WO2004022751A1 - Gene de cytotoxine de cnidaire et son expression et son application - Google Patents

Gene de cytotoxine de cnidaire et son expression et son application Download PDF

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WO2004022751A1
WO2004022751A1 PCT/CN2003/000538 CN0300538W WO2004022751A1 WO 2004022751 A1 WO2004022751 A1 WO 2004022751A1 CN 0300538 W CN0300538 W CN 0300538W WO 2004022751 A1 WO2004022751 A1 WO 2004022751A1
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sequence
src
protein
anemone
gene
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Anlong Xu
Xiaoyu Jiang
Lisheng Peng
Hongbin Tu
Huiping Chen
Wenli Yang
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Sun Yat Sen University
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Sun Yat Sen University
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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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a new gene, particularly a new gene for anemone cytotoxin.
  • the invention also relates to the expression of the above-mentioned genes and the application of the encoded protein to the preparation of a medicament for treating a tumor disease.
  • Anemone belongs to the coral class i ant ozod of the soelenterata, and is a relatively primitive animal in the ocean.
  • the tentacles of anemone tentacles can secrete a variety of anemone toxins, which are basically peptide toxins and have a variety of physiologically active effects on humans and animals.
  • Anemone toxins are classified into three categories based on their physiological functions: anemone neurotoxin, anemone cytolysin, and anemone potassium channel inhibitors.
  • Cytotoxic marine biotoxins have been used as one of the sources for screening anticancer and antiviral agents.
  • a variety of polypeptide cytolytic toxins have been found in marine life. These marine life include: anemone, jellyfish, seaweed, sea urchin, sea hare, sponge, etc.
  • Anemone cytolysin can destroy the structure of cells. It mainly acts on the cell membrane and binds with lipids or proteins of the cell membrane to make it penetrate and dissolve.
  • Anemone cytolysin is a class of basic proteins with a molecular weight of about 20 kDa, containing more than 30 strongly basic amino acids, lacking cysteine, and has cytolytic, cardiotoxic, and other activities.
  • Anemone cytolysin has at least two regions directly involved in membrane lipid (phosphatidylcholine, sphingosine, ganglioside) or membrane protein binding: amphiphilic alpha helix at the N terminus (amino acid residues 13-20) and The tryptophan-rich region (amino acid residues 105-120); the parental alpha helix is inserted into the cell membrane, while the tryptophan-rich region of the prolonged ES3 ⁇ 4 skin direction — stretches on the surface of the cell membrane, arginine, threonine and nearby Amino acids can interact with the polar ends of membrane lipids.
  • membrane lipid phosphatidylcholine, sphingosine, ganglioside
  • membrane protein binding amphiphilic alpha helix at the N terminus (amino acid residues 13-20) and The tryptophan-rich region (amino acid residues 105-120); the parental alpha helix is inserted into the
  • Anemone cytolysin can be used as a model of eukaryotic cytolysin. Anemone cytolysins often form oligomers, and their mode of action can be described as: Anemone cytolysins soluble in water-combined with cell membranes or membrane receptors-inserted into cell membranes-formed oligomers-formed membrane channels- Cell lysis.
  • a cytolysin Sticholysinll isolated from the sea anemone Stichodactyla helianthus ⁇ forms a tetramer, inserts into the cell membrane to form a cation channel, and destroys the cell.
  • Anemone cytolysins show a variety of biological activities; hemolytic activity, cytotoxicity, cardiac crest activation, block potassium channels, etc. Isolated from Anemone C Heteractis magnificat The half-lysed concentration of HmT on human red blood cells was 0.15ug / ml.
  • Equinatoxin ll isolated from anemone (Actinia equine ⁇ ) has a semi-lethal concentration of 35 ug / kg in mice, and the cause of death is myocardial ischemia.
  • Cytolysin III can kill Ehrlich ascitic tumor cells cultured in vitro, and also has a certain inhibitory effect on the tumors inoculated in mice. Further research on new cytotoxins from anemones is expected to obtain certain cardiovascular drugs or anticancer and antiviral agents.
  • Anemone cytolysin can be fused and expressed in E.Coli.
  • the cytolytic and cytotoxic activity of the recombinant protein is equivalent to that of the natural protein, but adding additional amino acids to the N-terminus reduces the activity of the recombinant protein. The more additional amino acids The lower the activity of the recombinant protein. The extra amino acids added at the N-terminus interfere with the interaction of the alpha helix with the cell membrane and reduce the activity of the toxin.
  • the object of the present invention is to provide a new anemone cytotoxin gene Src1.
  • Another object of the present invention is to provide expression of the above-mentioned novel genes.
  • Another object of the present invention is to provide an application of the above-mentioned new gene in the preparation of a medicament for preventing and treating tumors.
  • the sea anemone selected in the present invention is rose red green sea anemone (0 ⁇ rosea), which was collected from the sea area near Weizhou Island, Beihai City, Guangxi Zhuang Autonomous Region.
  • the invention constructs a rose red green anemone venom gland cDNA expression library: first, anemone tentacles are isolated, total RNA is extracted, and then double-stranded cDNA is obtained according to the instructions of the SMART TM cDNA Library Construction Kit of Clontech Company, and finally the double-stranded cDNA is ligated
  • the recombinant plasmid vector pcDNA3.0 was transformed into E.coli to construct a cDNA expression library of rose red green anemone venom.
  • a cDNA clone encoding the rose red green anemone cytolysin is obtained, and the number is Src I.
  • This cDNA sequence encodes a 216 amino acid toxin precursor protein, including a 19 amino acid signal peptide, a 19 amino acid propart motif, and a 178 amino acid mature protein.
  • the protein has an isoelectric point of 4.8 and a molecular weight of 19,500 Daltons. It is an acidic protein, which is the first acid anemone cytolysin reported.
  • the N-terminus of the mature protein has the typical characteristics of anemone cytolysin, that is, it has a parental alpha helix.
  • a pair of specific primers are designed, and a nucleotide sequence encoding a rosin-green-anemone cytolysin mature protein is amplified from a pcDNA3.0 vector by a PCR method, cloned into a prokaryotic expression vector PBV220, and constructed into The expression plasmid pBV220-Src I and transformed it into E. coli DH5 (i. After exploring and optimizing conditions such as culture time, culture temperature, induction time, etc., the expression level of the recombinant protein accounted for more than 15% of the total protein of the bacterial body, and basically The upper part is in an insoluble inclusion body state.
  • the above primers are synthesized based on the sequences of the mature protein encoded by the Src l gene and the multiple restriction sites of the prokaryotic expression vector pBV220.
  • the upstream primer contains the EcoR I digestion sequence (GAATTC) and the start codon (ATG), and the downstream primer contains BamH I digestion sequence (GGATCC) and stop codon (TTA), the sequence is as follows:
  • the invention also explores and optimizes the purification conditions of the recombinant Src I protein.
