CN1162541C - A kind of sea anemone cytotoxin gene, its encoded protein and application - Google Patents

Abstract

The present invention discloses a new sea anemone cytotoxin gene SrcI, which encodes toxin precursor proteins with 216 amino acids, signal peptide comprising 19 amino acids, propartmotif comprising 19 amino acids and mature proteins comprising 178 amino acids, wherein the molecular weight of the mature proteins is 19, 500 dalton, and the isoelectric point is 4.8. The gene of the present invention is acid sea anemone cytolysin. The present invention also discloses expression and an application of the new gene, which designs two pairs of primers which respectively constructs a recombinant prokaryotic expression carrier pBV220-SrcI and a recombinant eukaryotic expression carrier Adeno-SrcI. The recombinant sea anemone cytotoxin of the present invention has the anti-tumor activity. The present invention establishes a colibacillus expression system and an adenovirus expression system of rose and green sea anemone cytolysin, and can be used for developing new anti-tumor medicines.

Description

A kind of sea anemone cytotoxin gene, its encoded protein and application

(1) Technical field

The invention relates to a new gene of sea anemone cytotoxin. The present invention also relates to the expression of the above gene and the application of the encoded protein in the preparation of medicines for treating tumor diseases.

(2) Background technology

Sea anemones (anthopleura) belong to the coral class (anthozoa) of the coelenterate phylum (soelenterata), and are relatively primitive animals in the ocean. In the long-term biological evolution process, in order to resist predators and capture food, the nematocysts of sea anemone tentacles can secrete a variety of sea anemone toxins, basically polypeptide toxins, which have various physiological activities on humans and animals. Sea anemone toxins are divided into three categories according to their physiological functions: sea anemone neurotoxins, sea anemone cytolysins, and sea anemone potassium channel inhibitors.

Marine biotoxins with cytotoxicity have been used as one of the sources for screening anticancer and antiviral agents. A variety of polypeptide cytolytic toxins are found in marine organisms. These marine organisms include: sea anemones, jellyfish, seaweed, sea urchins, nudibranchs, sponges, etc. Sea anemone cytolysin can destroy the structure of the cell, mainly acting on the cell membrane, combining with the lipid or protein of the cell membrane to perforate and dissolve it. Sea anemone cytolysin is a type of basic protein with a molecular weight of about 20kDa, containing more than 30 strong basic amino acids, lacking cysteine, and having cytolytic, cardiotoxic and other activities. Its secondary structure contains α-helix, β-sheet, β-turn and random coil structure; after binding to the membrane, the α-helix and β-sheet increase, while the random coil structure decreases. Sea anemone cytolysins have at least two regions directly involved in the binding of membrane lipids (phosphatidylcholine, sphingosine, gangliosides) or membrane proteins: the N-terminal amphipathic alpha helix (amino acid residues 13-20) and Tryptophan-rich region (amino acid residues 105-120); the amphipathic α-helix inserts into the cell membrane, while the hydrophilic region of the tryptophan-rich region is an inverted β-sheet structure, stretching on the surface of the cell membrane, arginine, threonine Acids and nearby amino acids can interact with the polar ends of membrane lipids. The mode of action of membrane lipids is different from that of bacterial cytolysins (the α-helix of bacterial cytolysins is combined with cholesterol in the cell membrane to form a large pores, dissolve cell membranes); sea anemone cytolysin can be used as a model for eukaryotic cytolysin. Sea anemone cytolysin often forms oligomers to function, and its mode of action can be described as: sea anemone cytolysin soluble in water → binds to cell membrane or membrane receptors → inserts into cell membrane → forms oligomers → forms membrane channels → Cell lysis. For example: Sticholysin II, a cytolysin isolated from sea anemone (Stichodactyla helianthus), forms a tetramer, inserts into the cell membrane to form a cation channel, and destroys the cell.

Sea anemone cytolysins exhibit a variety of biological activities; hemolytic activity, cytotoxicity, cardiostimulatory activity, blocking potassium ion channels, etc. The half-dissolving concentration of HmT isolated from sea anemone (Heteractis magnifica) to human erythrocytes is 0.15ug/ml. The semi-lethal concentration of Equinatoxin II isolated from sea anemone (Actinia equina) to mice is 35ug/kg, and the cause of death is myocardial ischemia; experiments have shown that Equinatoxin II can directly act on the heart, and the toxicity is related to the concentration of the toxin, causing ventricular pressure Decrease, the threshold value for slowing blood exchange is 0.1-1nM. Stichlysin I, Stichlysin II, and Equinatoxin II have killing effects on the pathogen Giardia (a protozoan), with LC ₅₀ of 0.5nM, 1.6nM, and 0.8nM, respectively. In the presence of Ca++, Equinatoxin II (100nM) can significantly expand and rupture the neurocytoma NG108-15. Cytolysin III can kill Ehrlich ascitic tumor cells cultured in vitro, and also has a certain inhibitory effect on the tumor inoculated in mice. Further research on the new cytotoxins of sea anemones is expected to obtain some drugs with cardiovascular effects or anticancer and antiviral agents.

