the TNF family is that it forms a 12-16 aa insertion loop (AA'loop) at positions 137-152. Insertion of the TRAIL binding site of the receptor ensures specific binding of the receptor to TRAIL.
  • TRAIL 230th cysteine
  • cysteine mutated to serine or alanine
  • TRAIL will form an inactive dimeric structure, and the affinity with the receptor will decrease 200-fold, seriously affecting TRAIL.
  • Apoptosis-inducing activity (Jean et al. 2000).
  • the function of TRAIL is firstly used as a regulator of congenital or acquired immunity in organisms, and secondly plays an important role in the process of exogenous apoptotic pathways, and plays an important role in the anti-tumor process as immunosurveillance.
  • the greatest advantage of TRAIL is that it can selectively induce apoptosis of a variety of tumor cells with little toxicity to normal cells.
  • Apo2L/TRAIL is in vivo or in vitro for human tumor cell lines of various origins, including nodules, lung cancer, breast cancer, prostate cancer, pancreatic cancer, kidney cancer, central nervous system tumors, Thyroid cancer, lymphoma, leukemia, and multiple myeloma all have the effect of inducing apoptosis (Ashkenazi et al. 1999; Ashkenazi 2002).
  • TRAIL has been developed as an important potential anti-tumor drug in the past 20 years since the discovery of TRAIL.
  • the clinical trial of TRAIL has entered Phase II abroad and has completed Phase III in China.
  • a large number of in vitro and in vivo tests have confirmed that TRAIL has tumor-specific cytotoxicity, especially when it is combined with low-dose chemotherapeutic drugs, it shows significant synergy and synergy.
  • studies have found that TRAIL tolerance caused by loss of apoptotic machinery in the body is clearly associated with rapid growth and metastasis of tumor cells.
  • TRAIL works by binding to its corresponding specific death receptor.
  • DR 4 Death Receptor 4
  • DR 5 Death Receptor 5
  • Decoy receptor 1 DcR 1
  • DcR 2 Decoy receptor 2
  • OPG osteoprotegerin
  • the first four have been mapped to chromosome 8p22-21, suggesting that these receptors are genetically derived from more recent gene duplication events.
  • 8p21 also contains a number of sequences that are presumed to be tumor suppressor genes, and gene translocation at the 8p21 position is also frequently found in head and neck tumors.
  • Both DR 4 and DR 5 belong to the tumor necrosis factor receptor superfamily.
  • DR 4 and DR 5 are structurally similar and each contain a putative signal peptide sequence, two cysteine-rich pseudo-repeats, a transmembrane domain, and an intracellular death domain.
  • the DR 4 and DR 5 molecules consisted of 468 aa and 440 aa, respectively, with a sequence similarity of 58%.
  • Northern blot analysis showed that DR4 was expressed in most human tissues; DR 5 was also expressed in all tested tissues, especially in peripheral blood lymphocytes, spleen and ovarian tissues. Both DR 4 and DR 5 can be expressed on the surface of a variety of tumor cells.
  • Both segments contain a specific amino acid sequence associated with high affinity for the receptor-ligand (Hymowitz et al. 1999).
  • the DR 4 delivery death signal does not pass the recruitment of FADD, which indicates that DR4 may use a different bypass short-signal mechanism than DR 5 .
  • DR 5 is transmitted through the recruitment of intracellular death signaling aptamer molecule FADD.
  • DR 5 Unlike DR 4 , the binding of DR 5 to the receptor is temperature-controlled. At 4 ° C, TRAIL binds to all of its receptors, and at 37 ° C, TRAIL has the highest binding capacity to DR 5 . Therefore, at the optimal temperature (37 ° C) of the body and cells, DR 5 binds to TRAIL with the strongest affinity and plays the most important role in the TRAIL-induced apoptosis pathway (Truneh A et al) .2000).
  • Death receptor differ, not only decoy receptor competitive binding of TRAIL, a heterologous apoptotic effect trimer and thereby interfering DR 4 DR 5 can be formed with the induced DR 4, DR 5.
  • Decoy receptor 1 (DcR1) consists of 259 aa, and its extracellular region sequence is similar to DR 4 (similarity 69%) and DR 5 (similarity 52%), with no death domain. DcR 1 binds to the sugar chain of glycophospholipid inositol (GPI) by covalent attachment to form a "protein-sugar-fatty acid complex.” Due to the lack of intracellular death domain, DcR 1 binds to TRAIL and cannot induce apoptosis. Instead, it competitively inhibits the binding of TRAIL to DR 4 or DR 5 and plays a role in inhibiting apoptosis.
  • GPI glycophospholipid inositol
  • the mRNA of DcR 1 is expressed at high levels in peripheral blood lymphocytes, spleen, heart, lung, kidney, bone marrow and placenta, and is not expressed in the brain and colon (Sheridan et al. 1997; Pan et al. 1997).
  • Decoy receptor 2 (DcR 2 ) is composed of 386 aa, which contains an incomplete death domain. Therefore, DcR 2 binds to TRAIL and cannot mediate apoptosis, but can inhibit TRAIL through its extracellular domain. Induced apoptosis. DcR 2 mRNA is expressed in many tissues, including testis, peripheral blood lymphocytes, thymus, colon, small intestine, and prostate. An important difference between DcR 2 and DcR 1 is that DcR 2 contains a cytoplasmic domain that promotes the activation of the apoptotic transcription factor NF- ⁇ B (Marsters et al. 1997; Degli-Esposti et al. 1997), thereby Antagonize apoptosis.
  • the soluble TNF receptor family member Osteoprotegerin (OPG) consists of 401 aa, which was originally found to bind to TNF superfamily member RANKL and inhibit osteoclastogenesis and increase bone density in vivo (Simonet et al) .1997). It was later discovered that OPG binds to TRAIL and is a deceptive receptor for TRAIL that inhibits TRAIL-induced apoptosis (Emery et al. 1998). This may be an important reason why some tumor cells are resistant to TRAIL-induced apoptosis.
  • Hormone sensitive prostate cancer cell line PC 3, Du145 and hormone sensitive LNCaP cell line can produce anti-apoptotic effect (Holen et al.2002) generated by OPG.
  • MG63 osteoblast-like cell-derived OPG inhibits TRAIL-induced apoptosis of myeloma cells, which can be reversed by soluble NF- ⁇ B (Nuclear factor kappa B) receptor agonists (Shipman et al. 2003).
  • Apoptosis is a series of complex molecular events in which numerous pro-apoptotic factors and apoptosis-inhibiting factors interact to form a complex network of signaling processes.
  • Apoptotic signaling pathways mainly include extracellular (or death receptor) signaling pathways and intracellular (mitochondrial) signaling pathways.
  • the extracellular pathway plays an important role in the regulation of the immune system, including the binding of the death receptor to its ligand family member (tumor necrosis factor superfamily), which subsequently leads to receptor trimerization and aptamer molecules.
  • Recruitment of the death zone on the inside of the receptor cell membrane [aptamer molecules include FADD (FAS-associated death domain), TRA DD (TNF receptor-associated death region)].
  • the death receptors that have been elucidated include FasL/Fas R, TNF ⁇ /TNFR 1 , Apo3L/DR 3 , Apo2L/DR 4 and Apo2L/DR 5 systems.
  • DR 5 comprising the initial recruitment and activation of apoptosis aptamer molecules (of FADD) death induced by an enzyme region.
  • Caspases are a group of cysteine proteases, including apoptotic enzymes that act as initial actions, such as Caspase 2, 8, 9, 10 and apoptotic enzymes that act as effects, such as Caspase 3, 6, 7.
  • Aptamer molecules, such as FADD are recruited by their pre-caspase 8 in the death effector region and bind to the death receptor homotrimer to form a death signaling complex.
  • Activated Caspase 8 and Caspase 10 directly activate the proapoptotic molecule Bid in the effector Caspases or cleavage alternative pathways.
  • Bid is an important pro-apoptotic mitochondrial protein in the Bcl-2 family. Bid interacts with two other pro-apoptotic mitochondrial proteins, Bax and Bak, to promote mitochondrial release of cytochrome C and a second mitochondrial-derived apoptotic enzyme. Second mitochondria-derived activator of caspase (SMAC).
  • SMAC Second mitochondria-derived activator of caspase
  • Caspase 8, 10 directly activates the effect of apoptosis on Caspase 3, 6, and 7, which directly leads to the occurrence of apoptosis.
  • Cytochrome C activates another initial stage of the apoptotic enzyme Caspase 9, Caspase 9 to further enhance the activation of Caspase 3.
  • Caspase 9 is inhibited by an inhibitor of apoptosis proteins (IAPs).
  • IAPs inhibitor of apoptosis proteins
  • SMAC prevents the inhibition of Caspase 9 by IAPs by binding to IAPs, such as X-linked inhibitor of apoptosis protein (XIAP), thereby promoting the activation of Caspase 3, 6, and 7, thus in the cell.
  • Cross-linking is formed in the outer apoptotic signaling pathway.
  • Caspases are present in the cytoplasm by non-activated pro-enzyme formation, activated by N-terminal cleavage, and initiate Caspases sequential activation effects leading to apoptosis. Some Caspase are activated by polymerization with other Caspase. In the Caspase family, Caspase 3 is considered to be the most important effector molecule, which promotes DNA fragmentation by activating CAD. Caspase3 is normally left inactive by binding to its negative regulatory molecule CAD inhibitor (ICAD) in the nucleus, and ICAD is cleaved by Caspase 3 to release CAD.
  • ICAD negative regulatory molecule CAD inhibitor
  • the intracellular pathway is activated by initiating the release of pro-apoptotic proteins in the mitochondria.
  • Factors that stimulate mitochondrial release of pro-apoptotic proteins include cellular hypoxia, DNA damage, cellular stress, Ca2+ fluctuations, nitric oxide, fatty acids, and proteases. All of the above stimuli can lead to the formation of mitochondrial PT pores, changes in the inner mitochondrial membrane, PT pore opening, and release of Bcl-2 family proteins. Mitochondria and its interaction with Bcl-2 family proteins play a key role in regulating apoptosis. Apoptosis relies on changes in the integrity of the mitochondrial outer membrane, and as a result, pro-apoptotic proteins are released from the mitochondrial membrane space.
  • Bcl-2 family proteins which include two subfamilies of pro-apoptotic proteins and anti-apoptotic proteins.
  • anti-apoptotic Bcl-2 family members maintain the stability of the mitochondrial outer membrane and inhibit the release of intermembrane proteins from mitochondria, whereas pro-apoptotic proteins are just the opposite. Destabilization of the mitochondrial membrane leads to increased outer membrane permeability, formation of permeability transfer pores, loss of mitochondrial transmembrane potential leading to release of cytochrome c, SMAC/Diablo and HrA2/O mi proteins from mitochondria.
  • All apoptotic proteins contain a BH3 domain capable of forming a dimer with apoptosis-inhibiting Bcl-2 family, and pro-apoptotic proteins are further divided into a protein containing only one BH3 domain and a multi-domain including BH3 domain. protein.
  • the former cannot directly cause apoptosis of the mitochondrial membrane to cause apoptosis, but can activate a multidomain pro-apoptotic protein, and the latter can exert apoptosis-inducing activity.
  • P53 and its family member factors are also important for the apoptotic process.
  • P53 mutations occur in most malignant tumors, especially head and neck tumors, suggesting that they play a key role in tumorigenesis.