  • the recombinant Src I protein having a purity of more than 98% can be obtained.
  • the recombinant anemone cytotoxin obtained by the present invention has biological activity.
  • the recombinant Src I protein obtained by the present invention has a significant effect on hepatoma cell BEL-77401, gastric cancer cell BGC-823, and lung cancer cell Nsclc cultured in vitro.
  • IC 5Q is 31.2ug / ml, 3.1ug / mK 3.1ug / mL, respectively.
  • Intraperitoneal injection of recombinant Src I protein into NIH mice showed significant inhibitory effects on liver cancer solid tumors and ⁇ -180 solid tumors inoculated in mice.
  • the tumor inhibition rates were 39.5% and 26.5%, respectively. 0.6mg / kg, 1.2mg / kg.
  • the invention constructs an expression plasmid pBV220-Src I of the Src I anemone cytotoxin mature protein coding sequence, and the expression plasmid vector is digested with EcoRI I / BamH I to obtain a 534bp fragment, which is a rose red green anemone. Cytotoxin Src I mature peptide coding sequence.
  • the replication method of the expression plasmid vector of the present invention Refer to the method of Sambrook (Sambrook, et al. 1989, Molecular doing. Cold Spring Harbor Labroratory Press. USA), and use the CaCl 2 method in E. Coli. DH5a or BL21 (DE3) strain
  • the plasmid was transformed.
  • the bacteria were transformed with LB medium containing ampicillin (10 ( ⁇ g / mL)), and the plasmid was extracted by alkaline method.
  • the mature protein nucleotide sequence was amplified from the pcDNA3.0 vector by PCR method, cloned into the shuttle plasmid pshuttle, and a recombinant plasmid pshuttle-Src I was constructed.
  • This plasmid was digested with PI-SceI / 1-CeuI and purified.
  • the nuclear expression vector Adeno-X was ligated to construct a recombinant adenovirus Adeno-Src I plasmid.
  • the DNA of Adeno-Src I was digested with Pad and packaged into virus particles in human embryonic kidney cells HEK293. Recombinant adenovirus can be expressed in eukaryotic cells for the treatment of related diseases.
  • the above primers were synthesized based on the sequences of both ends of the mature protein encoded by the Src l gene and the multiple restriction sites of the shuttle plasmid pShuttle.
  • the upstream primers contained the Apa l digestion sequence (GGGCCC) and the start codon.
  • the downstream primer contains Notl digestion sequence (GCGGCCGC) and stop codon (TTA).
  • the sequence is as follows:
  • Figure 1 shows the results of total RNA electrophoresis of rose red green anemone tentacles.
  • Figure 2 shows the tentacle double-stranded cDNA electrophoresis results of rose red green anemone
  • Figure 3 shows the results of tentacle tentacle cDNA library total plasmid, Sfil digestion, and PCR detection electrophoresis
  • Figure 4 shows the results of PCR detection of tentacle cDNA library recombinants of rose red green anemone
  • Figure 5 shows the construction of the recombinant plasmid pBV220-Src I containing the gene Src I;
  • Figure 6 shows the electrophoresis results of the PCR products of the Src I gene of rose red green anemone
  • Figure 7 shows the results of digestion and PCR identification of pBV220-Src I expression plasmid containing gene Src l;
  • Figure 9 is the isoelectric point diagram of the Src l protein in the children group.
  • FIG. 10 is a hemolysis curve diagram of recombinant Src l protein
  • FIG. 11 is a light microscope photograph of human liver cancer cell BEL-7402 treated with recombinant Src l protein
  • FIG. 12 is a fluorescence photograph of recombinant human Src l protein acting on human liver cancer cell BEL-7402;
  • FIG. 13 is a light microscope photograph of human gastric cancer cell BGC-823 treated with recombinant Src l protein
  • FIG. 14 is a fluorescence photograph of human gastric cancer cell BGC-823 treated with recombinant Src l protein
  • FIG. 15 is the result of the effect of recombinant Src l protein on human lung cancer cell Nsclc;
  • Figure 16 shows the construction of the Adeno-Src I expression system
  • Figure 17 shows the identification of the recombinant adenovirus Adeno-Src I by PI-SceI / 1-Ceui digestion
  • Figure 18 is a Pad digestion of the recombinant adenovirus Adeno-Src I.
  • RA ladder Promega
  • 2 Rose red green anemone tentacles total RNA.
  • 1 l kb DNA ladder (Promega); 2: rose red green anemone tentacle dsDNA.
  • M 1 kb DNA ladder (Promega); 1-21: PCR detection of library recombinants.
  • 1 marker; 2: uninduced total cells; 3: total cells induced at 42 ° C; 4: ultrasonic supernatant; 5: ultrasonic precipitation; 6: washed supernatant of inclusion bodies; 7 : 0.3 M Nacl elution peak of refolded Src 1 after ion exchange chromatography; 8: 0.8 M Nac tritium of Src I after refolding by ion exchange chromatography.
  • RNA of venom gland extracted by one-step method of guanidine isothiocyanate was detected by 1% formaldehyde denaturing gel electrophoresis. Two distinct rRNA bands, 28S and 18S, were shown in Figure 1, which showed that the total RNA was in good integrity.
  • the SMART TM cDNA Library Construction ist-synthesized cDNA was electrophoresed on a 1% agarose gel. The results showed a uniform smear, as shown in Figure 2. The size ranges from 200bp to 8kb, mainly in the region below 3kb, at 500bp. More concentrated nearby, indicating good integrity of the cDNA.
  • the cDNA was inserted into the plasmid vector pcDNA3.0 to construct a cDNA expression library with a library clone number of 2.4 ⁇ 10 5 .
  • the total library plasmids were extracted for enzyme digestion and PCR analysis. As shown in Figure 3, the results showed that the size of the cDNA inserts fell within the range of 300bp-8kb, and 172 clones were picked to extract the plasmid.
  • the digestion and PCR identification showed that more than 95% of the clones were Recombinant, as shown in Figure 4, It shows that the cDNA expression library has better quality.
  • Example 2 Cloning, sequencing and analysis of tentacles of rosy red and green anemone tentacles.
  • Clones of tentative cDNA library of rosy and green anemone tentacles were selected and plasmid DNA was extracted according to the method of Omega Biotek Plasmid Miniprep Kit. 150 cDNA sequences were randomly determined. ABI PRISM 377 DNA Analyzer (Applied Biosystems) was used, and T7 and SP6 universal primers were used as sequencing primers to perform forward and reverse sequence determination. The sequences of cDNA fragments exceeding 1000bp were designed, and primers were designed based on the measured sequences to continue to test the cDNA.