Sea anemone cytolysin can be fused and expressed in E.Coli. The cytolytic activity and cytotoxic activity of the recombinant protein are comparable to those of the natural protein, but adding additional amino acids to the N-terminus will reduce the activity of the recombinant protein. The more additional amino acids The lower the activity of the recombinant protein. The extra amino acid added to the N-terminus interferes with the interaction of the α-helix with the cell membrane and reduces the activity of the toxin.

Currently, the secondary structures of some sea anemone cytolytic toxins have been determined, but the specific details of the higher order structure, molecular mechanism of action, membrane insertion and oligomer formation of these toxins are still unclear. Recently, however, high-resolution crystal structures of the toxin protein (including water-soluble state, membrane-bound state, and membrane pore-forming state) have been obtained, which are helpful for elucidating the high-level structure, molecular mechanism of action, membrane insertion, and oligomer formation of sea anemone cytolytic toxin. Formation is helpful.

(3) Contents of the invention

The object of the present invention is to provide a new sea anemone cytotoxin gene Src I.

Another object of the present invention is to provide the expression of the above-mentioned novel gene.

Another object of the present invention is to provide the application of the above-mentioned novel gene in the preparation of drugs for preventing or treating tumors.

The sea anemone selected in the present invention is rose red and green sea anemone (Sagartia rosea), which is collected from the sea area near Weizhou Island, Beihai City, Guangxi Zhuang Autonomous Region.

The present invention constructs the cDNA expression library of rose red and green sea anemone venom glands: first, isolate the tentacles of the sea anemone, extract the total RNA, then follow the instructions of Clontech's SMART ^TM cDNA LibraryConstruction Kit to obtain double-stranded cDNA, and finally connect the double-stranded cDNA to The transformed plasmid vector pcDNA3.0 was transformed into E.coli, thereby constructing the cDNA expression library of the venom gland of the rose red and green sea anemone.

The present invention obtains a cDNA clone encoding the cytolysin of the rose red-green sea anemone through sequence determination of the cDNA library clone of the venom gland of the rose red-green sea anemone, numbered 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 isoelectric point of the mature protein is 4.8 and the molecular weight is 19,500 Daltons , is an acidic protein, which is the first reported acidic sea anemone cytolysin. The N-terminus of the mature protein has the typical features of sea anemone cytolysins, that is, an α-helix with two parents.

In the present invention, by designing a pair of specific primers, the nucleotide sequence encoding the mature protein of rose red and green sea anemone cytolysin is amplified from the pcDNA3.0 vector by PCR method, cloned into the prokaryotic expression vector pBV220, and constructed to express Plasmid pBV220-Src I was transformed into Escherichia coli DH5α. After exploring and optimizing the culture time, culture temperature, induction time and other conditions, the expression of the recombinant protein accounts for more than 15% of the total protein of the bacteria, and is basically in the state of insoluble inclusion bodies.

The above primers were synthesized according to the sequence at both ends of the mature protein encoded by the Src I gene and the multi-restriction site of the prokaryotic expression vector pBV220, the upstream primer contained the EcoR I restriction sequence (GAATTC) and the initiation codon (ATG), and the downstream primer contained BamH I digestion sequence (GGATCC) and stop codon (TTA), the sequence is as follows:

Upstream primer, 5'G GAATTC ATC TCG GGT GGT ACT GTT ATT 3'

        EcoR I Restriction Sequence Initiation Codon

TGG CCA GAC GAC TTC AAT C 3’Downstream primer, 5'TA GGATCC

TGG CCA GAC GAC TTC AAT C 3'

        BamH I Restriction Sequence Termination Codon

The present invention also explores and optimizes the purification conditions of the recombinant Src I protein, and the recombinant Src I protein with a purity of more than 98% can be obtained through inclusion body washing, denaturation, renaturation and ion exchange chromatography.

The recombinant sea anemone cytotoxin obtained by the invention has biological activity.

The recombinant Src I protein obtained by the present invention has obvious effects on liver cancer cell BEL-7401, gastric cancer cell BGC-823, and lung cancer cell Nsclc cultured in vitro, with _IC50 being 31.2ug/ml, 3.1ug/ml, and 3.1ug/ml, respectively .