  • P53 protects cells from cell cycle arrest or apoptosis when they are facing an emergency stimulus.
  • P53 is primarily activated by three mechanisms. One is that radiation exposure increases double-strand DNA breaks leading to phosphorylation of P53, reducing its affinity for the negative regulator, MDM-2. Second, stimulation of tumor growth signals (such as Ras and Myc activation) leads to P14ARF-mediated isolation of MDM-2.
  • chemotherapeutic drugs UV and protein kinase inhibitors promote ATR and Cas ein Kinase II mediated MDM-2 phosphorylation.
  • pro-apoptotic genes such as Fas/CD95, Noxa, and apoptosis-inducing factor 1 (APAF-1) is stimulated.
  • TRAIL as an anti-tumor drug has been associated with its inherent limitations, and the presence of extensive resistance has prevented TRAIL from exerting a better therapeutic effect in clinical applications.
  • Roberta di pietro and Giorgia Zauli Robotta di pietro and Giorgia Zauli, 2004
  • TRAIL is sensitive to 61 of the 92 passages and primary tumor cells that have been studied, with a sensitivity rate of 66.30%, while the remaining 31
  • the drug resistance rate was 33.70%.
  • TRAIL was sensitive to 15 of the 29 passaged tumor cell lines involved in the trial, with a sensitivity rate of 51.72% and resistance to 14 strains with a resistance rate of 48.28%.
  • extensive resistance has become the biggest bottleneck in the clinical application of TRAIL.
  • Cummins et al. focused their research on the XIAP gene. By targeting gene deletions, Cummins et al. destroyed the XIAP gene in human colon cancer cells. Although the deletion of the XIAP gene does not affect the basal proliferation of the cells, it significantly increases the sensitivity of the cells to exogenous TRAIL. TRAIL can induce apoptosis in both wild-type tumor cells and XIAP-excised tumor cells, but the effect on the latter is significantly stronger than on wild-type cells. Proapoptotic effects in XIAP knockout tumor cells are associated with higher intracellular Caspase 3 levels. XIAP knockdown can reduce tumor cell survival and clonal proliferation. The high expression of XIAP in tumor cells is a key non-redundant regulator of many tumor cells tolerant to TRAIL (Cummins et al. 2004).
  • the second mitochondria-derived apoptotic enzyme activator is the second mitochondrial protein found (the first is cytochrome c).
  • the SMAC gene is located on chromosome 12q24.31, the putative SMAC-encoded protein is 239 aa polypeptide, the SMAC has 55 aa at the N-terminus, which is a mitochondrial targeting sequence (signal peptide), and the mature SMAC protein encodes 184 aa.
  • SMAC activates the inhibitory effect of IAPs on Caspase by binding to IAPs to activate the activity of Caspase 9, thereby increasing the sensitivity of cells to apoptosis and promoting cell apoptosis.
  • the object of the present invention is to provide a double-target fusion protein which enhances the anti-tumor activity of TRAIL, and the designed fusion protein targets both the TRAIL receptor DR 4 and DR 5 and the apoptosis antagonist XIAP.
  • TRAIL has a significant synergistic effect.
  • the fusion protein-encoding cDNA sequence was cloned using a prokaryotic vector with the fusion tag sequence removed, and the soluble expression level was high, and the protein recovery and purification efficiency was high.
  • the present invention adopts a technical solution of: a dual-target fusion protein that enhances TRAIL anti-tumor activity, and the therapeutic targets are TRAIL receptor DR 4 , DR 5 and apoptosis antagonist XIAP.
  • the double-target fusion protein is formed by fusing a transmembrane peptide sequence, a SMAC N7, an endogenous protease cleavage site and a TRAIL protein peptide.
  • transmembrane peptide sequence is R8.
  • amino acid sequence of the double-target fusion protein is shown in SEQ ID NO: 2.
  • cDNA sequence of the amino acid sequence of the double-target fusion protein is shown in SEQ ID NO: 1.
  • transmembrane peptide sequence is TAT.
  • amino acid sequence of the dual-target fusion protein is shown in SEQ ID NO: 5.
  • cDNA sequence of the amino acid sequence of the double-target fusion protein is shown in SEQ ID NO: 4.
  • a method for expressing a double target fusion protein in vitro for enhancing TRAIL antitumor activity comprises the following steps:
  • the vector constructed in the step (1) is expressed by the BL21 (DE3) host strain.
  • the expression induction temperature is 18 °C.
  • a third object of the present invention is to provide a use of a dual-target fusion protein which enhances TRAIL antitumor activity as an antitumor drug.
  • the beneficial technical effect of the present invention is that the fusion protein of the prokaryotic expression vector has higher soluble expression and the recovery and purification of the fusion protein compared with the prokaryotic expression vector encoding the cDNA sequence of the TRAIL protein soluble fragment (114-281aa) alone. higher efficiency.
  • In vitro bioactivity analysis showed that the obtained fusion protein can simultaneously activate the death receptor on the cell membrane and inhibit the expression of XIAP gene in the cytoplasm, and enhance the activity of inducing apoptosis of tumor cells through the dual target of apoptosis pathway, which has stronger resistance. Tumor effect.
  • the above-mentioned dual-target TRAIL fusion protein has great development potential and advantages, and is highly likely to develop into a new generation of tumor cell apoptosis-inducing drugs.
  • Figure 1 is an electrophoresis pattern of the PCR product after splicing in the pre-fusion stage; electrophoresis conditions: 3% Agarose, voltage 100V, 20min; lane 1 to 2: fusion product fragment 1 and 2 spliced PCR product electrophoresis band; M: DL500 molecular weight Marker; PCR The product loading was 5 ⁇ l;
  • Figure 2 is an electrophoresis pattern of the TRAIL (114-281aa) gene PCR product; electrophoresis conditions: 1% Agarose, voltage 100V, 20min; lane1: TRAIL (114-281aa) encoding cDNA sequence PCR product electrophoresis band; M: DM2000 molecular weight Marker, The amount of the sample was 5 ⁇ l; the amount of the PCR product was 5 ⁇ l;
  • Figure 3 is an electrophoresis pattern of fusion protein gene splicing PCR product; electrophoresis conditions: 1% Agarose, voltage 100V, 20min; lane1 ⁇ 2: fusion gene 1, 2 splicing PCR product electrophoresis band; M: DM2000 molecular weight Marker, Marker sample loading 5 ⁇ l; the amount of glue recovered product was 2 ⁇ l;
  • Figure 4 is a fusion protein gene pMD 19-T ligase digestion identification electrophoresis; electrophoresis conditions: 1% Agarose, voltage 100V, 20min; lane1 ⁇ 8: plate picking colony plasmid extraction enzyme digestion identification of electrophoresis bands; M: DM2000 molecular weight Marker, Marker sample loading 5 ⁇ l; enzyme digestion product loading is 2 ⁇ l;
  • Figure 5 is an electrophoresis map of the fusion protein gene pTWIN1 ligase digestion; electrophoresis conditions: 1% Agarose, voltage 100V, 20min; lane1 ⁇ 10: plate picking colony plasmid extraction enzyme digestion identification electrophoresis band; M: DM10000 molecular weight Marker, Marker The amount of the sample was 5 ⁇ l; the amount of the enzyme-cut product was 2 ⁇ l;
  • Figure 6 shows the SDS-PAGE electrophoresis pattern of pTWIN1/TRAIL protein expression; electrophoresis conditions: 15% gel, 200V, 35min; lane 1: TRAIL induced pre-electrophoresis band; lane 2: TRAIL induced post-electrophoresis band; lane3: TRAIL broken Post-bacterial supernatant electrophoresis band; lane4: TRAIL after precipitation, precipitation of electrophoresis bands; M: Unstained Protein Molecular Weight Marker (stripe molecular weight from top to bottom: 116.0KDa, 66.2KDa, 45.0KDa, 35.0KDa, 25.0 KDa, 18.4KDa, 14.4KDa), sample loading is 10 ⁇ l, Marker loading is 5 ⁇ l;
  • Figure 7 is a SDS-PAGE electrophoresis pattern of pTWIN1/fusion gene 1, 2 protein expression; electrophoresis conditions: 15% gel, 200V, 35min.
  • Lane 1 pTWIN1/fusion protein gene 1 induced pre-electrophoresis band
  • lane2 pTWIN1/fusion protein gene 1 induced post-electrophoresis band
  • lane 3 pTWIN1/fusion protein gene 1 after bacterial supernatant band
  • lane 4 pTWIN1/fusion protein gene 1 after denaturing precipitation electrophoresis band
  • lane5 pTWIN1/fusion protein gene 2 induced pre-electrophoresis band
  • lane 6 pTWIN1/fusion protein gene 2 induced post-electrophoresis band
  • lane 7 pTWIN1/fusion protein After the gene 2 was sterilized, the supernatant was electrophoresed; the lane8: pTWIN1/fusion protein gene 2 was precipitated and the electrophoresis band was precipitated;
  • M Unstained
  • Figure 8 is a SDS-PAGE electrophoresis pattern of pTWIN1/fusion gene 2 protein expression; electrophoresis conditions: 15% gel, 200V, 35min; M: Unstained Protein Molecular Weight Marker (strip molecular weight from top to bottom: 116.0KDa, 66.2 KDa, 45.0KDa, 35.0KDa, 25.0KDa, 18.4KDa, 14.4KDa), the sample loading was 10 ⁇ l, and the Marker loading was 5 ⁇ l.
  • the target of the TRAIL protein (114-281aa) in the designed fusion protein targets the TRAIL receptor DR 4 , DR 5 , and SMAC N7 peptides against the apoptosis antagonistic factor XIAP, in order to have a significant synergistic effect on TRAIL.
  • the full-length fusion gene sequences to be synthesized were divided into two parts: the fusion front sequence (see sequences 3, 6) and the TRAIL sequence (see sequence 7).
  • the fusion anterior sequence includes (R8+SMAC N7+AP) (see sequence 3) and (TAT+SMAC N7+AP) (see sequence 6) peptide-encoding cDNA sequences, and the pre-fusion sequence is entrusted to the Huada gene synthesis.
  • segment double-stranded splicing primers (see SEQ ID NO: 8-18) were used as raw materials, and the synthesized double-stranded splicing primers were reacted together, and the fused anterior segment sequence (R8+SMAC N7+AP) and (TAT) were obtained without Taq DNA Polymerase. +SMAC N7+AP) encodes cDNA.
  • the TRAIL protein (114-281aa)-encoding cDNA sequence was amplified by using TRAIL upstream and downstream primers (see Sequences 19 and 20) using a plasmid containing TRAIL gene cDNA (laboratory) as a template, and finally the fusion anterior sequence (sequence) 3, 6) Ligation of the TRAIL sequence (SEQ ID NO: 7) to obtain a full-length fusion gene sequence (sequences 1, 4).
  • the full length fusion gene sequence I includes the sequence combination and sequence linkage of the cDNA sequence encoding the R8+SMAC N7+AP+TRAIL (114-281aa).
  • the fusion protein type I amino acid sequence is as follows (sequence 2: 186 aa):
  • the full-length fusion gene sequence 1 was divided into two parts, a fusion front sequence (see sequence 3) and a TRAIL sequence (see sequence 7).