  • the sequence listing encodes a protein of 216 amino acids, including a signal peptide of 19 amino acid residues, a propart motif of 19 amino acid residues, and a 178 amino acid residue.
  • the mature protein the N-terminus of the mature protein, has a heliolysin-specific alpha helix.
  • the molecular weight of the mature protein is 19,500 Daltons, and the isoelectric point of the protein is 4.8, which is an acidic cytolysin.
  • all reported anemone cytolysins are strongly basic proteins with isoelectric points above 9.0. It is the first time that acid cytolysins have been found in anemones.
  • the nucleotide sequence and predicted amino acid sequence of Src I are shown in the Sequence Listing.
  • design 3 'end primers based on the obtained cytolysin cDNA sequence
  • use SMART TM cDNA Library Construction Kit library construction primers as 5' end primers, and perform RT-PCR.
  • the expected specific amplification band appeared at 800bp, and this band was recovered.
  • the recovered PCR products were ligated to pGEM-T Easy Vector, transformed into DH5a E. coli, and positive clones were selected for sequencing. Sequencing analysis confirmed that it was the desired gene we wanted to obtain.
  • the 8 Henglong determined were the same cDNA sequence.
  • PCR amplification and gene cloning were performed by conventional methods.
  • the PCR product is about 600bp and encodes 178 amino acid residues, as shown in Figure 6.
  • the target gene was cloned into the prokaryotic expression vector pBV220, and the expression plasmid pBV220-Src I was constructed. The construction process is shown in Figure 5. Enzyme digestion and sequencing analysis showed that the cloned gene was the target gene as shown in Figure 7.
  • the promoter also contains the cl regulatory gene, and a foreign gene with a start codon can be inserted into multiple restriction sites downstream of the promoter to express a non-fusion protein, and the product is available for clinical use.
  • the expression plasmid pBV220-Src I was transformed into E. coli DH5a.
  • the bacterial cells were collected and analyzed by SDS-PAGE electrophoresis, which showed that the genetically engineered bacteria had obvious specific expression product bands after induction, and the molecular weight was consistent with the predicted value of 20kD, as shown in Figure 8.
  • Thin-layer scanning analysis showed that: Under this condition, the expression of recombinant protein accounted for more than 17% of the total protein of the bacterial body, and it was basically in an insoluble inclusion body state.
  • the induced bacterial cells were sonicated, and the pellet was centrifuged. The pellet was washed with different buffer solutions to remove foreign proteins. The purity of the inclusion bodies after washing was 80%.
  • the solutions used in the inclusion body washing are: ultrasound buffer (10mM Tris-Hcl, pH7.0, ImMEDTA), buffer KO.IM Tris-Hcl, pH8.0, 10mM EDTA, 0.5% Triton X 100).
  • Buffer 2 50mM PB, 0.5M Nacl, 3M urea
  • buffer 3 0.1 M Tris-Hcl, pH8.5, 10MMEDTA, 3M urea
  • the washed inclusion bodies were dissolved in a denaturing solution (8M urea, 10 mM Tris-Hcl, pH 8.0, 10 mM DTT).
  • the denatured protein was renatured in renaturation solution (3M urea, 20mM Tris-Hcl, pH8.0, O.lmM oxidized glutathione, 0.9mM reduced glutathione) by dialysis.
  • Refolded proteins can be obtained by ion exchange chromatography and hydrophobic chromatography with a purity of more than 99%, as shown in Figure 8.
  • the isoelectric point of the recombinant protein was determined by plate electrophoresis, and isoelectric focusing polyacrylamide gel electrophoresis was performed according to a conventional method.
  • the determined result is: pH 4.81, as shown in Figure 9. It is basically consistent with the predicted isoelectric point.
  • Example 8 Effect of Recombinant Anemone Cytolysin Protein on Tumor Cells Cultured in vitro Tumor cells were seeded in a 24-well plate at 1.0 ⁇ 10 6 cells / ml, and samples were added when the cells grew to 60% -70% confluence. Four parallel experiments were performed for each sample concentration, and Hank's was used as a negative control.
  • Fluorescence detection of apoptosis (Hoeches 55258 staining method): Fix cells with 4% paraformaldehyde at 4 ° C for 20 minutes, wash twice with Hank's, add Hoeches 55258 staining solution, the final concentration is 5-10ug / ml Stain for 10 minutes at room temperature, wash twice with Hank's, and observe under a fluorescence microscope.
  • Recombinant proteins of different concentrations (0.31 ug / mU 3.1 ug / mK 7.8 ug / mK 15.6 ug / ml, 31.2 ug / mK 72.1 ug / ml) were added to human liver cancer cell BEL-7402 and observed within 0-36 hours
  • the sample at 0.31-15.6 ug / ml has no effect on the cells, and can cause obvious changes in cell morphology at a concentration of 31.2 ug / ml. As shown in Figure 11, the cells are significantly elongated.
  • the recombinant protein should be able to interact with the cell membrane. From the phenomenon of the recombinant protein acting on the liver cancer cell BEL-7402, the recombinant protein may not only interact with the cell membrane, but may also interact with the protein on the membrane, causing signal changes Changes in microtubules, microfilaments, etc., cause changes in cell morphology.
  • Recombinant proteins of different concentrations (0.31 ug / ml, 3.1 ug / ml, 7.8 ug / ml, 15.6 ug / mK 31.2 ug / mK 72.1 ug / ml) were added to human low-differentiated gastric cancer cell BGC-823, after adding the sample After observation, after 6 hours, the sample concentration of 3.1 ug / ml can make the cells round and smaller, as shown in Figure 13. After fluorescent staining, it can be seen that the nucleus is significantly smaller, and the cytoplasm has only a thin layer wrapped outside the nucleus. See Figure 14.
  • Recombinant proteins of different concentrations (0.31 ug / mU 3.1 ug / ml. 7.8 ug / mU 15.6 ug / ml, 31.2 ug / ml, 72.1 ug / ml) were added to human non-small cell lung cancer Nsclc. ⁇ After 6 hours, the sample concentration of 3.1 ug / ml can make the cells swell and rupture. As shown in Figure ⁇ 5, staining with Hoeches 55 5S shows that the cells are not apoptotic, and most of the cells are necrotic.
  • the tumor suppression test of the recombinant protein was performed according to conventional methods; the animals were NIH mice, provided by the Experimental Animal Center of the First Military Medical University (2000A037); the administration method was intraperitoneal injection; the negative control was physiological saline, and the positive control was cyclophosphamide; The test was completed by Guangdong Occupational Health Inspection Center; the test results are shown in Table 1-2.
  • Intraperitoneal injection of purified recombinant protein into NIH mice has an inhibitory effect on solid tumors and S-180 solid tumors inoculated in mice.