The recombinant Src I protein obtained in the present invention is injected intraperitoneally into NIH mice, and has obvious inhibitory effect on liver cancer solid tumors and S-180 solid tumors inoculated in mice, and the tumor inhibition rates are 39.5% and 26.5% respectively. They are 0.6mg/kg and 1.2mg/kg respectively.

The present invention constructs the expression plasmid pBV220-Src I of the Src I sea anemone cytotoxin mature protein coding sequence, and the expression plasmid vector can be digested by EcoRI I/BamH I to obtain a 534bp fragment, which is the rose red and green sea anemone Cytotoxin Src I mature peptide coding sequence.

In the present invention, by designing another pair of specific primers, the nucleotide sequence of the mature protein encoding rose red and green sea anemone cytolysin is amplified from the pcDNA3.0 vector by PCR method, cloned into the shuttle plasmid pshuttle, and the recombinant plasmid is constructed pshuttle-Src I, the plasmid was digested by PI-Sce I/I-Ceu I and connected to the eukaryotic expression vector Adeno-X to construct a recombinant adenovirus Adeno-Src I plasmid, and the DNA of Adeno-Src I was passed through Pac After digesting with enzyme I, it was 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 according to the sequence at both ends of the mature protein encoded by the Src I gene and the multiple restriction sites of the shuttle plasmid pShuttle. The upstream primer contained the Apa I restriction sequence (GGGCCC) and the initiation codon (ATG), and the downstream primer contained the Not I Restriction sequence (GCGGCCGC) and stop codon (TTA), the sequence is as follows:

ATCTCGGGTGGTACTGTTATTG 3’Upstream primer, 5'GG GGGCCC

ATCTCGGGTGGTACTGTTATTG 3'

               Apa I Restriction Sequence = Initiation Codon

TGGCCAGACGACTTCAATC 3’Downstream primer, 5'GTCAT GCGGCCG C

TGGCCAGACGACTTCAATC 3'

        Not I Restriction Sequence Termination Codon

The replication method of the expression plasmid vector of the present invention: with reference to Sambrook (Sambrook, etal.1989, Molecular cloing.Cold Spring Harbor Laboratory Press.USA) method, transform in E.Coli.DH5α or BL21 (DE3) bacterial strain by _CaCl method For the plasmid, the bacteria were transformed with LB medium containing ampicillin (100 μg/mL), and the plasmid was extracted by alkaline method.

(4) Description of drawings

Figure 1 is the electrophoresis result of the total RNA of the tentacle of the rose red and green sea anemone;

Fig. 2 is the double-stranded cDNA electrophoresis result of the tentacle of rose red and green sea anemone;

Fig. 3 is the electrophoresis result of the total plasmid of the rose red and green sea anemone tentacles cDNA library, Sfi I digestion and PCR detection;

Fig. 4 is the PCR detection electrophoresis result of the cDNA library recombinant of rose red and green sea anemone tentacles;

Fig. 5 is the construction of the recombinant plasmid pBV220-Src I expression plasmid containing gene Src I;

Fig. 6 is the electrophoresis result of the PCR product of rose red and green sea anemone Src I gene;

Figure 7 is the pBV220-Src I expression plasmid digestion and PCR identification electrophoresis results containing gene Src I;

Figure 8 is the expression of recombinant Src I protein, inclusion body denaturation, refolding and SDS-PAGE of ion exchange chromatography;

Fig. 9 is the isoelectric point diagram of recombinant Src I protein;

Fig. 10 is the hemolysis curve figure of recombinant Src I protein;

Figure 11 is a light microscope photo of human liver cancer cell BEL-7402 acted on by recombinant Src I protein;

Fig. 12 is the fluorescent photo of recombinant Src I protein acting on human liver cancer cell BEL-7402;

Figure 13 is a light micrograph of human gastric cancer cell BGC-823 acted on by recombinant Src I protein;

Fig. 14 is the fluorescent photo of recombinant Src I protein acting on human gastric cancer cell BGC-823;

Figure 15 is the result of recombinant Src I protein acting on human lung cancer cell Nsclc;

Figure 16 is the construction of Adeno-Src I expression system;

Figure 17 is the PI-Sce I/I-Ceu I enzyme digestion identification of recombinant adenovirus Adeno-Src I;

Figure 18 shows the Pac I digestion of recombinant adenovirus Adeno-Src I.

In Fig. 1, 1: RNA ladder (Promega); 2: total RNA of tentacle of rose red and green sea anemone.

In Figure 2, 1: 1kb DNA ladder (Promega); 2: dsDNA of rose red and green sea anemone tentacles.