  • the fusion anterior sequence includes the (R8+SMAC N7+AP) (see sequence 3) peptide-encoding cDNA sequence, and the fusion anterior sequence is synthesized using a segmented double-stranded splicing primer (see Sequence 8-12) that is entrusted to the Huada gene synthesis.
  • the double-stranded splicing primers were reacted together, and the fused front-end fusion sequence (R8+SMAC N7+AP) was first obtained without Taq DNA Polymerase.
  • the TRAIL protein (114-281aa)-encoding cDNA sequence was amplified by using TRAIL upstream and downstream primers (see Sequences 19 and 20) using a plasmid containing TRAIL gene cDNA (laboratory) as a template, and finally the fusion anterior sequence (sequence) 3) Ligation of the TRAIL sequence (SEQ ID NO: 7) to obtain a full-length fusion gene sequence 1.
  • the TRAIL protein (114-281aa) encodes the cDNA sequence (sequence 7: 510 bp) as follows:
  • the full-length fusion gene cDNA sequence II includes the sequence combination and sequence linkage of the TAT+SMAC N7+AP+TRAIL (114-281aa)-encoding cDNA sequences.
  • the full-length fusion gene sequence II encodes the amino acid sequence as follows (sequence 5: 189 aa):
  • the full-length fusion gene sequence 2 was divided into two parts, a fusion front sequence (see sequence 6) and a TRAIL sequence (see sequence 7).
  • the fusion anterior sequence includes the (TAT+SMAC N7+AP) (see sequence 6) peptide-encoding cDNA sequence, and the fusion anterior sequence is a split double-stranded splicing primer (see SEQ ID NO: 13-18) that is commissioned by the Huada gene to synthesize the double strand.
  • the splicing primers were reacted together, and the fused front-end fusion sequence (TAT+SMAC N7+AP) was first obtained without Taq DNA Polymerase.
  • the TRAIL protein (114-281a a) encoding cDNA sequence was amplified by using TRAIL upstream and downstream primers (see Sequences 19 and 20) using a plasmid containing TRAIL gene cDNA (laboratory) as a template, and finally the fusion front sequence ( Sequence 6) was ligated with the TRAIL sequence (SEQ ID NO: 7) to obtain a full-length fusion gene sequence 2.
  • the TRAIL protein (114-281aa) encodes a cDNA sequence amplification primer (see sequences 19, 20), and a PstI cleavage site sequence and a protective base GGT sequence are added before the downstream primer.
  • Upstream primer (sequence 19): GTTCGTGAACGTGGTCCGCAGCGTGTTGCTGCT
  • Downstream primer (sequence 20): GGTCTGCAGTTATTACAAAACAAGGAAAGCACC
  • Synthetic primer 20120328-YJ1 laboratory-provided TLP (TRAIL cDNA) plasmid template, batch number 20120406.
  • Primer dissolution Dissolve into 100 pmol/ ⁇ l in ultramolec water according to the molar ratio provided on the primer data, and set aside. Then, 5 ⁇ l of 45 ⁇ l of ultrapure water was added and diluted to 10 pmol/ ⁇ l for amplification.
  • the PCR tube was placed in a PCR machine to carry out a PCR reaction, and the reaction conditions are shown in Table 3.
  • step 5 The purified product of step 5 is reused with the upstream and downstream primers of the fusion front sequence (for the fusion front sequence 1, ie, the sequence 3 upstream and downstream primers are sequence 8, sequence 12; for the fusion front sequence 2, ie, the sequence 6 upstream and downstream primers are respectively Sequence 13 and sequence 18) perform a PCR to increase the copy number of the sequence.
  • the PCR reaction system is shown in Table 4, and each tube is made.
  • the TRAIL sequence (sequence 7) PCR amplification reaction system was prepared; the TRAIL downstream primer contained a PstI cleavage site convenient for ligation to the expression vector, and the TRAIL sequence was made into 2 tubes.
  • the PCR product was electrophoresed, and 1% of the gel was used to observe the result. After the target band was used, the gel was recovered. The gel recovery procedure is described in the kit protocol and finally eluted with 30 ⁇ l of water for use.
  • the fusion recovery product of the fusion anterior sequence (pre-fusion phase 1, ie, sequence 3; pre-fusion phase 2, ie, sequence 6) was subjected to PCR splicing reaction with the TRAIL sequence recovered by gel (ie, sequence 7), and the splicing reaction system and reaction conditions are as follows. Table 8, Table 9, finally obtained full-length fusion gene sequences 1, 2 (ie, sequence 1, sequence 4).
  • PCR results after merging the anterior sequence 1 and 2 are shown in Fig. 1.
  • the fusion front sequence 1 and 2 genes of about 80 bp were amplified, respectively.
  • TRAIL (114-281aa) coding cDNA sequence PCR results are shown in Figure 2. As can be seen, a TRAIL (114-281aa)-encoding cDNA sequence of about 500 bp in size was amplified.
  • the full-length fusion gene sequences 1, 2 were first ligated and transformed with the pMD19-T vector, and then the successfully transformed clones were subcloned into the expression vectors pTWIN1 and pET-32a to construct Prokaryotic expression vector of long fusion gene sequences 1, 2.
  • the pMD19-T Vector ligation reaction system was connected for 4.5 hours at 16 °C.
  • Fig. 4 is an electrophoresis map of the fusion protein gene pMD 19-T ligase digestion.
  • the prokaryotic expression vectors pTWIN 1 and pET-32a were used, and the vector pTWIN1, pET-32a vector and the positive clone vector pMD19/full-length fusion gene sequence 1 and pMD19/full-length fusion gene sequence 2 were digested with NdeI and PstI, respectively.
  • pTWIN1 removed the two cohesive ends of the Intern region (about 1549 bp), and inserted the full-length fusion gene sequences 1 and 2 in the same region, and pET-32a obtained two regions in which the Trx fusion protein tag expression region was removed.
  • the cohesive terminus, in which the same double-digested full-length fusion gene sequence 1, 2 was inserted. Therefore, NdeI and PstI were digested, and ligated into the Top10 competent cells of the Tiangen organism by ligation with the TaKaRa ligation kit.
  • Positive clone vector pMD19/full length fusion gene sequence 1 and pMD19/full length fusion gene sequence 2 were derived from Example 2, plasmid lot number 20120515, pTWIN1 lot number 20120406, and derived from 20120406-YJ2.
  • NdeI, PstI double-digestion vector pTWIN1 or pET-32a and pMD19/full-length fusion gene sequence 1, 2 plasmid the enzyme digestion system is shown in Table 13, and the reaction system is 100 ⁇ l.
  • connection system is shown in Table 14, and each tube is made.
  • the experiment used TB medium to culture and induce expression of pTWIN1/full-length fusion gene sequence 1, pTWIN1/full-length fusion gene sequence 2 and compared with wild-type TRAIL prokaryotic expression vector pTWIN1/TRAIL expression. Since a small amount of sample is required for purification of the protein of interest, it is prepared using a large number of shake flasks. Previously, the medium used for experimentally culturing and inducing expression of the TRAIL series protein was LB medium. Therefore, it is proposed to select the TB medium whose main carbon source is glycerol. The feasibility of using this medium is tested in this experiment.
  • SDS-PAGE electrophoresis related solution was prepared at 20120428-XQ2, pTWIN1/full-length fusion gene sequence 1, pTWIN1/full-length fusion gene sequence 2 strains were purchased from Invitrogen.
  • Reagent name specification lot number Manufacturer Tryptone 500g 829408 OXOID Yeast Extract 500g 990951 OXOID NaCl AR 500g 20110113 Chengdu Kelon Chemical Reagent Factory Glycerol AR 500g 20110113 Chengdu Kelon Chemical Reagent Factory K2HPO4 ⁇ 3H2O AR 500g 20110301 Chengdu Kelon Chemical Reagent Factory KH2PO4 AR 500g 20110113 Chengdu Kelon Chemical Reagent Factory IPTG 100g P13/342/129 INALCO SPA MILANO ITALY Ammonium persulfate ⁇ 98%, 100g 215589 SIG-ALD, Biodee dispensing TEMED ⁇ 99%, 100ml T22500 SIG-ALD, Biodee dispensing Unstained Protein Molecular Weight Marker 1ml 00078434 Thermo Scientific Ampicillin sodium for injection 0.5g E1005102 North China Pharmaceutical Co., Ltd.
  • the cells were resuspended in 5 ml of H 2 O and disrupted by ultrasound.
  • the bacteriostatic conditions were as follows: 150W pulsed for 5s, paused for 8s, and cycled 60 times.
  • the bacterial suspension was centrifuged at 12000 rpm for 10 min, the supernatant was separated and precipitated, and the pellet was resuspended in 1 ml of H 2 O. 25 ⁇ l of each of the supernatant and the pellet was added to 25 ⁇ l of 2 ⁇ loading buffer to prepare an electrophoresis sample.
  • the prepared electrophoresis sample was placed in a boiling water bath for 10 min, centrifuged at 12000 rpm for 10 min, and then the supernatant was subjected to 10 ⁇ l electrophoresis.
  • the expression of the target protein of the wild-type TRAIL prokaryotic expression vector is weak, and the expression of the target protein is mainly distributed in the precipitate, which is not conducive to purification.
  • the newly constructed pTWIN1/full-length fusion gene sequence1, pTWIN1/full-length fusion gene sequence 2 has strong expression of the target protein, and the target protein is mainly present in the supernatant.
  • the expression protein of pTWIN1/full-length fusion gene sequence 1 is almost completely soluble, and the soluble protein of pTWIN1/fusion protein gene 2 accounts for about 60%.
  • the medium protein content of the target can also meet the requirements of convenient purification.
  • the expression of wild-type TRAIL is still poor and may be related to the spatial structure of its protein.
  • Fig. 8 is a SDS-PAGE electrophoresis pattern of pTWIN1/fusion gene 2 protein expression.
  • a large amount of protein of interest was induced, but the protein was poorly soluble. Only a small amount of the target protein was present in the supernatant.
  • the temperature was lowered to 30 °C, the expression intensity of the target protein was not affected by the temperature during the induction period, and there was still a high level. The expression, while the solubility has improved.
  • the induction temperature was lowered to the limit of 18 ° C, the expression intensity of the target protein was weakened, but the expressed target protein was completely soluble.
  • the protein After changing the medium from LB medium to more nutritious TB medium with glycerol as the main carbon source, the purpose of expression
  • the protein is about 60% soluble. Although the result is less than 18 °C, it is obviously better than 30 °C.
  • the target protein content in the supernatant can also meet the requirements of convenient purification.
  • the same concentration of drug-filled wells were observed under light microscope for 48 hours. It was obvious that the number of cells in the four groups was different. The cell morphology of the blank control group did not appear obvious. Changes, for breast cancer cell lines, the number of cells in the three drug-added groups was significantly reduced, and the wild-type TRAIL protein group still had some normal-shaped spindle-shaped MDA-MB-231, and most of the fusion protein type I and type II cells. Rounded and lost the original normal form. The wild type TRAIL protein has a weak killing effect on SPCA1 cells, and few cells have morphological changes. Compared with the control group, the number of cells is not significantly reduced.
  • MDA-MB-231 is a wild-type TRAIL protein-sensitive cell line, fusion protein type I, type II and wild-type TRAIL protein in the range of 50-0.02 ug/ml, observed after 48 hours, three to MDA-MB-231 All of them have a dose-effect relationship.