  • the inhibitory rate on solid tumors of liver cancer is 39.1% and the effective concentration is 0.6mg / kg ;
  • the tumor inhibition rate of S-180 solid tumors is 26.9%, and the effective concentration is o
  • the operation method is according to Adeno-X TM Expression System User of Clontech
  • PCR amplification, genomic cloning, plasmid extraction, etc. are performed according to conventional methods.
  • the PCk product was cloned into the shuttle plasmid pShuttle to construct the pShuttle-Src I plasmid;
  • pShuttle-Src I was digested with PI-SceI / 1-CeuI and ligated with an adenovirus vector to construct a recombinant adenovirus Adeno-Src I;
  • Recombinant adenovirus Adeno-Src I was digested by Pad, as shown in Figure 18, and then transfected into human embryonic kidney HEK293 cells, which were packaged into virus particles in HEK293 cells.
  • the extracted recombinant adenovirus can be used for in vitro cell and in vivo tests. Products are available Clinical use.
  • the detailed construction process of recombinant adenovirus is shown in Figure 16. Enzymatic digestion and sequencing analysis showed that the cloned gene was the target gene, as shown in Figure 17.

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Description

一种海葵细胞毒素基因及其表达、 应用
(一） 技术领域
本发明涉及一种新基因， 特别是一种海葵细胞毒素新基因。 本发明 还涉及上述基因的表达及其编码的蛋白在制备治疗肿瘤疾病药物中的应 用。
(二） 背景技术
海葵 （ anthopleura ) 属于腔肠动物门 （ soelenterata ) 的珊瑚纲 i ant ozod ， 是海洋中较原始的动物类。 在长期的生物进化过程中， 为 了抵御敌害及捕获食物， 海葵触手的刺丝囊能够分泌多种海葵毒素， 基 本上是多肽类毒素， 对人和动物有多种生理活性作用。 根据海葵毒素的 生理功能将其分为 3 类： 海葵神经毒素、 海葵溶细胞素及海葵钾离子通 道抑制剂。
具有细胞毒作用的海洋生物毒素已作为筛选抗癌及抗病毒制剂的来 源之一。 在海洋生物中发现多种多肽溶细胞毒素。 这些海洋生物包括： 海葵、 水母、 海藻， 海胆、 海兔、 海绵等。 海葵溶细胞素可以破坏细胞 的结构， 主要是作用于细胞膜， 与细胞膜的脂类或蛋白质结合， 使之穿 孔溶解。 海葵溶细胞素是一类碱性蛋白， 分子量约 20kDa， 含有 30多个 强碱性氨基酸， 缺乏半胱氨酸， 具有溶细胞性、 心脏毒性及其他活性。 其二级结构含有 α螺旋、 β折叠、 β转角和随机卷曲结构； 与膜结合后 α螺旋、 β折叠则有所增加， 随机卷曲结构则减少。 海葵溶细胞素至少 有两个区域直接参与膜脂 （磷脂酰胆碱、 鞘氨醇、 神经节苷脂） 或膜蛋 白的结合： Ν末端的双亲 α螺旋 （氨基酸残基 13-20 ) 和富含色氨酸区域 (氨 残基 105-120 ) ； 双亲 α螺旋插入细胞膜， 而富含色氨酸区域的 亲永 ES¾皮向— 伸展在细胞膜表面， 精氨酸、 苏氨酸及附 近的氨基酸能与膜脂的极性端作用， 这种和膜脂的作用方式与细菌类溶 细胞素不同 （细菌类溶细胞素的 α螺旋是与细胞膜中的胆固醇结合， 形 成大的孔洞， 溶解细胞膜） ； 海葵溶细胞素可作为真核生物溶细胞素的 模型。 海葵溶细胞素往往形成寡聚体起作用， 其作用方式可描述为： 溶 于水的海葵溶细胞素一与细胞膜或膜受体结合一插入细胞膜一形成寡聚 体一形成膜孔道一细胞裂解。 例如： 从海葵 Stichodactyla helianthus^ 分离的一种溶细胞素 Sticholysinll, 形成四聚体， 插入细胞膜形成阳离子 通道， 破坏细胞。
海葵溶细胞素表现出多种生物学活性； 溶血活性、 细胞毒性、 心脏 剌激活性、 阻断钾离子通道等。 从海葵 C Heteractis magnificat 中分离的 HmT对人红细胞的半溶解浓度为 0.15ug/ml。 从海葵 (Actinia equine^ 分 离的 Equinatoxin ll对老鼠的半致死浓度为 35ug/kg， 致死原因是心肌缺 血； 实验表明， Equinatoxin ll可直接作用于心脏， 毒性与毒素的浓度相 关， 引起心室压降低， 使血液交换减慢的域值为 0.1-lnM。 Stichlysin l、 Stichlysin II、 Equinatoxin II对病原体 G¾ i¾z (—种原生动物） 有杀伤作 用， LC50分别为 0.5nM、 1.6nM、 0.8nM。在 Ca++存在时， Equinatoxin ll
( lOOnM) 可明显使神经细胞瘤 NG108-15膨大破裂。 Cytolysin III可杀 伤体外培养的 Ehrlich ascitic肿瘤细胞， 对接种于小鼠的该肿瘤也有一定 的抑制作用。 对海葵新的细胞毒素的进一步的研究， 有望获得某些具有 心血管作用的药物或抗癌及抗病毒制剂。
海葵溶细胞素可以在 E.Coli 中进行融合表达， 重组蛋白的溶细胞活 性和细胞毒活性与天然蛋白活性相当， 但在 N末端加上额外氨基酸就降 低重组蛋白的活性，额外氨基酸越多重组蛋白活性越低。 N末端加上的额 外氨基酸干扰了 α螺旋与细胞膜的作用， 降低毒素的活性。
目前， 已经测定了一些海葵溶细胞毒素的二级结构， 但是这些毒素 的高级结构、 分子作用机制、 膜插入和寡聚体的形成的具体细节还不清 楚。 不过， 最近得到了高分辨率的毒素蛋白晶体结构 （包括水溶状态、 膜结合状态和膜孔形成状态） ， 对阐明海葵溶细胞毒素的高级结构、 分 子作用机制、 膜插入和寡聚体的形成是有帮助的。