In Fig. 3, 1: 1kb DNA ladder (Promega); 2: PCR result of total library plasmid; 3: total library plasmid; 4: Sfi I digestion result of total library plasmid.

In Fig. 4, M: 1kb DNA ladder (Promega); 1-21: PCR detection of library recombinants.

In Fig. 6, 1: 1kb DNA marker (NEB company); 2: the PCR product of Src I gene of rose red and green sea anemone.

In Fig. 7, 1: 1kb DNA marker (NEB Company); 2: EcoR I/BamH I double digestion of pBV220-Src I; 3: PCR identification of pBV220-Src I.

In Figure 8, 1: marker; 2: total uninduced bacteria; 3: total bacteria induced at 42°C; 4: ultrasonic supernatant; 5: ultrasonic precipitation; 6: supernatant of inclusion bodies after washing; 7: 0.3M Nacl elution peak of Src I after renaturation by ion exchange chromatography; 8: 0.8M Nacl elution peak of Src I after renaturation by ion exchange chromatography.

In Fig. 9, 1: marker; 2: Src I recombinant protein.

Among Fig. 17, 1: PI-Sce I/I-Ceu I digestion of Adeno-Src I; 2: 1kb DNA marker (NEB company).

In Fig. 18, 1: Pac I digestion of Adeno-Src I; 2: 1kb DNA marker (NEB company).

(5) Specific implementation methods

The present invention will be further described below in conjunction with the accompanying drawings, which will help those of ordinary skill in the art understand the present invention, but the present invention is not limited in any form.

Example 1 Construction of a cDNA library of rose red and green sea anemone venom glands

The extraction of the total RNA from the venom glands of rose red and green sea anemones was carried out by referring to the one-step rapid hot phenol extraction method in "Modern Molecular Biology Experimental Technology"; the synthesis of cDNA was operated according to the instructions of SMART ^TM cDNA Library Construction Kit of Clontech Company.

Two rRNA bands, 28S and 18S, were clearly seen in the poisonous gland total RNA extracted by the one-step method of guanidine isothiocyanate through 1% formaldehyde denaturing gel electrophoresis, as shown in Figure 1, indicating that the integrity of the total RNA was good. The cDNA synthesized by SMART ^TM cDNA Library Construction Kit was electrophoresed on 1% agarose gel, and the result showed a uniform smear, as shown in Figure 2. The size ranged from 200bp to 8kb, mainly in the region below 3kb, at 500bp Nearby is more concentrated, indicating that the integrity of the cDNA is good. The cDNA was inserted into the plasmid vector pcDNA3.0 to construct a cDNA expression library, and the number of library clones was 2.4×10 ⁵ . Extract the total library plasmid for enzyme digestion and PCR analysis, as shown in Figure 3, the results show that the cDNA insert size falls within the range of 300bp-8kb, pick 172 clones to extract the plasmid, enzyme digestion and PCR identification show that more than 95% of the clones are The recombinants, as shown in Figure 4, indicate that the cDNA expression library has good quality.

Example 2 Cloning, sequencing and analysis of the cDNA library of the tentacle of the rose red and green sea anemone

The clones of the tentacle cDNA library of rose red and green sea anemone were selected, and the plasmid DNA was extracted according to the method of Omega Biotek PlasmidMiniprep Kit. 150 cDNA sequences were randomly determined. Using ABI PRISM 377 DNA Analyzer (Applied Biosystems), using T7 and SP6 universal primers as sequencing primers, for forward and reverse sequence determination, for sequences of cDNA fragments exceeding 1000bp, design primers based on the measured sequences, and continue to measure cDNA. The sequencing work was completed by the Central Laboratory of the School of Life Sciences, Sun Yat-sen University, Guangzhou. The obtained sequences were preliminarily analyzed by Blast X. The results showed that the rose red and green sea anemone venom gland cDNA library contained a variety of genes, among which the abundance of toxin genes was high. There were three cDNA sequences encoding sea anemone neurotoxins and one cDNA sequence encoding sea anemone cytolysin. The cDNA number of cytolysin is Src I, as shown in the sequence table, it 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 protein of 178 amino acid residues. Mature protein, the N-terminus of the mature protein has the characteristic α-helix of sea anemone cytolysin. The molecular weight of the mature protein is 19,500 Daltons, the isoelectric point of the protein is 4.8, and it is an acidic cytolysin. At present, all reported sea anemone cytolysins are strongly basic proteins with an isoelectric point above 9.0, and it is the first time that acidic cytolysins have been found in sea anemones.