  • the fusion protein type I has a significant inhibitory effect on growth, IC50 is 4.04 ⁇ 10 -6 ng/ml, and the fusion protein type II has a significant inhibitory effect on IC50 of 6.67 ⁇ 10 -6 ng/ml.
  • the wild type TRAIL protein IC50 was 9.45 ng/ml.
  • SPCA1 is not sensitive to wild-type TRAIL protein, and the growth inhibition is not obvious in the range of 50-0.003 ug/ml, and the IC50 is 428.05 ug/ml.
  • the fusion protein 1, 2 has a significant growth inhibition effect in the same concentration range, wherein the IC50 of the fusion protein type I is 4.64 ug/ml, and the IC50 of the fusion protein type II is 8.78 ug/ml, which is largely reversed. Tolerance of tumor cells to wild-type TRAIL, the results of cytotoxicity test are shown in Table 16.
  • the fusion protein type I, type II and wild type TRAIL were all 10 ng/ml, and were detected 6 hours after drug administration. Apoptosis, the results showed that in both cell lines, fusion protein 1, 2 induced apoptosis is stronger than wild-type TRAIL protein, especially in lung cancer cell lines that are not sensitive to TRAIL, the fusion protein can still play a stronger role. The killing effect. There was a statistically significant difference in apoptotic rate between the two groups (P ⁇ 0.01).
  • Results embodiment of the present embodiment (Table 17), further validated against experimental design using role TRAIL receptors DR 4, DR 5 antagonize apoptosis and XIAP factor two targets apparent efficiency of TRAIL.

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Abstract

本发明公开了一种增强TRAIL抗肿瘤活性的双靶点融合蛋白，由穿膜肽、SMAC N7、内源性蛋白酶酶切位点与TRAIL蛋白肽段顺序融合而成。所述融合蛋白具有更高的原核表达的可溶性和更强的抗肿瘤作用。

Description

增强TRAIL抗肿瘤活性的双靶点融合蛋白及其体外表达方法和应用 技术领域
本发明涉及基因工程药物技术领域，具体来说是一种增强TRAIL抗肿瘤活性的双靶点融合蛋白和该融合蛋白的体外表达方法及应用。
背景技术
1.TRAIL蛋白结构及分子生物学
肿瘤坏死因子相关凋亡诱导配体(TNF-related apoptosis-inducing ligand；TRAIL)为肿瘤坏死因子(Tumor necrosis factor，TNF)超家族的成员，其基因序列分别于1995年由Wiley等人(Wiley et al.1995)和1996年由Pitti等人(Pitti et al.1996)独立克隆获得。后者将其命名为凋亡素2配体(Apo2ligand，Apo2L)，后来的研究证实Apo2L与TRAIL实为同一种蛋白，因此习惯上可将其称为Apo2L/TRAIL。
人Apo2L/TRAIL的编码基因定位于染色体3q26(Miriani and Krammer.1998)，完整的Apo2L/TRAIL的cDNA序列编码全长为281个氨基酸的前体蛋白，其相对分子质量为32.5kDa。天然的Apo2LTRAIL蛋白表现为II型跨膜蛋白，分细胞外C端区域、跨膜区、细胞内N端区域等三部分。Apo2LTRAIL的N端1-14aa为短小的胞内段，无信号肽序列；第15-40位aa为疏水区，形成跨膜结构；C端第41-281位aa为胞外区，保守性强。C端能形成几个β折叠，再形成典型的β夹心结构，进而形成同源三聚体的亚单位，胞外区为蛋白发挥功能的主要结构区。Apo2L/TRAIL前体蛋白在特定的蛋白酶作用下水解形成可溶性TRAIL，其第114-281aa为蛋白质的可溶性片段(Miriani and Krammer.1998)。
人Apo2L/TRAIL的C端(胞外区)序列与TNF和Fas配体一样高度保守，人TRAIL分子的胞外区与Fas配体、肿瘤坏死因子α、淋巴毒素-α和淋巴毒素-β的同源性分别为28％、23％和22％(Wiley et al.1995)。TRAIL与TNF家族其他成员最独特的区别在于其第137-152位形成一个12-16个aa的插入环(AA’loop)，此结构可 插入受体的TRAIL结合位点，保证受体与TRAIL的特异性结合。结构突变研究证实，此插入环在TRAIL田胞毒性中起着关键的作用。晶体结构研究表明，Apo2L/TRAIL分子为一个同源三聚体分子，内部含有一个Zn原子，Zn原子同时与三个配体亚单位的第230位半胱氨酸连接，通过相互作用以维持分子的稳定(Hymow itz，et al.2000)。TRAIL是TNF家族中唯一的具有一个Cys残基的成员，Zn原子的结合对于同源三聚体的稳定性和生物活性至关重要(Bodmer et al.2000；Hymowitz，et al.2000)。Jean等研究证实，如将第230位半胱氨酸(Cys)突变为丝氨酸或丙氨酸后，TRAIL将形成无活性的二聚体结构，与受体的亲和力将下降200倍，严重影响TRAIL诱导细胞凋亡的活性(Jean et al.2000)。TRAIL的功能首先是作为生物体先天性或获得性免疫的调节剂，其次在细胞外源性凋亡途径过程中发挥重要作用，作为免疫监视在抗肿瘤过程中发挥重要作用。TRAIL的最大优点是可以选择性地诱导多种肿瘤细胞凋亡而对正常细胞几乎没有毒性。研究资料表明，Apo2L/TRAIL无论在体内，还是体外对于各种来源的人肿瘤细胞系，包括结(直)肠癌、肺癌、乳腺癌、前列腺癌、胰腺癌、肾癌、中枢神经系统肿瘤、甲状腺癌、淋巴瘤、白血病以及多发性骨髓瘤等都具有诱导凋亡的作用(Ashkenazi et al.1999；Ashkenazi 2002)。
从TRAIL发现至今近20年时间里，TRAIL一直被作为一个重要的潜在抗肿瘤药物开发，TRAIL的临床试验在国外已进入II期，在国内已完成III期。大量体内外试验均证实，TRAIL具有肿瘤特异性细胞毒性，尤其当它与小剂量化疗药物联用时即表现出明显的协同和增效作用。相反，研究发现机体中凋亡机制的缺失导致的TRAIL耐受与肿瘤细胞的快速生长和转移明确相关。
2.TRAIL受体结构及分子生物学
TRAIL发挥作用是通过与其相应特异性死亡受体结合而实现的。目前发现的人TRAIL田胞膜上受体共5种(LeBlanc and Ashkenazi 2003)，它们分别被称为死亡受体4(Death receptor 4，DR4)、死亡受体5(Death receptor 5，DR5)、诱骗受体1(Decoy receptor 1，DcR1)、诱骗受体2(Decoy receptor 2，DcR2)和护骨素(osteoprotegerin，OPG)。前四种已定位于染色体8p22-21上，这表明上述受体在遗传学上均起源于较近的基因复制事件。8p21还包含有许多推定可能为抑癌基因的序列，8p21位置的基因转位在头颈部肿瘤中亦较常发现。
DR4和DR5均属肿瘤坏死因子受体超家族。DR4和DR5结构类似，均包含一个推定的信号肽序列，两个富含半胱氨酸的假重复序列，一个跨膜结构域，一个细胞内死亡结构域。DR4和DR5分子分别由468个aa和440个aa组成，两者序列相似性为58％。Northern blot分析表明，DR4在大多数人体组织中表达；DR5亦在所有检测组织中表达，尤其最高表达于外周血淋巴细胞、脾和卵巢组织。DR4和DR5均可表达于多种肿瘤细胞表面。
Pan等(Pan et al.1997)研究证实，与FAS、TNFR和DR3一样，DR4过表达可诱导细胞凋亡。Walczak等(Walczak et al.1997)也发现，与DR4一样，DR5过表达也可引起凋亡酶依赖的细胞凋亡途径活化。Apo2LTRAIL-DR5复合物的晶体结构分析表明，在配体三聚体的每一个单体分子之间，3个受体分子相互靠近形成一个长的狭缝，这种接触面又可分为两个区段，一个区段位于受体细胞表面复合物的底部，一个区段位于复合物的顶部。两个区段均含有与受体-配体高亲和力相关的特定氨基酸序列(Hymowitz et al.1999)。DR4传递死亡信号不通过FADD的募集，这表明DR4可能使用与DR5不同的旁路短截信号机制。与DR4不同，DR5通过细胞内适配体分子FADD的募集而传递死亡信号。
与DR4不同，DR5与受体的结合是受温度控制的，在4℃时，TRAIL与其所有受体结合力均相似，而在37℃时，TRAIL与DR5的结合力最高。因此在机体及细胞最适宜的生存温度(37℃)，DR5相对于其他受体来讲，以最强的亲和力与TRAIL结合并在TRAIL诱导的凋亡通路中发挥最大作用(Truneh A et al.2000)。
与死亡受体不同，诱骗受体不仅能竞争性结合TRAIL，也能与DR4、DR5形成异源三聚体从而干扰DR4和DR5诱导的细胞凋亡作用。
诱骗受体1(Decoy receptor1，DcR1)由259个aa组成，其胞外区序列类似于DR4(相似度69％)和DR5(相似度52％)，无死亡结构域。DcR1通过共价连接与糖磷脂肌醇(GPI)的糖链结合，形成“蛋白-糖-脂肪酸复合物”。因缺乏胞内死亡域，DcR1与TRAIL结合不能诱导细胞凋亡，反而竞争性抑制TRAIL与DR4或DR5的结合，起到一定的凋亡抑制作用。DcR1的mRNA在外周血淋巴细胞、脾、心、肺、肾、骨髓及胎盘呈高水平表达，在脑及结肠中未见表达(Sheridan et al.1997；Pan et al.1997)。