(三） 发明内容
本发明的目的在于提供一种新的海葵细胞毒素基因 Src l。
本发明的另一目的在于提供上述新基因的表达。
本发明的另一目的在于提供上述新基因在制备预防和治疗肿瘤药物 中的应用。 ―
本发明所选择的海葵为玫瑰红绿海葵0¾^^ rosea),采自广西壮族 自治区北海市涠洲岛附近海域。
本发明构建了玫瑰红绿海葵毒腺 cDNA表达文库： 首先分离海葵触 手， 提取总 RNA， 然后按 Clontech 公司 SMART™ cDNA Library Construction Kit说明书的操作进行， 获得双链 cDNA， 最后将双链 cDNA 连接到改造的质粒载体 pcDNA3.0上并转化 E.coli, 从而构建成玫瑰红绿 海葵毒腺的 cDNA表达文库。
本发明通过对玫瑰红绿海葵毒腺的 cDNA文库克隆的序列测定， 从 中得到了一个编码玫瑰红绿海葵溶细胞素的 cDNA克隆， 编号为 Src I。 这个 cDNA序列编码 216个氨基酸的毒素前体蛋白， 包括 19个氨基酸的 信号肽、 19个氨基酸的 propart motif 和 178个氨基酸的成熟蛋白， 成熟 蛋白的等电点为 4.8， 分子量为 19， 500道尔顿， 是一种酸性蛋白， 这是 首次报道的酸性海葵溶细胞素。 成熟蛋白的 N末端具有海葵溶细胞素的 典型特征， 即具有双亲的 α螺旋。
本发明通过设计了一对特异引物， 将编码玫瑰红绿海葵溶细胞素成 熟蛋白的核苷酸序列用 PCR方法从 pcDNA3.0载体上扩增出来， 克隆到 原核表达载体 PBV220上， 构建成表达质粒 pBV220-Src I并将其转化大 肠杆菌 DH5 (i。 经过对培养时间、 培养温度、 诱导时间等条件的摸索和 优化， 重组蛋白的表达量占菌体总蛋白的 15%以上， 并且基本上处于不 溶的包涵体状态。
上述的引物根据 Src l基因编码的成熟蛋白两端序列和原核表达载体 pBV220的多酶切位点合成， 上游引物含有 EcoR I酶切序列 （GAATTC)和起 始密码子（ ATG)，下游引物含有 BamH I酶切序列（ GGATCC )和终止密码子（ TTA)， 序列如下：
上游引物， 5， G GAATTC ATG ATC TCG GGT GGT ACT GTT ATT 3 '
EcoR I酶切序列 起始密码子
下游引物， 5， TA GGATCC TTA TGG CCA GAC GAC TTC AAT C 3 '
BamH .1酶切序列 终止密码子
本发明还摸索和优化了重组 Src I蛋白的纯化条件， 通过包涵体的洗 涤、 变性、 复性和离子交换层析， 可得到纯度达 98%以上的重组 Src I蛋 白。
本发明获得的重组海葵细胞毒素具有生物活性。
本发明获得的重组 Src I蛋白对体外培养的肝癌细胞 BEL-7401、胃癌 细胞 BGC-823、 肺癌细胞 Nsclc有明显的作用， IC5Q分别为 31.2ug/ml、 3.1ug/mK 3.1ug/mL
¾明 重组 Src I蛋白对 NIH小鼠进行腹腔注射，对接种于小 鼠的肝癌实体瘤和 δ-180实体瘤有明显的抑制作用，抑瘤率分别为 39.5%、 26.5%, 使用浓度分别为 0.6mg/kg、 1.2mg/kg。
本发明构建了 Src I 海葵细胞毒素成熟蛋白编码序列的表达质粒 pBV220-Src I, 由该表达质粒载体经 EcoRI I /BamH I双酶切， 可得到 534bp的片段， 即为玫瑰红绿海葵细胞毒素 Src I成熟肽编码序列。
本发明的表达质粒载体的复制方法： 参照 Sambrook ( Sambrook, et al.1989, Molecular doing. Cold Spring Harbor Labroratory Press. USA) 方 法， 按 CaCl2法在 E.Coli. DH5a或 BL21 (DE3) 菌株中转化质粒， 用含 氨苄青霉素 （10(^g/mL) 的 LB培养基转化细菌， 碱法提取质粒。
本发明通过设计另一对特异引物， 将编码玫瑰红绿海葵溶细胞素的 成熟蛋白核苷酸序列用 PCR方法从 pcDNA3.0载体上扩增出来， 克隆到 穿梭质粒 pshuttle 上， 构建重组质粒 pshuttle-Src I， 该质粒通过 PI-SceI/1-CeuI双酶切后与真核表达载体 Adeno-X相连， 构建成重组腺病 毒 Adeno-Src I质粒， Adeno-Src I的 DNA通过 Pad酶切后在人胚肾细胞 HEK293包装成病毒粒子。重组腺病毒可在真核细胞中表达， 用于相关疾 病的治疗。
上述引物根据 Src l基因编码的成熟蛋白两端序列和穿梭质粒 pShuttle 的多酶切位点合成， 上游引物含有 Apa l酶切序列（GGGCCC )和起始密码子
( ATG) ， 下游引物含有 Notl酶切序列 （GCGGCCGC ) 和终止密码子 （TTA) ， 序 列如下：
上游引物， 5， GG GGGCCC A1GATCTCGGGTGGTACTGTTATTG 3 '
Apa l酶切序列 起始密码子
下游引物， 5' GTCAT GCGGCCG C TTATGGCCAGACGACTTCAATC 3 '
Notl酶切序列 终止密码子
(四） 附图说明
图 1为玫瑰红绿海葵触手总 RNA电泳结果；
图 2为玫瑰红绿海葵触手双链 cDNA电泳结果；
图 3为玫瑰红绿海葵触手 cDNA文库总质粒、 Sfil酶切和 PCR检测电泳 结果；
图 4为玫瑰红绿海葵触手 cDNA文库重组子的 PCR检测电泳结果； 图 5为含基因 Src I的重组质粒 pBV220-Src I表达质粒构建；
图 6为玫瑰红绿海葵 Src I基因的 PCR产物电泳结果；
图 7为含基因 Src l的 pBV220-Src I表达质粒酶切和 PCR鉴定电泳结果；
§ 8 组 Src I 蛋白的表达、 包涵体变性、 复性和离子交换层析的
SDS-PAGE;
图 9为童组 Src l蛋白等电点图；
图 10为重组 Src l蛋白的溶血曲线图；
图 11为重组 Src l蛋白作用人肝癌细胞 BEL-7402的光镜照片；
图 12为重组 Src l蛋白作用人肝癌细胞 BEL-7402的荧光照片；
图 13为重组 Src l蛋白作用人胃癌细胞 BGC-823的光镜照片；
图 14为重组 Src l蛋白作用人胃癌细胞 BGC-823的荧光照片；
图 15为重组 Src l蛋白作用人肺癌细胞 Nsclc的结果；
图 16为 Adeno-Src I表达系统的构建；
图 17为重组腺病毒 Adeno-Src I的 PI-SceI/1-Ceui酶切鉴定；
图 18为重组腺病毒 Adeno-Src I的 Pad酶切。 图 1中， 1 : R A ladder (Promega公司）； 2: 玫瑰红绿海葵触手总 RNA。 图 2中， 1 : l kb DNA ladder (Promega公司） ； 2: 玫瑰红绿海葵触手 dsDNA。
图 3中， 1 : 1 kb DNA ladder (Promega公司） ； 2: 文库总质粒的 PCR 结果； 3: 文库总质粒； 4: 文库总质粒的 Sfil酶切结果。