See the Sequence Listing for the nucleotide sequence and deduced amino acid sequence of Src I. Using the total RNA of rose red and green sea anemone tentacles as the starting material, the 3' end primer was designed according to the obtained cytolysin cDNA sequence, and the library construction primer of SMART ^TM cDNA Library Construction Kit was used as the 5' end primer for RT-PCR. An expected specific amplification band appeared at 800bp, and this band was recovered. The recovered PCR product was connected to pGEM-T Easy Vector, transformed into DH5α Escherichia coli, and positive clones were selected for sequencing. It was confirmed by sequencing analysis that it was the desired target gene, and the 8 clones determined were the same cDNA sequence.

Example 3 Construction of Recombinant Rose Anemone Cytolysin Expression Plasmid

According to the sequence at both ends of the mature protein encoded by the Src I gene and the multi-restriction site of the prokaryotic expression vector pBV220, a pair of primers were synthesized, the sequence of which is as follows:

ATC TCG GGT GGT ACT GTT ATT 3’Upstream primer, 5'G GAATTC

ATC TCG GGT GGT ACT GTT ATT 3'

   The single underlined part is the EcoR I restriction sequence, and the double underlined part is the start codon

Downstream primer, 5'TA GGATCC TGG CCA GAC GAC TTC AAT C 3'

   The single underline is the BamH I restriction sequence, and the double underline is the stop codon

PCR amplification and gene cloning were carried out according to conventional methods. The PCR product is about 600bp, encoding 178 amino acid residues, as shown in Figure 6. The target gene was cloned into the prokaryotic expression vector pBV220 to construct the expression plasmid pBV220-Src I. The construction process is shown in Figure 5. Identification by enzyme digestion and sequencing analysis showed that the cloned gene was the target gene as shown in Figure 7.

The prokaryotic expression vector pBV220 contains the P _R P _L promoter and cI regulatory gene. The foreign gene with the start codon can be inserted into the multiple restriction site downstream of the promoter to express the non-fusion protein. The product is available for clinical use .

Example 4 Expression of Recombinant Rose Anemone Cytolysin Protein

The expression plasmid pBV220-Src I was transformed into Escherichia coli DH5α. When the engineered bacteria containing the target gene grow to OD ₆₀₀ =0.5 at 30°C, they are immediately induced at 42°C for 4 hours. The bacteria 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. TLC scanning analysis showed that: under this condition, the expression amount of the recombinant protein accounted for more than 17% of the total bacterial protein, basically in the state of insoluble inclusion bodies.

Example 5 Purification of recombinant rose red and green sea anemone cytolysin protein

The induced bacteria were disrupted by ultrasound, and the precipitate was collected by centrifugation. The precipitate was washed with different buffer solutions to remove foreign proteins, and the purity of the inclusion body after washing was 80%. The solutions used in inclusion body washing are: ultrasonic buffer (10mM Tris-Hcl, pH7.0, 1mM EDTA), buffer1 (0.1M Tris-Hcl, pH8.0, 10mM EDTA, 0.5% Triton X 100), buffer 2 (50mMPB, 0.5M Nacl, 3M urea) and buffer 3 (0.1M Tris-Hcl, pH8.5, 10mM EDTA, 3M urea).

The inclusion bodies after washing were dissolved in denaturing solution (8M urea, 10mM Tris-Hcl, pH8.0, 10mM DTT). The denatured protein was refolded by dialysis in refolding solution (3M urea, 20mM Tris-Hcl, pH8.0, 0.1mM oxidized glutathione, 0.9mM reduced glutathione).

Refolding proteins can obtain recombinant proteins with a purity of more than 99% through ion exchange chromatography and hydrophobic chromatography, as shown in Figure 8.

Example 6 Determination of the isoelectric point of recombinant rose red and green sea anemone cytolysin protein

The isoelectric point of the recombinant protein was determined by plate electrophoresis, and the isoelectric focusing polyacrylamide gel electrophoresis was performed according to a conventional method. The measured result is: pH 4.81, as shown in Figure 9. Basically consistent with the predicted isoelectric point.

Example 7 Identification of hemolytic activity of recombinant sea anemone cytolysin protein

Take 5ml of human blood, add 3.8% sodium citrate (sodium citrate:blood ratio 1:9), centrifuge at room temperature, 3000rpm for 5 minutes. Remove the supernatant, wash the red blood cells with Hank's solution until the supernatant is clear (about 2-3 times with Hank's), make the red blood cells into a 0.5% or 1% (v/v) cell suspension with Hank's, add the sample to Incubate in 2ml of erythrocyte suspension at 37°C for 20 minutes, centrifuge at room temperature at 3000rpm for 5 minutes, take the supernatant, and measure OD ₅₄₀ . With Hank's as negative control, 100% red blood cells were hemolyzed with saponin at a final concentration of 0.1 mg/ml. Three parallel experiments were performed for each concentration of samples. The result is shown in Figure 10.