诱骗受体2(Decoy receptor 2，DcR2)由386个aa组成，胞内含有不完整的死亡结构域，因此，DcR2与TRAIL结合不能介导凋亡，但可以通过其胞外区抑制TRAIL诱导的细胞凋亡。DcR2的mRNA在许多组织中均有表达，包括睾丸、外周血淋巴细胞、胸腺、结肠、小肠及前列腺。DcR2和DcR1的重要区别在于，DcR2含有一个胞浆结构域，该结构域可以促进凋亡转录因子NF-κB的活化(Marsters et al.1997；Degli-Esposti et al.1997)，从而拮抗凋亡。
可溶性TNF受体家族成员护骨素(Osteoprotegerin，OPG)由401个aa组成，最初发现其能够与TNF超家族成员RANKL结合，在体内具有抑制破骨细胞发生、增加骨密度的作用(Simonet et al.1997)。后来发现OPG能够与TRAIL结合，也是一种TRAIL的诱骗受体，可抑制TRAIL诱导的细胞凋亡(Emery et al.1998)。这可能是部分肿瘤细胞对抗TRAIL诱导细胞凋亡的重要原因。激素不敏感的前列腺癌细胞系PC3、Du145和激素敏感的细胞系LNCaP可通过产生OPG而产生抗凋亡效应(Holen et al.2002)。MG63造骨样细胞来源的OPG可抑制TRAIL诱导的骨髓瘤细胞凋亡，这一作用能被可溶性NF-κB(Nuclear factor kappa B)受体激动剂逆转(Shipman et al.2003)。
3.TRAIL诱导细胞凋亡的机制
凋亡是一系列的复杂的分子事件，众多的促凋亡因子和凋亡抑制因子相互作用形成一个信号传递过程的复杂网络。凋亡信号传递通路主要包括细胞外(或死亡受体)信号传导途径和细胞内(线粒体)信号传导途径。细胞外途径对于免疫系统的调节起着重要的作用，其作用过程包括死亡受体与其配体家族成员(肿瘤坏死因子超家族)的结合，随后导致受体的三聚化和适配体分子在受体细胞膜内侧死亡区域的募集[适配体分子包括FADD(FAS相关死亡结构域)，TRA DD(TNF受体相关死亡区域)]。迄今为止，已经阐明的死亡受体包括FasL/Fas R，TNFα/TNFR1，Apo3L/DR3，Apo2L/DR4和Apo2L/DR5等系统。
当Apo2L/TRAIL与细胞膜上的两种死亡受体DR4或DR5结合后，DR5通过死亡区域包含适配体分子(FADD)的募集和活化起始凋亡诱导酶。凋亡酶(Caspases)是一组半胱氨酸蛋白酶，包括作为起始作用的凋亡酶，如Caspase2、8、9、10和作为效应作用的凋亡酶，如Caspase3、6、7。适配体分子，如FADD通过其死亡效应区域前Caspase 8的募集并与死亡受体同源三聚体结合进而形成死亡信号复合物。活化的Caspase 8和Caspase 10可直接活化效应Caspases或裂解替代途径中的促凋亡分子Bid。Bid为Bcl-2家族中重要的促凋亡线粒体蛋白，Bid又与另外两种促凋亡线粒体蛋白Bax及Bak相互作用，共同促使线粒体释放细胞色素C和第二个线粒衍生的凋亡酶激活剂(Second mitochondria-derived activator of caspase，SMAC)。一方面Caspase 8、10直接活化凋亡效应作用Caspase3、6、7，后者直接导致细胞凋亡的发生。细胞色素C与Apaf-1一起，活化另一个起始阶段凋亡酶Caspase 9，Caspase 9进一步增强Caspase 3的活化。Caspase 9受一种凋亡抑制因子(Inhibitor of apoptosis proteins，IAPs)的抑制。SMAC通过与IAPs，如X-连锁凋亡蛋白抑制因子(X-linked inhibitor of apoptosis protein，XIAP)的结合阻止IAPs对Caspase 9的抑制，从而促进Caspase 3、6、7的活化，于是在细胞内、外凋亡信号传递途径中形成交叉联结。
Caspases以非活化的前酶形成存在于细胞浆中，通过N端裂解而活化，启动Caspases顺序活化效应而导致细胞凋亡。一些Caspase通过与其他Caspase形成聚合而活化。在Caspase家族中，Caspase 3被认为是最重要的效应分子，该分子通过活化CAD而促进DNA片段化。Caspase3通常通过与其阴性调节分子CAD抑制剂(ICAD)结合存在于核内而保持非活化状态，ICAD被Caspase 3裂解继而释放CAD。
细胞内途径是通过启动线粒体内促凋亡蛋白的释放而活化。刺激线粒体释放促凋亡蛋白的因素包括细胞缺氧、DNA损伤、细胞应激、Ca2+波动、一氧化氮、脂肪酸和蛋白酶等。上述所有刺激均可导致线粒体PT孔的形成，线粒体膜内层的改变，PT孔开放以及Bcl-2家族蛋白的释放。线粒体及其与Bcl-2家族蛋白的相互作用在调节凋亡过程中发挥关键作用。凋亡有赖于线粒体外膜完整性的改变，作为结果，促凋亡蛋白从线粒体膜间隙释放出来。上述事件受Bcl-2家族蛋白的调控，Bcl-2家族蛋白包括促凋亡蛋白和抗凋亡蛋白两个亚家族。总体上讲，抗凋亡Bcl-2家族成员维持线粒体外膜的稳定性，抑制膜间蛋白从线粒体中释放出来，而促凋亡蛋白正好相反。线粒体膜的去稳定性可导致外膜通透性增高，通透性转移孔形成，线粒体跨膜电势丧失从而导致细胞色素c、SMAC/Diablo和HrA2/O mi蛋白从线粒体中释放出来。
所有凋亡蛋白均包含能够与凋亡抑制Bcl-2家族形成二聚体的BH3结构域，促凋亡蛋白又分为仅含一个BH3结构域的蛋白和包含BH3结构域在内的多结构域蛋白。前者不能直接导致线粒体膜通透性升高而引起细胞凋亡，但可以活化多结构域促凋亡蛋白，通过后者而发挥诱导凋亡的活性。
转录因子和肿瘤抑制因子P53及其家族成员因子，如P63、P73等对于凋亡过程也十分重要。P53基因突变发生于大多数恶性肿瘤中，尤其是头颈部肿瘤，提示其在肿瘤发生过程中扮演着关键的作用。P53在细胞面临应急刺激时通过导致细胞周期停滞或凋亡而起着保护作用。P53主要通过三种机制而活化。一是放射线照射增加双链DNA断裂可导致P53磷酸化，降低其与阴性调节剂，MDM-2的亲和力。其次可通过肿瘤生长信号(如Ras和Myc活化)刺激活化而导致P14ARF介导的MDM-2的隔离。第三，化疗药物、紫外线和蛋白激酶抑制剂促进ATR和Cas ein KinaseII介导的MDM-2磷酸化。P53活化后，刺激促凋亡基因，如Fas/CD95、No xa和凋亡诱导因子1(APAF-1)的转录。
TRAIL作为抗肿瘤药物的优越性与其固有的局限一直相伴存在，而广泛耐药的存在妨碍了TRAIL在临床应用过程中发挥更好的疗效。根据Roberta di pietro和Giorgia Zauli的综述(Roberta di pietro and Giorgia Zauli，2004)，TRAIL对于业已研究的92种传代及原代肿瘤细胞中的61种敏感，敏感率为66.30％，而对其余的31种耐药，耐药率为33.70％。我们先前进行的研究中，TRAIL对于参与试验的29株传代肿瘤细胞系中的15株敏感，敏感率为51.72％，而对14株耐药，耐药率为48.28％。无疑，广泛耐药成了TRAIL临床应用的最大瓶颈。
尽管有关TRAIL耐药的机理业已进行了广泛和深入的研究，但Cummins等人把研究的重点集中在XIAP基因上。通过靶向性基因缺失，Cummins等人破坏了人结肠癌细胞中的XIAP基因。尽管XIAP基因的缺失并不影响细胞的基础增殖，但却明显增加了细胞对外源性TRAIL的敏感性。TRAIL对野生型肿瘤细胞和XIAP敲除的肿瘤细胞均可诱导细胞凋亡，但对后者的作用明显强于对野生型细胞。在XIAP基因敲除的肿瘤细胞中促凋亡作用与其细胞内较高的Caspase 3水平相关。XIAP基因敲除可以减少肿瘤细胞的存活和克隆增殖，XIAP在肿瘤细胞的高表达是诸多肿瘤细胞对TRAIL耐受的关键非冗余调节因素(Cummins et al.2004)。
第二个线粒体衍化的凋亡酶激活剂(SMAC)是第二个被发现的线粒体蛋白(第一个是细胞色素c)。SMAC基因定位于染色体12q24.31，推定的SMAC编码蛋白为239个aa的多肽，SMAC的N端有55个aa，为线粒体靶向序列(信号肽)，成熟SMAC蛋白编码184aa。SMAC通过与IAPs结合解除IAPs对Caspase的抑制效应从而活化Caspase 9的活性，进而增加细胞对凋亡刺激的敏感性，促进细胞的凋亡。Chai等人研究(Chai et al.2000)表明，SMAC的N端7肽在体外能促进前Caspase 3的活化。一些学者(Liu et al.2000)通过构效研究证实SMAC的N端4个氨基酸(Ala-Val-Pro-Ile)通过与XIAP表面BIR3沟结合从而解除IAPs对Caspase 9的抑制。
技术问题
本发明的目的是提供一种增强TRAIL抗肿瘤活性的双靶点融合蛋白，设计的融合蛋白的作用靶点既针对TRAIL受体DR4、DR5，又同时针对凋亡拮抗因子XIAP，以期对于TRAIL具有明显的增效作用。融合蛋白编码cDNA序列采用去除了融合标签序列的原核载体进行克隆，可溶性表达水平较高，蛋白回收及纯化效率较高。
技术解决方案
为实现上述目的，本发明采用的技术方案是：一种增强TRAIL抗肿瘤活性的双靶点融合蛋白，所述治疗的靶点为TRAIL受体DR4、DR5和凋亡拮抗因子XIAP。
进一步的，所述双靶点融合蛋白由穿膜肽序列、SMAC N7、内源性蛋白酶酶切位点与TRAIL蛋白肽段顺序融合而成。
进一步的，：所述穿膜肽序列为R8。
进一步的，所述双靶点融合蛋白的氨基酸序列如SEQ ID NO：2所示。
进一步的，所述双靶点融合蛋白的氨基酸序列的cDNA序列如SEQ ID NO：1所示。
进一步的，所述穿膜肽序列为TAT。
进一步的，所述双靶点融合蛋白的氨基酸序列如SEQ ID NO：5所示。
进一步的，所述双靶点融合蛋白的氨基酸序列的cDNA序列如SEQ ID NO：4所示。
一种增强TRAIL抗肿瘤活性的双靶点融合蛋白体外表达方法，包括如下步骤：
(1)选择表达载体pET32a或pTWIN 1，将表达载体pET32a中切除Trx标签序列，或者将表达载体pTWIN 1中切除Intein内含肽序列，然后将编码融合蛋白的基因cDNA序列克隆于原核表达载体上；
(2)步骤(1)中构建的载体采用BL21(DE3)宿主菌表达。
进一步的，所述步骤(2)中，宿主菌表达时，表达诱导温度为18℃。
本发明的第三目的是提供一种增强TRAIL抗肿瘤活性的双靶点融合蛋白用作抗肿瘤药物的用途。
有益效果
本发明的有益技术效果是：本发明与单独TRAIL蛋白可溶性片段(114-281aa)编码cDNA序列的原核表达载体相比，原核表达载体的融合蛋白具有更高的可溶性表达，融合蛋白的回收及纯化效率更高。体外生物活性分析表明，所获融合蛋白能同时激动细胞膜上死亡受体和抑制细胞质内XIAP基因的表达，通过凋亡途径双靶点的作用增强诱导肿瘤细胞凋亡的活性，具有更强的抗肿瘤作用。上述双靶点TRAIL融合蛋白极具发展潜力和优势，极有可能发展成为新一代肿瘤细胞凋亡诱导药物。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1是融合前段拼接后PCR产物电泳图；电泳条件：3％Agarose，电压100V，20min；lane 1～2：融合前段序列1、2拼接后PCR产物电泳条带；M：DL500分子量Marker；PCR产物上样量均为5μl；
图2是TRAIL(114-281aa)基因PCR产物电泳图；电泳条件：1％Agarose，电压100V，20min；lane1：TRAIL(114-281aa)编码cDNA序列PCR产物电泳条带；M：DM2000分子量Marker，上样量5μl；PCR产物上样量均为5μl；