图 4中， M: l kb DNA ladder (Promega公司）； 1-21 : 文库重组子的 PCR 检测。
图 6中， 1 : l kb DNA marker (NEB公司） ； 2: 玫瑰红绿海葵 Src l基因 的 PCR产物。
图 7中， 1： 1 kb DNA markerC NEB公司）； 2 : pBV220-Src I的 EcoRI/BamHI 双酶切； 3: pBV220-Src I的 PCR鉴定。
图 8中， 1 : marker; 2： 未诱导的总菌体； 3 : 42°C诱导的总菌体； 4: 超 声上清； 5: 超声沉淀； 6: 包涵体经洗涤的上清； 7: 复性后的 Src l经离 子交换层析的 0.3 M Nacl洗脱峰； 8: 复性后的 Src I经离子交换层析的 0.8 M Nac 冼脱峰。
图 9中， 1: marker; 2： Src l重组蛋白。
图 17中， 1： Adeno-Src I的 PI-SceI/1-CeuI酶切； 2: 1 kb DNA marker (NEB 公司） 。
图 18中， 1: Adeno-Src I的 Pad酶切； 2: 1 kb DNA marker (NEB公司）。
(五)具体实施方式
下面结合附图对本发明作进一步说明， 将有助于本领域的普通技术 人员理解本发明， 但不以任何形式限制本发明。
实施例一 玫瑰红绿海葵毒腺 cDNA文库的构建
玫鸨 ¾： §§¾腺总 RNA昀提取参考 现代分子生物学实验技术》 的一步决 ^^ 法进行； cDNA 的合成按 Clontech公司 SMART™ cDNA Library Construction Kit说明书操作。
采用异硫氰酸胍一步法提取的毒腺总 RNA经 1%甲醛变性胶电泳检 测可见清晰的 28S、 18S两条 rRNA条带， 如图 1 , 表明总 RNA完整性良 好。采用 SMARTTMcDNA Library Construction ist合成的 cDNA在 1 %的 琼脂糖凝胶上电泳，结果呈现均匀的 smear,如图 2，大小在 200bp到 8kb 的范围内，主要是在 3kb以下的区域，在 500bp附近更为集中，表明 cDNA 的完整性良好。将 cDNA插入质粒载体 pcDNA3.0上构建成 cDNA表达文 库， 文库克隆数为 2.4xl05。 提取总文库质粒进行酶切和 PCR分析， 如图 3， 结果表明 cDNA插入片段大小落在 300bp-8kb的范围内， 挑取 172个 克隆提取质粒， 酶切和 PCR鉴定表明超过 95%的克隆为重组子， 如图 4， 表明该 cDNA表达文库具有较好的质量。
实施例二 玫瑰红绿海葵触手 cDNA文库的克隆、 序列测定和分析 挑选玫瑰红绿海葵触手 cDNA文库的克隆， 按 Omega Biotek Plasmid Miniprep Kit的方法提取质粒 DNA。 随机测定了 150个 cDNA序列。 使 用 ABI PRISM 377 DNA Analyzer (Applied Biosystems), 采用 T7和 SP6 通用引物为测序引物， 进行正反向序列测定， 对 cDNA片段超过 lOOObp 的序列， 根据已测得的序列设计引物， 继续测通 cDNA。 测序工作由广州 市中山大学生命科学学院中心实验室完成。所得序列经 BlastX初步分析。 结果表明玫瑰红绿海葵毒腺 cDNA文库包含的基因多种多样， 其中毒素 基因的丰度较高， 有三个编码海葵神经毒素的 cDNA序列和一个编码海 葵溶细胞素的 cDNA序列， 其中编码溶细胞素的 cDNA编号为 Src I， 如 序列表所示， 它编码 216个氨基酸的蛋白， 其中包括 19个氨基酸残基的 信号肽、 19个氨基酸残基的 propart motif和 178个氨基酸残基的成熟蛋 白， 成熟蛋白的 N末端具有海葵溶细胞素特征性 α螺旋。 成熟蛋白的分 子量为 19， 500道尔顿， 蛋白的等电点为 4.8， 是酸性溶细胞素。 目前， 所有报道的海葵溶细胞素为强碱性蛋白， 等电点在 9.0以上， 在海葵中发 现酸性溶细胞素尚属首次。
Src I的核苷酸序列及推测的氨基酸序列见序列表。 以玫瑰红绿海葵 触手总 R A为出发材料，根据已获得的溶细胞素 cDNA序列设计 3'端引 物， 以 SMART™ cDNA Library Construction Kit的文库构建引物为 5'端 引物， 进行 RT-PCR, 在 800bp处出现预期的特异扩增带， 回收此带。 将 回收的 PCR产物连接到 pGEM-T Easy Vector, 转化 DH5a大肠杆菌， 挑 选阳性克隆测序。 经测序分析证实， 是我们所希望得到的目的基因， 测 定的 8个享隆， 为同一 cDNA序列。
实^^三 ί¾玫瑰红绿海^溶细胞素表达质粒的构建
根据 Src I基因编码的成熟蛋白两端序列和原核表达载体 pBV220的 多酶切位点， 合成一对引物， 序列如下：
上游引物， 5' G GAATTC ATG ATC TCG GGT GGT ACT GTT ATT 3'
单下划线部分为 EcoR I酶切序列， 双下划线为起始密码子 下游引物， 5' TA GGATCC TTA TGG CCA GAC GAC TTC AAT C 3'
单下划线部分为 BamH I酶切序列， 双下划线为终止密码子
PCR扩增、 基因克隆皆按常规方法进行。 PCR产物约 600bp， 编码 178个氨基酸残基，如图 6。将目的基因克隆到原核表达载体 pBV220上， 构建成表达质粒 pBV220-Src I。 构建过程见图 5。 经酶切鉴定和测序分析 表明克隆的基因为目的基因如图 7。 原核表达载体？8¥225)含？？！^启动子， 同时含有 cl调控基因， 带有 起始密码子的外源基因可 t入启动子下游的多酶切位点， 表达非融合蛋 白， 产品可供临床使用。
实施例四 重组玫瑰红绿海葵溶细胞素蛋白的表达
将表达质粒 pBV220-Src I转化大肠杆菌 DH5a。含目的基因的工程菌 在 30°C生长到 OD6( )=0.5时， 立即在 42°C诱导 4小时。 收集菌体， 经 SDS-PAGE 电泳分析表明： 基因工程菌经诱导后有明显的特异表达产物 带， 分子量与预测值 20kD相符， 如图 8。 薄层扫描分析表明： 在此条件 下重组蛋白的表达量占菌体总蛋白的 17%以上， 基本上处于不可溶的包 涵体状态。
实施例五 重组玫瑰红绿海葵溶细胞素蛋白的纯化
诱导后的菌体通过超声破菌， 离心取沉淀， 沉淀通过不同的缓冲溶 液洗涤， 去除杂蛋白， 洗涤后的包涵体纯度为 80%。 在包涵体洗涤中用 到的溶液有： 超声 buffer (10mMTris-Hcl， pH7.0, ImMEDTA) 、 buffer KO.IM Tris-Hcl, pH8.0, lOmM EDTA, 0.5% Triton X 100). buffer 2 (50mM PB, 0.5MNacl， 3M尿素)和 buffer 3 (0.1 M Tris-Hcl， pH8.5, lOmMEDTA, 3M尿素） 0