Example 8 The effect of recombinant sea anemone cytolysin protein on tumor cells cultured in vitro

Tumor cells were inoculated in 24-well plates at 1.0×10 ⁶ cell/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 the cells with 4% paraformaldehyde at 4°C for 20 minutes, wash with Hank's twice, add Hoeches 55258 staining solution, the final concentration is 5-10ug/ml, Stained at room temperature for 10 minutes, washed twice with Hank's, and observed under a fluorescent microscope.

Different concentrations of recombinant protein (0.31ug/ml, 3.1ug/ml, 7.8ug/ml, 15.6ug/ml, 31.2ug/ml, 72.1ug/ml) were added to human liver cancer cells BEL-7402, at 0- Observed within 36 hours, the sample with a concentration of 0.31-15.6ug/ml has no effect on the cells, and the concentration of 31.2ug/ml can cause a significant change in the shape of the cells, as shown in Figure 11, the cells are obviously elongated, this phenomenon occurs after adding protein 8 It became more obvious after 1 hour, and it remained until 36 hours (no observation after 36 hours). There was no necrosis observed by light microscopy, and no apoptosis was observed by Hoeches 55258 staining, as shown in Figure 12. Judging from the hemolysis phenomenon of the recombinant protein, the recombinant protein should be able to interact with the cell membrane. Judging from the phenomenon of the recombinant protein acting on the liver cancer cell BEL-7402, the recombinant protein not only interacts with the cell membrane, but may also interact with the protein on the membrane, causing a change in the signal , leading to changes in microtubules, microfilaments, etc., causing changes in cell morphology.

Add different concentrations of recombinant protein (0.31ug/ml, 3.1ug/ml, 7.8ug/ml, 15.6ug/ml, 31.2ug/ml, 72.1ug/ml) to human poorly differentiated gastric cancer cell BGC-823, add After the sample was observed, after 6 hours, the sample concentration of 3.1ug/ml could make the cells round and smaller, as shown in Figure 13. After fluorescent staining, it can be seen that the nucleus of the cell became significantly smaller, and only a thin layer of cytoplasm remained outside the nucleus. layer, as shown in Figure 14.

Add different concentrations of recombinant protein (0.31ug/ml, 3.1ug/ml, 7.8ug/ml, 15.6ug/ml, 31.2ug/ml, 72.1ug/ml) to human non-small cell lung cancer Nsclc, after adding the sample After observation, 6 hours later, the sample concentration of 3.1ug/ml can cause the cells to expand and rupture, as shown in Figure 15, it can be seen by Hoeches 55258 staining that the cells are not apoptosis, but most of the cells are necrotic.

Example 9 Anti-tumor test of recombinant sea anemone cytolysin protein on mouse liver cancer (Heps) and sarcoma S-180

The tumor inhibition test of the recombinant protein was carried out according to conventional methods; the animals were NIH mice, provided by the Experimental Animal Center of First Military Medical University (2000A037); the administration method was intraperitoneal injection; the negative control was normal saline, and the positive control was cyclophosphamide; The test was completed by the Guangdong Provincial Occupational Health Inspection Center; the test results are shown in Tables 1 and 2.

The purified recombinant protein was injected intraperitoneally into NIH mice, and it had inhibitory effects on both liver cancer solid tumors and S-180 solid tumors inoculated in mice. kg; the tumor inhibition rate for S-180 solid tumors was 26.9%, and the effect concentration was 1.16mg/kg.

Table 1 The inhibitory effect of recombinant Src I protein on mouse liver cancer solid tumor (Heps)

Dose group Number of animals Body weight Tumor weight Tumor inhibition rate P value

(mg/ml) Start End Start End (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

P value: compared with the control group, analysis of variance.

Table 2 Inhibitory effect of recombinant Src I protein on mouse sarcoma (S-180)

Dose group Number of animals Weight gain Tumor weight Inhibition rate Thymus index Spleen index Liver index

(mg/m1) Start End (g) (g) (%) (1/1000) (1/1000) (1/100)

0 10 10 2.58±2.24 1.53±0.44 - 3.28±0.85 4.93±1.62 5.35±0.62

Cp, 20 10 10 1.23±1.29 0.64±0.25 58.33 ^▲▲ 2.16±0.40 ^▲▲ 4.07±1.02 5.80±0.56

0.116 10 10 3.26±1.07 1.12±0.58 26.87 ^▲ 3.41±0.78 6.54±1.76 ^▲ 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 value: compared with the control group, analysis of variance. ^▲ P＜0.05, ^▲▲ P＜0.01