图3是融合蛋白基因拼接PCR产物电泳图；电泳条件：1％Agarose，电压100V，20min；lane1～2：融合基因1、2拼接PCR产物电泳条带；M：DM2000分子量Marker，Marker上样量5μl；胶回收产物上样量均为2μl；
图4是融合蛋白基因pMD 19-T连接酶切鉴定电泳图；电泳条件：1％Agarose，电压100V，20min；lane1～8：平板挑取菌落质粒提取酶切鉴定电泳条带；M：DM2000分子量Marker，Marker上样量5μl；酶切产物上样量均为2μl；
图5是融合蛋白基因pTWIN1连接酶切鉴定电泳图；电泳条件：1％Agarose，电压100V，20min；lane1～10：平板挑取菌落质粒提取酶切鉴定电泳条带；M：DM10000分子量Marker，Marker上样量5μl；酶切产物上样量均为2μl；
图6是pTWIN1/TRAIL蛋白表达SDS-PAGE电泳图；电泳条件：15％凝胶，200V，35min；lane 1：TRAIL诱导前电泳条带；lane 2：TRAIL诱导后电泳条带；lane3：TRAIL破菌后上清电泳条带；lane4：TRAIL破菌后沉淀电泳条带；M：Unstained Protein Molecular Weight Marker (条带分子量从上到下依次为：116.0KDa、66.2KDa、45.0KDa、35.0KDa、25.0KDa、18.4KDa、14.4KDa)，样品上样量均为10μl，Marker上样量为5μl；
图7是pTWIN1/融合基因1、2蛋白表达SDS-PAGE电泳图；电泳条件：15％凝胶，200V，35min。lane 1：pTWIN1/融合蛋白基因1诱导前电泳条带；lane2：pTWIN1/融合蛋白基因1诱导后电泳条带；lane 3：pTWIN1/融合蛋白基因1破菌后上清电泳条带；lane 4：pTWIN1/融合蛋白基因1破菌后沉淀电泳条带；lane5：pTWIN1/融合蛋白基因2诱导前电泳条带；lane 6：pTWIN1/融合蛋白基因2诱导后电泳条带；lane 7：pTWIN1/融合蛋白基因2破菌后上清电泳条带；lane8：pTWIN1/融合蛋白基因2破菌后沉淀电泳条带；M：Unstained Protein Molecular Weight Marker(条带分子量从上到下依次为：116.0KDa、66.2KDa、45.0KDa、35.0KDa、25.0KDa、18.4KDa、14.4KDa)，样品上样量均为10μl，Marker上样量为5μl；
图8是pTWIN1/融合基因2蛋白表达SDS-PAGE电泳图；电泳条件：15％凝胶，200V，35min；M：Unstained Protein Molecular Weight Marker(条带分子量从上到下依次为：116.0KDa、66.2KDa、45.0KDa、35.0KDa、25.0KDa、18.4KDa、14.4KDa)，样品上样量均为10μl，Marker上样量为5μl。
本发明的最佳实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
下面结合实施例对本发明作进一步详细的说明。
实施例1
编码融合蛋白I、II型基因的cDNA序列拼接合成
设计的融合蛋白中的TRAIL蛋白(114-281aa)的作用靶点针对TRAIL受体DR4、DR5，SMAC N7肽段针对凋亡拮抗因子XIAP，以期对于TRAIL具有明显的增效作用。
将所要合成的全长融合基因序列(见序列1、4)分为融合前段序列(见序列3、6)和TRAIL序列(见序列7)两部分。融合前段序列包括(R8+SMAC N7+AP)(见序列3)及(TAT+SMAC N7+AP)(见序列6)肽段编码cDNA序列两种，融合前段序列采用委托华大基因合成的分段双链拼接引物(见序列8-18)为原料，将合成的双链拼接引物一起反应，在无Taq DNA Polymerase的条件下，先得到拼接的融合前段序列(R8+SMAC N7+AP)及(TAT+SMAC N7+AP)编码cDNA。同时通过以含有TRAIL基因cDNA的质粒(实验室自备)作为模板，采用TRAIL上下游引物(见序列19、20)扩增TRAIL蛋白(114-281aa)编码cDNA序列，最后将融合前段序列(序列3、6)分别与TRAIL序列(序列7)进行连接PCR反应得到全长融合基因序列(序列1、4)。
(一)全长融合基因cDNA序列I、II的设计与拼接合成
1.全长融合基因cDNA序列I的组成及拼接合成
全长融合基因序列I包括R8+SMAC N7+AP+TRAIL(114-281aa)编码cDNA序列的顺序组合及序列连接。
拟合成及拼接的全长融合基因cDNA序列I全长如下(序列1：564bp)：
ATGCGTCGTCGTCGTCGTCGTCGTCGTGCTGTTCCGATTGCGCAGAAAGCTCCGGTTCGTGAACGTGGTCCGCAGCGTGTTGCTGCTCACATCACTGGTACTCGTGGTCGTTCTAACACTCTTTCTTCTCCGAACTCTAAAAACGAAAAAGCTCTTGGTCGTAAAATCAACTCTTGGGAATCTTCTCGTTCTGGTCACTCTTTCCTTTCTAACCTTCACCTTCGTAACGGTGAACTTGTTATCCACGAAAAAGGTTTCTACTACATCTACTCTCAGACTTACTTCCGTTTCCAGGAAGAAATCAAAGAAAACACTAAAAACGATAAACAGATGGTTCAGTACATCTACAAATACACCTCTTACCCGGACCCGATCCTTCTTATGAAATCTGCTCGTAACTCTTGCTGGTCTAAAGATGCTGAATACGGTCTTTACTCTATCTACCAGGGTGGTATCTTCGAACTTAAAGAAAACGATCGTATCTTCGTTTCTGTTACTAACGAACACCTTATCGATATGGATCACGAGGCTTCTTTCTTCGGTGCTTTCCTTGTTGGTTAATAA
融合蛋白I型氨基酸序列如下(序列2：186aa)：
MRRRRRRRRAVPIAQKAPVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG
将全长融合基因序列1分为融合前段序列(见序列3)和TRAIL序列(见序列7)两部分。融合前段序列包括(R8+SMAC N7+AP)(见序列3)肽段编码cDNA序列，融合前段序列采用委托华大基因合成的分段双链拼接引物(见序列8-12)为原料，将合成的双链拼接引物一起反应，在无Taq DNA Polymerase的条件下，先得到拼接的融合前段序列(R8+SMAC N7+AP)编码cDNA。同时通过以含有TRAIL基因cDNA的质粒(实验室自备)作为模板，采用TRAIL上下游引物(见序列19、20)扩增TRAIL蛋白(114-281aa)编码cDNA序列，最后将融合前段序列(序列3)与TRAIL序列(序列7)进行连接PCR反应得到全长融合基因序列1。
考虑在融合前段序列的5’端加上NdeI酶切位点序列及保护碱基GGT，在序列3’端加上与TRAIL蛋白(114-281aa)编码cDNA序列相重叠的部分序列GTT CGT GAA CGT GGT CGTG，组合在一起的融合前段序列(序列3：79bp)即变成：
GGTCATATGCGTCGTCGTCGTCGTCGTCGTCGTGCTGTTCCGATTGCGCAGAAAGTCCCGGTTCGTGAACGTGGTCGTG
TRAIL蛋白(114-281aa)编码cDNA序列(序列7：510bp)如下：
GTTCGTGAACGTGGTCCGCAGCGTGTTGCTGCTCACATCACTGGTACTCGTGGTCGTTCTAACACTCTTTCTTCTCCGAACTCTAAAAACGAAAAAGCTCTTGGTCGTAAAATCAACTCTTGGGAATCTTCTCGTTCTGGTCACTCTTTCCTTTCTAACCTTCACCTTCGTAACGGTGAACTTGTTATCCACGAAAAAGGTTTCTACTACATCTACTCTCAGACTTACTTCCGTTTCCAGGAAGAAATCAAAGAAAACACTAAAAACGATAAACAGATGGTTCAGTACATCTACAAATACACCTCTTACCCGGACCCGATCCTTCTTATGAAATCTGCTCGTAACTCTTGCTGGTCTAAAGATGCTGAATACGGTCTTTACTCTATCTACCAGGGTGGTATCTTCGAACTTAAAGAAAACGATCGTATCTTCGTTTCTGTTACTAACGAACACCTTATCGATATGGATCACGAGGCTTCTTTCTTCGGTGCTTTCCTTGTTGGTTAATAA
设计融合蛋白I融合前段序列基因拼接引物：
CPP-1-1F(序列8：40bp)：GGTCATATGCGTCGTCGTCGTCGTCGTCGTCGTGCTGTTC
CPP-1-2F(序列9：39bp)：CGATTGCGCAGAAAGCTCCGGTTCGTGAACGTGGTCGTG
CPP-1-3R(序列10：19bp)：CACGACCACGTTCACGAAC
CPP-1-4R(序列11：40bp)：CGGAGCTTTCTGCGCAATCGGAACAGCACGACGACGACGA
CPP-1-5R(序列12：20bp)：CGACGACGACGCATATGACC
2.全长融合基因cDNA序列II的组成及拼接合成
全长融合基因cDNA序列II包括TAT+SMAC N7+AP+TRAIL(114-281aa)编码cDNA序列的顺序组合及序列连接。
拟合成及拼接的全长融合基因cDNA序列II全长如下(序列4：573bp)：
CCGGTTCGTGAACGTGGTCCGCAGCGTGTTGCTGCTCACATCACTGGTACTCGTGGTCGTTCTAACACTCTTTCTTCTCCGAACTCTAAAAACGAAAAAGCTCTTGGTCGTAAAATCAACTCTTGGGAATCTTCTCGTTCTGGTCACTCTTTCCTTTCTAACCTTCACCTTCGTAACGGTGAACTTGTTATCCACGAAAAAGGTTTCTACTACATCTACTCTCAGACTTACTTCCGTTTCCAGGAAGAAATCAAAGAAAACACTAAAAACGATAAACAGATGGTTCAGTACATCTACAAATACACCTCTTACCCGGACCCGATCCTTCTTATGAAATCTGCTCGTAACTCTTGCTGGTCTAAAGATGCTGAATACGGTCTTTACTCTATCTACCAGGGTGGTATCTTCGAACTTAAAGAAAACGATCGTATCTTCGTTTCTGTTACTAACGAACACCTTATCGATATGGATCACGAGGCTTCTTTCTTCGGTGCTTTCCTTGTTGGTTAATAA
全长融合基因序列II编码氨基酸序列如下(序列5：189aa)：
MYGRKKRRQRRRAVPIAQKAPVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMUHEASFFGAFLVG
将全长融合基因序列2分为融合前段序列(见序列6)和TRAIL序列(见序列7)两部分。融合前段序列包括(TAT+SMAC N7+AP)(见序列6)肽段编码cDNA序列，融合前段序列采用委托华大基因合成的分段双链拼接引物(见序列13-18)，将合成的双链拼接引物一起反应，在无Taq DNA Polymerase的条件下，先得到拼接的融合前段序列(TAT+SMAC N7+AP)编码cDNA。同时通过以含有TRAIL基因cDNA的质粒(实验室自备)作为模板，采用TRAIL上下游引物(见序列19、20)扩增TRAIL蛋白(114-281a a)编码cDNA序列，最后将融合前段序列(序列6)与TRAIL序列(序列7)进行连接PCR反应得到全长融合基因序列2。
考虑在融合前段序列的5’端加上NdeI酶切位点序列及保护碱基GGT，在序列3’端加上与TRAIL蛋白(114-281aa)编码cDNA序列相重叠的部分序列GTT CGT GAA CGT GGT CGTG，组合在一起的融合前段序列(序列6：88bp)即变成：GGTCATATGTACGGCCGTAAAAAGCGTCGTCAGCGTCGTCGTGCTGTTCCGATTGCGCAGAAAGCTCCGGTTCGTGAACGTGGTCGTG
设计融合蛋白2融合前段序列基因拼接引物：
CPP-2-1F(序列13：37bp)：GGTCATATGTACGGCCGTAAAAAGCGTCGTCAGCGTC
CPP-2-2F(序列14：30bp)：GTCGTGCTGTTCCGATTGCGCAGAAAGCTC
CPP-2-3F(序列15：21bp)：CGGTTCGTGAACGTGGTCGTG
CPP-2-4R(序列16：37bp)：CACGACCACGTTCACGAACCGGAGCTTTCTGCGCAAT
CPP-2-5R(序列17：35bp)：CGGAACAGCACGACGACGCTGACGACGCTTTTTAC
CPP-2-6R(序列18：16bp)：GGCCGTACATATGACC
TRAIL蛋白(114-281aa)编码cDNA序列扩增引物(见序列19、20)，在下游引物之前加上PstI酶切位点序列和保护碱基GGT序列。
上游引物(序列19)：GTTCGTGAACGTGGTCCGCAGCGTGTTGCTGCT
下游引物(序列20)：GGTCTGCAGTTATTACAAAACAAGGAAAGCACC
(二)实验材料、试剂与仪器设备
1.材料：合成的引物20120328-YJ1、实验室自备的TLP(TRAIL cDNA)质粒模板、批号20120406。
2.试剂见表1所示：
表1.融合基因序列拼接合成所用试剂
试剂名称 规格 批号 生产厂家
TaKaRa Ex Taq 250U 5U/μl CKA4201A TaKaRa
25mM MgCl2 1ml AA1601A TaKaRa
10X Ex Taq Buffer(Mg2+ free) 1ml A2701A TaKaRa
dNTP Mixture（2.5mM each） 180μl BG8201A TaKaRa
DL2000 DNA Marker 500μl B6701A TaKaRa
DL500 DNA Marker 500μl B901A TaKaRa
GelRedTM 10000×in water 0.5ml 11G0127 BIOTIUM
Agarose G-10 100g 111860 GENE COMPANY LTD
(三)实验方法及步骤
1.引物溶解：用超纯水按引物资料上提供的摩尔比，溶解成100pmol/μl，备用。再取5μl加45μl的超纯水，稀释成10pmol/μl做扩增用。
2.根据表2配制融合前段序列拼接反应体系，每个片段各做1管。
表2.融合前段序列拼接反应体系
试剂 30μl反应体系
编号 融合前段1（序列3） 融合前段2（序列6）
合成的各5μl引物（100pmol/μl） 25μl 30μl
RNase-Free Water 5μl 0μl
3.涡旋震荡混匀，短暂离心，将溶液收集到管底。
4.将PCR管置于PCR仪中，进行PCR反应，反应条件见表3。
表3.融合前段序列拼接反应条件
5.将以上PCR产物做纯化，纯化步骤见操作规程，最后用30μl纯水洗脱，备用。
6.将步骤5纯化产物再用融合前段序列上下游引物(对于融合前段序列1，即序列3上下游引物分别为序列8、序列12；对于融合前段序列2，即序列6上下游引物分别为序列13、序列18)做一次PCR，以增加序列的拷贝数，PCR反应体系见表4，各做1管。
表4.融合前段序列PCR反应体系
7.涡旋震荡混匀，短暂离心，将溶液收集到管底。
8.PCR扩增反应条件见表5。
表5.融合前段序列PCR反应条件
步  骤 温 度 时 间  
预变性 94℃ 1min  
变性 94℃ 15s
退火 58℃ 15s 25cycles
延伸 72℃ 30s  
终延伸 72℃ 2min  
9.将以上PCR产物电泳，由于片段在80bp左右，因此用3％胶电泳，观察电泳结果后做胶回收。胶回收步骤见操作规程，最后用35μl纯水洗脱，备用。
10.根据表6配制TRAIL序列(序列7)PCR扩增反应体系；TRAIL下游引物含有方便与表达载体连接的PstI酶切位点，TRAIL序列共做2管。
表6.TRAIL序列PCR反应体系
试剂 50μl反应体系
TLP质粒模板（稀释10倍） 1μl
10X Ex Taq Buffer（Mg2+ free） 5μl
dNTP Mix，2.5mM each 4μl
25mM MgCl 2 4μl
TaKaRa Ex Taq 0.5μl
上游引物（10pmol/μl） 1μl
下游引物（10pmol/μl） 1μl
RNase-Free Water 34.5μl
11.涡旋震荡混匀，短暂离心，将溶液收集到管底。
12.PCR扩增反应条件见表7。
表7.TRAIL序列PCR反应条件
步  骤 温 度 时 间  
预变性 94℃ 3min  
变性 94℃  
退火 58℃ 30s 25cycles
延伸 72℃ 1min  
终延伸 72℃ 3min  
13.PCR产物电泳，用1％的胶，观察结果，有目的条带后做胶回收。胶回收步骤见试剂盒操作规程，最后用30μl水洗脱，备用。
14.融合前段序列的胶回收产物(融合前段1，即序列3；融合前段2，即序列6)分别与胶回收的TRAIL序列(即序列7)进行PCR拼接反应，拼接反应体系及反应条件见表8、表9，最后得到全长融合基因序列1、2(即序列1、序列4)。
表8.全长融合基因序列PCR拼接反应体系
15.漩涡震荡混匀后短暂离心，将溶液收集到管底。
16.再继续PCR扩增，反应条件见表9。
表9.全长融合基因序列PCR拼接反应条件
步  骤 温 度 时 间  
连接的变性 94℃  
连接的退火 55℃ 30s 5cycles
连接的延伸 72℃ 1min  
变性 94℃  
退火 58℃ 30s 25cycles
延伸 72℃ 1min  
终延伸 72℃ 3min  
17.将以上PCR产物电泳，用1％的胶，观察结果，有目的条带后做胶回收。合并4管PCR产物胶回收，胶回收步骤见操作规程，最后用30μl纯水洗脱，备用。
(四)实验结果
1.融合前段序列1、2拼接后PCR结果见图1。图中可见，分别扩增得到约80bp的融合前段序列1、2基因。
2.TRAIL(114-281aa)编码cDNA序列PCR结果见图2。图中可见，扩增得到大小约500bp的TRAIL(114-281aa)编码cDNA序列。
3.全长融合基因序列拼接PCR结果见图3。图中可见，拼接扩增得到大小约580bp的全长融合基因序列1、2。
最后，试验成功获得全长融合基因序列1、2，可用于后续与pMD19-T载体的连接。
实施例2
全长融合基因序列1、2与pMD 19-T载体的连接
为增加表达载体构建的成功率，首先将全长融合基因序列1、2与pMD19-T载体连接及转化，然后再将成功转化的克隆子亚克隆到表达载体pTWIN1和pET-32a上，构建全长融合基因序列1、2的原核表达载体。
(一)实验材料、试剂与仪器设备
1.材料：全长融合基因序列1、2来源于实施例1的结果。
2.试剂见表10。
表10.全长融合基因序列1、2与pMD19-T载体连接所用试剂
试剂名称 规格 批号 生产厂家
pMD19-T Vector 20T CK2401AA TAKARA
Top10感受态细胞 100μl 111108 天根生物
Tryptone 500g 829408 OXOID
Yeast Extract 500g 990951 OXOID
NaCl AR 500g 20110113 成都市科龙化工试剂厂
(二)实验方法及步骤
1.按照表11配制连接反应体系，各做1管。
表11.全长融合基因序列1、2与pMD19-T载体连接反应体系
2.pMD19-T Vector连接反应体系使用16℃连接4.5小时。
3.往连接产物中加入50μl感受态细胞，冰浴30分钟。
4.在水浴42℃热击45秒。
5.置冰上孵育2分钟。
6.加入600μl SOC培养基，37℃振荡培养60分钟。
7.转化的感受态细胞离心后，在超净工作台，弃去大部分培养基，余约100μl培养基，将细菌吹匀，全部涂布于含氨苄青霉素的LB固体培养基上，37℃培养过夜。
(三)实验结果
2种连接片段的平板均长出较多菌落。菌种接种经细菌培养、质粒提取及酶切鉴定均有多个阳性克隆，保存菌种，送测序。全长融合基因序列1、2均获得测序完全正确质粒。如图4所示，图4是融合蛋白基因pMD 19-T连接酶切鉴定电泳图。
实施例3
全长融合基因序列1、2与原核表达载体的连接
选用原核表达载体pTWIN 1和pET-32a，分别用NdeI和PstI双酶切载体pTWIN1、pET-32a载体及阳性克隆载体pMD19/全长融合基因序列1和pMD19/全长融合基因序列2。pTWIN1得到去除了Intern区域(约1549bp)两个粘性末端，再在此区域插入经同样双酶切的全长融合基因序列1、2；而pET-32a得到去除Trx融合蛋白标签表达区域的两个粘性末端，再在此区域插入经同样双酶切的全长融合基因序列1、2。因此采用NdeI和PstI双酶切，用TaKaRa连接试剂盒连接后转化入天根生物的Top10感受态细胞。
(一)实验材料、试剂与仪器设备
1.材料：阳性克隆载体pMD19/全长融合基因序列1和pMD19/全长融合基因序列2来源于实施例2，质粒批号20120515，pTWIN1批号20120406、来源于20120406-YJ2。pET-32a批号20120406、来源于20120406-YJ2。
2.试剂见表12
表12.全长融合基因序列1、2与原核表达载体的连接所用试剂
试剂名称 规格 批号 生产厂家
GelRedTM 10000×in water 0.5ml 11G0127 BIOTIUM
Agarose G-10 100g 111860 GENE COMPANY LTD
GeneRuler 1kb DNA Ladder 0.5μg/μl 00013872 Fermentas
DL2000 500μl B6701A TaKaRa
Pst I 3000U，15U/μl CK707A TaKaRa
NdeI 400U，10U/μl CKA201A TaKaRa
10×H Buffer 500μl CA2101B TaKaRa
Sol I 75μl CD1401A TaKaRa
Gel Extraction Kit(50) 50次 00D2500010000K27K065 OMEGA
Top10感受态细胞 100μl 111108 天根生物
(二)实验方法及步骤