洗涤后的包涵体在变性液（8M尿素， lOmM Tris-Hcl， pH8.0, lOmM DTT) 中溶解。 将变性蛋白在复性液（3M尿素， 20mMTris-Hcl， pH8.0, O.lmM氧化型谷胱甘肽， 0.9mM还原型谷胱甘肽） 中通过透析复性。
复性蛋白通过离子交换层析和疏水层析可获得纯度在 99%以上的重 组蛋白， 如图 8。
实施例六 重组玫瑰红绿海葵溶细胞素蛋白等电点的测定
通过平板电泳测定重组蛋白的等电点， 等电聚焦聚丙烯酰胺凝胶电 泳按常规方法进行。 ^定的结果为： pH 4.81， 如图 9。 与预测的等电点 基本相符。
实施例七 重组海葵溶细胞素蛋白的溶血活性鉴定
取人血 5ml， 加入 3.8%的柠檬酸钠 （柠檬酸钠： 血液为 1: 9) ， 室 温， 3000rpm， 离心 5分钟。 去上清， 用 Hank's液洗涤红细胞， 直到上 清清亮 （约需用 Hank's洗涤 2-3次） ， 用 Hank's将红细胞配成 0.5%或 1% (v/v)的细胞悬液， 将样品加入到 2ml的红细胞悬液中， 37°C温育 20 分钟， 室温， 3000rpm， 离心 5 分钟， 取上清， 测定 OD54o。 以 Hank's 为阴性对照， 用终浓度 O.lmg/ml皂苷使红细胞 100%溶血。 每个浓度的 样品做三个平行实验。 结果如图 10。
实施例八 重组海葵溶细胞素蛋白对体外培养的胂瘤细胞的作用 将肿瘤细胞按 1.0xl06cell/ml接种于 24孔板， 当细胞生长到 60%-70 %汇合度时加入样品， 每个样品浓度做 4个平行实验， 以 Hank's作为阴 性对照。
细胞凋亡的荧光检测 （Hoeches 55258染色法） ： 用 4 %的多聚甲醛 在 4°C将细胞固定 20分钟， 用 Hank's洗涤 2次， 加入 Hoeches 55258染 液， 终浓度为 5-10ug/ml， 在室温染色 10分钟， 用 Hank's洗涤 2次， 在 荧光显微镜下观察。
将不同浓度的重组蛋白（0.31 ug/mU 3.1 ug/mK 7.8 ug/mK 15.6 ug/ml、 31.2 ug/mK 72.1 ug/ml)加入到人肝癌细胞 BEL-7402中， 在 0-36小时内 观察， 0.31-15.6 ug/ml浓度的样品对细胞没有影响， 在 31.2 ug/ml的浓度 时能引起细胞形态明显的改变， 如图 11， 细胞明显拉长， 此现象在加入 蛋白 8小时后就比较明显， 一直维持到 36小时 （36小时后没有观察） ， 光镜观察没有坏死现象，通过 Hoeches 55258染色法染色没有观察到细胞 凋亡的现象， 如图 12。 从重组蛋白的溶血现象看， 重组蛋白应能与细胞 膜作用， 从重组蛋白作用肝癌细胞 BEL-7402的现象来看， 重组蛋白不仅 与细胞膜作用， 还可能与膜上的蛋白作用， 引起信号的改变， 导致微管、 微丝等的变化， 引起细胞形态的改变。
将不同浓度的重组蛋白（0.31 ug/ml、3.1 ug/ml、 7.8 ug/ml、 15.6 ug/mK 31.2 ug/mK 72.1 ug/ml) 加入到人低分化胃癌细胞 BGC-823中， 加入样 品后进行观察， 6小时后， 3.1 ug/ml的样品浓度就可使细胞变圆变小， 如 图 13， 经荧光染色， 可见细胞核明显变小， 胞质仅剩包裹在核外的一薄 层， 如图 14。
将不同浓度的重组蛋白（0.31 ug/mU 3.1 ug/ml. 7.8 ug/mU 15.6 ug/ml、 31.2 ug/ml, 72.1 ug/ml) 加入到人非小细胞肺癌 Nsclc中， 加入样品后进 行观 ^， 6小时后， 3.1 ug/ml的样品浓度就可使细胞膨大、 破裂， 如图 Ϊ5, 经 Hoeches 55 5S染色可知， 细胞并非凋亡， 绝大多数细胞坏死。
实施例九 重组海葵溶细胞素蛋白对小鼠肝癌 （Heps) 和肉瘤 S-180 的抗肿瘤试验
重组蛋白的抑瘤试验按常规方法进行； 动物为 NIH小鼠， 由第一军 医大学试验动物中心提供 （2000A037) ； 给药方法为腹腔注射； 阴性对 照为生理盐水， 阳性对照为环磷酰胺； 试验由广东省职业卫生检验中心 完成； 试验结果见表 1-2。
纯化的重组蛋白对 NIH小鼠进行腹腔注射， 对接种于小鼠的肝癌实 体瘤和 S-180实体瘤均有抑制作用.， 对肝癌实体瘤的抑瘤率为 39.1%， 作 用浓度为 0.6mg/kg; 对 S-180 实体瘤的抑瘤率为 26.9%， 作用浓度为 o
1.16mg/kg。
重组 Src I蛋白对于小鼠肝癌实体瘤 (Heps)的抑制作用
剂量组 动物数 体重 瘤重 抑瘤率 Ρ值
(mg/ml) 开始结束 开始 结束 (Mean + Sd) (%)
0 9 9 18.2 22.4 1.89±0·65 ― ―
0.065 9 9 18.0 21.6 1.15 + 0.56 39.15 〈0， 05
Cp, 20 10 10 18.5 19.4 0.72±0.27 61.90 〈0.01
Ρ值： 与对照组比较， 方差分析。 重组 Srcl蛋白对小鼠肉瘤 （S-180) 的抑制作用
剂量组 动物数 增加体重 瘤重 抑瘤率 胸腺指数 脾指数 肝指数
(mg/ml) 开始 结柬 (g) (g) ( ) (1/1000) (1/1000) (1/100)
0 10 10 2.58±2· 24 1.53土 0.44 ― 3.28 + 0.85 4.93±1.62 5.35+0.62
Cp, 20 10 10 1.23土 1.29 0.64士 0.25 58.33" 2.16 + 0.40AA 4.07±1.02 5.80土 0.56
10 10 3.26土 1.07 1.12土 0.58 26.87* 3.41±0.78 6.54±1.76A 5.54±0.55
0.031 10 10 3.49±1.14 1.19±0.47 15.56 3.29±0.60 5.79 + 0.78 5.26±0.52
P值： 与对照组比较， 方差分析。 AP<0.05， "P<0.01
实施例十 重组腺病毒 Adeno-Src I的构建
操作方法按 Clontech公司的 Adeno-XTM Expression System User
Manual进行。 根据 Src I基因编码的成熟蛋白两端序列和穿梭质粒 pShuttle 的多酶切位点， 合成一对引物， 序列如下：
上游引物， 5， GG GGGCCC AlfiATCTCGGGTGGTACTGTTATTG 3'
单下划线部分为 Apal酶切序列， 双下划线为起始密码子
下游引物， 5' GTCAT GCGGCCG C HATGGCCAGACGACTTCAATC 3，
单下划线部分为 Notl酶切序列， 双下划线为终止密码子
PCR扩增、 基园克隆、 质粒提取等按常规方法进行。 将 PCk产物克 隆到穿梭质粒 pShuttle上，构建成 pShuttle-Src I质粒； pShuttle-Src I通过 PI-SceI/1-CeuI双酶切后与腺病毒载体连接，构建成重组腺病毒 Adeno-Src I；重组腺病毒 Adeno-Src I经过 Pad酶切，如图 18,后转染人胚肾 HEK293 细胞， 在 HEK293 细胞中包装成病毒粒子， 提取的重组腺病毒可用于体 外细胞和体内试验， 产品可供临床使用。 重组腺病毒的构建详细过程见. 图 16。 经酶切鉴定和测序分析表明克隆的基因为目的基因， 如图 17。