Example 10 Construction of recombinant adenovirus Adeno-Src I

The operation method is carried out according to Adeno-X ^TM Expression System User Manual of Clontech Company. According to the sequence at both ends of the mature protein encoded by the Src I gene and the multiple restriction site of the shuttle plasmid pShuttle, a pair of primers were synthesized, the sequence of which is as follows:

ATCTCGGGTGGTACTGTTATTG 3’Upstream primer, 5'GG GGGCCC

ATCTCGGGTGGTACTGTTATTG 3'

   The single underline is the Apa I restriction sequence, and the double underline is the start codon

TGGCCAGACGACTTCAATC 3’Downstream primer, 5'GTCAT GCGGCG C

TGGCCAGACGACTTCAATC 3'

   The single underline part is the Not I restriction sequence, and the double underline is the stop codon

PCR amplification, gene cloning, plasmid extraction, etc. were carried out according to conventional methods. The PCR product was cloned into the shuttle plasmid pShuttle to construct the pShuttle-Src I plasmid; pShuttle-Src I was digested with PI-Sce I/I-Ceu I and then ligated with the adenovirus vector to construct the recombinant adenovirus Adeno-SrcI ; Recombinant adenovirus Adeno-Src I was digested with PacI, as shown in Figure 18, and then transfected into human embryonic kidney HEK293 cells, packaged into virus particles in HEK293 cells, and the extracted recombinant adenovirus can be used for in vitro cell and in vivo tests, and the product can be For clinical use. The detailed process of constructing the recombinant adenovirus is shown in FIG. 16 . Identification by enzyme digestion and sequencing analysis showed that the cloned gene was the target gene, as shown in Figure 17.

Sequence Listing

<110> Sun Yat-Sen University

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Arg Leu Thr Leu Asp Leu Leu Lys Thr Leu Leu Gly Thr Leu Gly Ser

15 20 25

atc tct aga aag att gca att ggt gtt gac aac gag acg ggt ggg cta 296

Ile Ser Arg Lys Ile Ala Ile Gly Val Asp Asn Glu Thr Gly Gly Leu

30 35 40

att aca gga aat aac gta tat ttc cgt tcc ggg acc tct gat gac atc 344

Ile Thr Gly Asn Asn Val Tyr Phe Arg Ser Gly Thr Ser Asp Asp Ile

45 50 55

ctc cct cat cgt gtg gaa act ggt gaa gcg ctt ctc tat aca gct cgc 392

Leu Pro His Arg Val Glu Thr Gly Glu Ala Leu Leu Tyr Thr Ala Arg

60 65 70 75

aaa act aaa ggc cca gtc gca aca ggt gcc gtt gga gta ttt act tat 440

Lys Thr Lys Gly Pro Val Ala Thr Gly Ala Val Gly Val Phe Thr Tyr

80 85 90

tac ttg agc gat gga aac aca ctg gca gtg tta ttc agc gtc ccc ttt 488

Tyr Leu Ser Asp Gly Asn Thr Leu Ala Val Leu Phe Ser Val Pro Phe

95 100 105

gat tat aac ttc tac agc aac tgg tgg aat gtc aag atc tat tca gga 536

Asp Tyr Asn Phe Tyr Ser Asn Trp Trp Asn Val Lys Ile Tyr Ser Gly

110 115 120

aaa cgg aat gcg gac tat gat atg tac cat gag ctg tac tat gat gcg 584

Lys Arg Asn Ala Asp Tyr Asp Met Tyr His Glu Leu Tyr Tyr Asp Ala

125 130 135

aat cca ttc gag ggg gac gat acc tgg gag tat aga tac ctt gga tat 632

Asn Pro Phe Glu Gly Asp Asp Thr Trp Glu Tyr Arg Tyr Leu Gly Tyr

140 145 150 155

gga atg agg atg gaa ggt tac atg aac agc ccc gga gaa gcg att ctt 680

Gly Met Arg Met Glu Gly Tyr Met Asn Ser Pro Gly Glu Ala Ile Leu

160 165 170

aag atc acg gtg atg ccc gat tgaagtcgtc tggccaaagc aaaaaaaaaa 731

Lys Ile Thr Val Met Pro Asp

175

aaaaaaaaaa aaaaaaaa 749

<210>2

<211>216

<212>PRT

<213>Rose anemone (Sagartia rosea sp)