1.酶切载体和目的片段
(1)NdeI、PstI双酶切载体pTWIN1或pET-32a与pMD19/全长融合基因序列1、2质粒，酶切体系见表13，反应体系100μl。
表13.载体pTWIN1、pET-32a与pMD19/全长融合基因序列1、2质粒酶切反应体系
(2)将EP管放入多用恒温箱中，37℃，2.5小时。
(3)纯化酶切产物，用OMEGA的Cycle-Pure Kit。用30μl超纯水洗脱，电泳，照相，备用。
2.载体pTWIN1或pET-32a与融合蛋白基因1、2的连接并转化
(1)连接体系见表14，各做1管。
表14.全长融合基因序列1、2与载体pTWIN1或pET-32a连接反应体系
(2)16℃连接过夜。
(3)将连接产物5μl分别加入50μl Top10感受态细胞，冰浴30分钟。
(4)在水浴42℃热击90秒。
(5)置冰上孵育2分钟。
(6)加入500μl SOC培养基，37℃振荡培养45分钟。
(7)转化的感受态细胞离心后，在超净工作台，弃去400μl，余约100μl培养基，将细菌吹匀，全部涂布于含Amp的LB固体培养基上，37℃培养过夜。
(三)实验结果
转化入Top10的感受态细胞，每一种连接产物的平板均有菌落长出。此次实验比较成功，有待进一步鉴定转化子。菌种接种经细菌培养、质粒提取及酶切鉴定均有多个阳性克隆，保存菌种，送测序。融合蛋白基因序列1、2在两种载体均获得测序完全正确重组质粒。电泳结果如图5所示。
实施例4
pTWIN1/全长融合基因序列1、pTWIN1/全长融合基因序列2载体的表达研究
试验使用TB培养基培养及诱导表达pTWIN1/全长融合基因序列1、pTWIN1/全长融合基因序列2并与野生型TRAIL原核表达载体pTWIN1/TRAIL表达进行对比研究。由于需要制备少量样品用于纯化目的蛋白，使用大量摇瓶方式进行制备。此前实验培养及诱导表达TRAIL系列蛋白使用的培养基均为LB培养基。因此拟选择主要碳源为甘油的TB培养基，本实验试验使用该培养基的可行性。
(一)实验材料、试剂与仪器设备
1.SDS-PAGE电泳相关溶液配制于20120428-XQ2，pTWIN1/全长融合基因序列1、pTWIN1/全长融合基因序列2菌种购自Invitrogen公司。
2.试剂见表15。
表15.融合基因序列1、2载体表达研究所用试剂
试剂名称 规格 批号 生产厂家
Tryptone 500g 829408 OXOID
Yeast Extract 500g 990951 OXOID
NaCl AR 500g 20110113 成都市科龙化工试剂厂
丙三醇 AR 500g 20110113 成都市科龙化工试剂厂
K2HPO4·3H2O AR 500g 20110301 成都市科龙化工试剂厂
KH2PO4 AR 500g 20110113 成都市科龙化工试剂厂
IPTG 100g P13/342/129 INALCO SPA MILANO ITALY
Ammonium persulfate ≥98%，100g 215589 SIG-ALD，Biodee分装
TEMED ≥99%，100ml T22500 SIG-ALD，Biodee分装
Unstained Protein Molecular Weight Marker 1ml 00078434 Thermo Scientific
注射用氨苄西林钠 0.5g E1005102 华北制药股份有限公司
(二)实验方法及步骤
1.取pTWIN1/全长融合基因序列1、pTWIN1/全长融合基因序列2及pTWIN 1/TRAIL甘油菌各500μl接入4ml LB(Amp+)培养基中，37℃，220rpm振摇活化过夜。
2.取活化过夜的菌液各1ml接入50ml TB培养基中，37℃，220rpm振摇培养2h(OD600大约1)，降温至28℃，加入0.1M IPTG 500μl诱导培养，诱导前取样1ml离心弃去上清，加入25μl H2O重悬后加入25μl 2×loading buffer制成诱导后电泳样品。
3.诱导6h后收菌，测量pH值，取样300μl离心弃去上清，加入30μl H2O重悬后加入50μl 2×loading buffer制成诱导后电泳样品，剩余菌液10000rpm离心2min，弃去上清后保存。
4.菌体使用5ml H2O重悬，超声波破菌。破菌条件为：150W脉冲破菌5s后暂停8s，循环60次。
5.破菌液取1ml 12000rpm离心10min，分离上清和沉淀，沉淀使用1ml H2O重悬，上清和沉淀重悬液各取25μl加入25μl 2×loading buffer，制成电泳样品。
6.将制成的电泳样品置于沸水浴中处理10min，12000rpm离心10min，然后取上清10μl电泳。
(三)实验结果
1.由SDS-PAGE蛋白电泳图6可见，野生型TRAIL原核表达载体目的蛋白的表达较弱，且目的蛋白主要存在于沉淀中。
2.由SDS-PAGE蛋白电泳图7可见，pTWIN1/全长融合基因序列1、pTWIN1/全长融合基因序列2目的蛋白的表达较强，且目的蛋白主要存在于上清中。
从实验结果可以看到，野生型TRAIL原核表达载体目的蛋白的表达较弱，且目的蛋白的表达主要分布在沉淀中，不利于纯化。新构建的pTWIN1/全长融合基因序列1、pTWIN1/全长融合基因序列2目的蛋白的表达较强，且目的蛋白主要存在于上清中。其中pTWIN1/全长融合基因序列1表达蛋白几乎完全可溶，pTWIN1/融合蛋白基因2可溶性蛋白大约占60％，这个结果虽不及18℃诱导结果，但明显优于30℃诱导结果，上清中目的蛋白含量也能够达到方便纯化的要求。野生型TRAIL的表达依然不佳，可能与其蛋白质的空间结构相关。
实施例5
pTWIN1/全长融合基因序列2不同温度、不同培养基表达研究
实验材料和方法同实施例3，如图8所示，图8是pTWIN1/融合基因2蛋白表达SDS-PAGE电泳图。37℃条件下诱导即有大量目的蛋白表达，但蛋白可溶性很差，上清中仅存在少量目的蛋白；降低温度至30℃诱导发现目的蛋白表达强度未受诱导期间温度影响，依然有较高水平的表达，而可溶性有所改善。将诱导温度降至极限18℃，目的蛋白表达强度有所减弱，但表达的目的蛋白完全可溶。将培养基由LB培养基换成以甘油为主要碳源的营养更丰富的TB培养基后，表达的目的 蛋白约60％可溶，这个结果虽不及18℃结果，但明显优于30℃结果，上清中目的蛋白含量也能够达到方便纯化的要求。
实施例6
融合蛋白I型、II型及TRAIL蛋白生物活性检测
融合蛋白I型、II型、野生型TRAIL蛋白及空白对照作用48小时后取相同浓度加药孔在光镜下观察，明显可以观察出四组在细胞数量上的不同，空白对照组细胞形态未发生明显变化，对于乳腺癌细胞株而言，三个加药组细胞数量明显减少，野生型TRAIL蛋白组尚残存部分形态正常的梭形MDA-MB-231，融合蛋白I型、II型组大部分细胞变圆，失去原有正常形态。野生型TRAIL蛋白对SPCA1细胞的杀伤作用微弱，极少细胞出现形态变化，与对照组相比细胞数量减少不明显。
1.SRB法肿瘤细胞毒性增殖抑制作用
MDA-MB-231为野生型TRAIL蛋白敏感细胞株，融合蛋白I型、II型和野生型TRAIL蛋白在50-0.02ug/ml范围内，作用48小时后观察，三者对MDA-MB-231均具有量效关系，融合蛋白I型对生长抑制作用较明显，IC50为4.04×10-6ng/ml，融合蛋白II型对生长抑制作用也较明显IC50为6.67×10-6ng/ml，野生型TRAIL蛋白IC50为9.45ng/ml。
SPCA1对野生型TRAIL蛋白不够敏感，在50～0.003ug/ml范围内生长抑制作用不明显，IC50为428.05ug/ml。而融合蛋白1、2在相同浓度范围内，具有较明显的生长抑制作用，其中融合蛋白I型的IC50为4.64ug/ml，融合蛋白II型的IC50为8.78ug/ml，较大程度逆转了肿瘤细胞对野生型TRAIL的耐受，细胞毒性试验结果见表16。
表16.融合蛋白I型、II型及野生型TRAIL对肿瘤细胞SRB细胞毒性试验结果
蛋白 细胞 IC50
融合蛋白1 MDA-MB-231 4.04×10-6 ng/ml
融合蛋白2 MDA-MB-231 6.67×10-6 ng/ml
融合蛋白1 SPCA1 4.64 ug/ml
融合蛋白2 SPCA1 8.78 ug/ml
天然TRAIL MDA-MB-231 9.45 ng/ml
天然TRAIL SPCA1 428.05 ug/ml
(IC50＜10ug/ml为敏感)
2.AnnexinV-FITC/PI荧光标记流式细胞术检测凋亡
融合蛋白I型、II型和野生型TRAIL作用浓度皆为10ng/ml，在加药后6小时检测 凋亡，结果显示在两种细胞株中，融合蛋白1、2诱导凋亡作用都强于野生型TRAIL蛋白，特别是在对TRAIL不敏感的肺癌细胞株中，融合蛋白仍然能起到较强的杀伤作用。两组凋亡率差异有统计学意义(P＜0.01)。
本实施例的结果(表17)，进一步验证了实验设计的采用针对TRAIL受体DR4、DR5和凋亡拮抗因子XIAP双靶点对于TRAIL的明显增效作用。
表17.融合蛋白I型、II型及野生型TRAIL对肿瘤细胞作用流式分析结果
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (11)

  1. 增强TRAIL抗肿瘤活性的双靶点融合蛋白，其特征在于：所述治疗的靶点为TRAIL受体DR4、DR5和凋亡拮抗因子XIAP。
  2. 根据权利要求1所述的增强TRAIL抗肿瘤活性的双靶点融合蛋白，其特征在于，所述双靶点融合蛋白由穿膜肽序列、SMAC N7、内源性蛋白酶酶切位点与TRAIL蛋白肽段顺序融合而成。
  3. 根据权利要求2所述的增强TRAIL抗肿瘤活性的双靶点融合蛋白，其特征在于：所述穿膜肽序列为R8。
  4. 根据权利要求3所述的增强TRAIL抗肿瘤活性的双靶点融合蛋白，其特征在于：所述双靶点融合蛋白的氨基酸序列如SEQ ID NO：2所示。
  5. 根据权利要求4所述的增强TRAIL抗肿瘤活性的双靶点融合蛋白，其特征在于：所述双靶点融合蛋白的氨基酸序列的cDNA序列如SE Q ID NO：1所示。
  6. 根据权利要求2所述的增强TRAIL抗肿瘤活性的双靶点融合蛋白，其特征在于：所述穿膜肽序列为TAT。
  7. 根据权利要求6所述的增强TRAIL抗肿瘤活性的双靶点融合蛋白，其特征在于：所述双靶点融合蛋白的氨基酸序列如SEQ ID NO：5所示。
  8. 根据权利要求7所述的增强TRAIL抗肿瘤活性的双靶点融合蛋白，其特征在于：所述双靶点融合蛋白的氨基酸序列的cDNA序列如SE Q ID NO：4所示。
  9. 增强TRAIL抗肿瘤活性的双靶点融合蛋白体外表达方法，其特征在于，包括如下步骤：
    (1)选择表达载体pET32a或pTWIN
    1，将表达载体pET32a中切除Trx标签序列，或者将表达载体pTWI N 1中切除Intein内含肽序列，然后将编码融合蛋白的基因cDNA序列克隆于原核表达载体上；
    (2)步骤(1)中构建的载体采用BL21(DE3)宿主菌表达。
  10. 根据权利要求9所述的增强TRAIL抗肿瘤活性的双靶点融合蛋白体外表达方法，其特征在于，所述步骤(2)中，宿主菌表达时，表达诱导温度为18℃。
  11. 增强TRAIL抗肿瘤活性的双靶点融合蛋白用作抗肿瘤药物的用途。
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