Claims

权 利 要 求
1、一种海葵细胞毒素基因 Src I， 来源于玫瑰红绿海葵毒腺 cDNA文 库， 核苷酸序列如序列表所示。
2、 如权利要求 1所述的基因， 其特征在于编码 216个氨基酸的毒素 前体蛋白， 包括 19个氨基酸的信号肽、 19个氨基酸的 propart motif 和 178个氨基酸的成熟蛋白， 成熟蛋白的等电点为 4.8， 分子量为 19， 500 遣尔顿， 是一种酸性蛋白， 成熟蛋白的 N末端具有海葵溶细胞素的典型 特征， 即具有双亲的 α螺旋， 氨基酸序列如序列表所示。
3、 一种重组原核表达载体， 其特征在于含有权利要求 1所述的基因 Src I， 是将 Src I基因克隆到原核表达载体 pBV220上构建成的非融合表 达载体 pBV220-Src I。
4、如权利要求 3所述的重组原核表达载体，其特征在于 Src I基因在 大肠杆菌中以包涵体的形式表达。
6、 一种重组真核表达载体， 其特征在于含有如权利要求 1所述的基 因 Src I， 是可在哺乳动物细胞表达的 Adeno-Src I。
7、 一种用作引物的 DNA片段， 其特征在于由权利要求 1所述核苷 酸序列的一部分序列组成， 上游引物含有 EcoR I酶切序列 （GAATTC) 和起始密码子 （ATG) ， 下游引物含有 BamH I酶切序列 （GGATCC)和 终止密码子 （TTA) , 序列如下：
上游引物， 5， G GAATTC ATG ATC TCG GGT GGT ACT GTT ATT 3 '
EcoR I酶切序列 起始密码子
下游引物， 5， TA GGATCC TTA TGG CCA GAC GAC TTC AAT C 3，
BamH I酶切序列 终止密码子
8、 一种用作引物的 DNA片段， 其特征在于由权利要求 1所述核苷酸 序列的一部分序列组成， 上^引物含有 Apa l酶切 ^列 （GGGCCC) 和¾ 始密码子 （ATG) ， 下游引物含有 Notl酶切序列 （GCGGCCGC) 和终止 密码子 （TTA) ， 序列如下：
上游引物， 5 ' GG GGGCCC A1QATCTCGGGTGGTACTGTTATTG 3，
Apa l酶切序列 起始密码子
下游引物， 5， GTCAT GCGGCCG C imTGGCCAGACGACTTCAATC 3 '
Notl酶切序列 终止密码子
9、 含有如权利要求 2所述蛋白在制备预防和治疗肿瘤的药物中的应 用。
PCT/CN2003/000538 2002-09-03 2003-07-07 Gene de cytotoxine de cnidaire et son expression et son application Ceased WO2004022751A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2789626A3 (en) * 2008-09-24 2015-06-17 Tel Hashomer Medical Research Infrastructure and Services Ltd. Peptides and compositions for prevention of cell adhesion and methods of using same

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* Cited by examiner, † Cited by third party
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998023639A2 (en) * 1996-11-27 1998-06-04 University Of Florida ShK TOXIN COMPOSITIONS AND METHODS OF USE
WO1999013895A1 (en) * 1997-09-17 1999-03-25 University Of Florida Polypeptide compositions that inhibit potassium channel activity and uses therefor

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998023639A2 (en) * 1996-11-27 1998-06-04 University Of Florida ShK TOXIN COMPOSITIONS AND METHODS OF USE
WO1999013895A1 (en) * 1997-09-17 1999-03-25 University Of Florida Polypeptide compositions that inhibit potassium channel activity and uses therefor

Cited By (4)

* Cited by examiner, † Cited by third party
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EP2789626A3 (en) * 2008-09-24 2015-06-17 Tel Hashomer Medical Research Infrastructure and Services Ltd. Peptides and compositions for prevention of cell adhesion and methods of using same
US9777050B2 (en) 2008-09-24 2017-10-03 Tel Hashomer Medical Research, Infrastructure And Services Ltd. Peptides and compositions for prevention of cell adhesion and methods of using same
EP3670524A1 (en) * 2008-09-24 2020-06-24 Tel HaShomer Medical Research Infrastructure and Services Ltd. Peptides and compositions for prevention of cell adhesion and methods of using same
US11001612B2 (en) 2008-09-24 2021-05-11 Amir Zlotkin Peptides and compositions for prevention of cell adhesion and methods of using same

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