<400>2

Met Ser Arg Leu Ile Val Val Cys Ile Val Ile Ser Met Ile Cys Gly

-35 -30 -25

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

-20 -15 -10

Asp Glu Asp Glu Lys Pro Lys Ile Ser Gly Gly Thr Val Ile Ala Ala

-5 -1 -1 1 5 10

Gly Arg Leu Thr Leu Asp Leu Leu Lys Thr Leu Leu Gly Thr Leu Gly

15 20 25

Ser Ile Ser Arg Lys Ile Ala Ile Gly Val Asp Asn Glu Thr Gly Gly

30 35 40

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

45 50 55

Ile Leu Pro His Arg Val Glu Thr Gly Glu Ala Leu Leu Tyr Thr Ala

60 65 70

Arg Lys Thr Lys Gly Pro Val Ala Thr Gly Ala Val Gly Val Phe Thr

75 80 85 90

Tyr Tyr Leu Ser Asp Gly Asn Thr Leu Ala Val Leu Phe Ser Val Pro

95 100 105

Phe Asp Tyr Asn Phe Tyr Ser Asn Trp Trp Asn Val Lys Ile Tyr Ser

110 115 120

Gly Lys Arg Asn Ala Asp Tyr Asp Met Tyr His Glu Leu Tyr Tyr Asp

125 130 135

Ala Asn Pro Phe Glu Gly Asp Asp Thr Trp Glu Tyr Arg Tyr Leu Gly

140 145 150

Tyr Gly Met Arg Met Glu Gly Tyr Met Asn Ser Pro Gly Glu Ala Ile

155 160 165 170

Leu Lys Ile Thr Val Met Pro Asp

175

Claims (7)

1, a kind of Cnidarian cytotoxin gene Src I derives from rose-red green sea anemone poison gland cDNA library, and nucleotide sequence is shown in SEQ ID NO:1 in the sequence table.

2, the coded albumen of a kind of Cnidarian cytotoxin gene Src I, it is characterized in that this albumen is to have 216 amino acid whose toxin precursor proteins, comprise 19 amino acid whose signal peptides, 19 amino acid whose propartmotif and 178 amino acid whose maturation proteins, the iso-electric point of maturation protein is 4.8, molecular weight is 19,500 dalton, it is a kind of acidic protein, the N-terminal of maturation protein has the characteristic feature of sea anemone cytolysin, the α spiral that promptly has parents, aminoacid sequence is shown in SEQ ID NO:2 in the sequence table.

3, a kind of recombinant prokaryotic expression vector is characterized in that containing right and requires 1 described gene Src I, is that it is cloned into the non-fusion expression carrier pBV220-Src I that is built on the prokaryotic expression carrier pBV220.

4, a kind of recombinant eukaryon expression vector, it is characterized in that containing gene Src I as claimed in claim 1, be that it is cloned on the shuttle plasmid pshuttle, construction recombination plasmid pshuttle-Src I, and pass through to link to each other the recombinant eukaryon expression vector Adeno-Src I that is built into carrier for expression of eukaryon Adeno-X behind the PI-SceI/I-CeuI double digestion.

5, a kind of dna fragmentation as primer, it is characterized in that cutting the site by the multienzyme of described gene encoding mature albumen two terminal sequences of claim 1 and prokaryotic expression carrier pBV220 synthesizes, upstream primer contains the EcoRI enzyme and cuts sequence (GAATTC) and initiator codon (ATG), downstream primer contains the BamHI enzyme and cuts sequence (GGATCC) and terminator codon (TTA), sequence is as follows: upstream primer, 5 ' G GAATTC ATC TCG GGT GGT ACT GTT ATT 3 '

The EcoRI enzyme is cut the ATG code of sequence downstream primer, 5 ' TA GGATCC

TGG CCA GAC GAC TTC AATC 3 '

The BamHI enzyme is cut the sequence terminator codon.

6, a kind of dna fragmentation as primer, it is synthetic to it is characterized in that cutting the site by the multienzyme of maturation protein two terminal sequences of the described genes encoding of claim 1 and shuttle plasmid pShuttle, upstream primer contains the ApaI enzyme and cuts sequence (GGGCCC) and initiator codon (ATG), downstream primer contains the NotI enzyme and cuts sequence (GCGGCCGC) and terminator codon (TTA), sequence is as follows: upstream primer, 5 ' GG GGGCCC

ATCTCGGGTGGTACTGTTATTG 3 '

The ApaI enzyme is cut the ATG code of sequence downstream primer, 5 ' GTCAT GCGGCCGC TGGCCAGACGACTTCAATC 3 '

The NotI enzyme is cut the sequence terminator codon.

7, as the application of albumen as described in the claim 2 in the medicine of preparation prevention or treatment tumour.

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