CN100564517C - A kind of anti-glioma peptide of scorpion and its production and application - Google Patents
A kind of anti-glioma peptide of scorpion and its production and application Download PDFInfo
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- CN100564517C CN100564517C CNB200610124582XA CN200610124582A CN100564517C CN 100564517 C CN100564517 C CN 100564517C CN B200610124582X A CNB200610124582X A CN B200610124582XA CN 200610124582 A CN200610124582 A CN 200610124582A CN 100564517 C CN100564517 C CN 100564517C
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Abstract
The invention discloses a kind of anti-glioma peptide of scorpion and its production and application, anti-glioma peptide of scorpion recombination bacillus coli Escherichia coli DE3/rBmKCT/pGEX-5X, CCTCC NO:M206015, it is characterized in that: at first be the design primer, cDNA sequence with screening anti-glioma peptide of scorpion gene from scorpion venom gland cDNA library is a template, carry out pcr amplification, acquisition contains the gene or the gene fragment of the anti-glioma peptide of scorpion of cutting site sequence of restriction enzyme; Next is with restriction enzyme PCR product and expression vector plasmid to be carried out enzyme to cut; The 3rd is to use T
4Dna ligase makes up recombinant expression vector rBmKCT/pGEX; The 4th is with recombinant expression vector rBmKCT/pGEX transformed competence colibacillus intestinal bacteria DE3, is built into genetic engineering bacterium (intestinal bacteria DE3/rBmKCT/pGEX-5X).The inventive method is easy, and is safe, low production cost, and purifying is simple and product purity is high, and anti-glioma peptide of scorpion is in preparation treatment or prevent application in the gliomatous medicine.
Description
Technical field
The invention belongs to biological technical field, the method that the present invention relates to a kind of anti-glioma peptide of scorpion and produce anti-glioma peptide of scorpion, specifically, the present invention relates to anti-glioma peptide of scorpion recombination bacillus coli and construction process thereof, the preparation method who also relates to a kind of anti-glioma peptide of scorpion of efficient bio-active, adopt the method for genetic engineering means generation anti-glioma peptide of scorpion, the application of the anti-glioma peptide of scorpion of generation in the medicine of preparation treatment or prevention anti-glioma.
Background technology
Scorpion venom has complicated composition and character and produces multiple physiology, pharmacologically active, antitumor, treatment rheumatism, anti-epileptic and cardiovascular disorder there is the critical treatment effect, but because scorpion venom complicated component, structural similitude, be difficult to separate, many composition roles even opposite, and effective constituent often content is low, these have all limited the research and the application of scorpion venom undoubtedly.Produce effective scorpion venom composition by engineered method and then become very necessary.Worldwide, obtained scorpion venom gene hundreds of bar, yet can successfully express the gene that produces the effective buthotoxin composition and but only have tens, its difficult point mainly is that output is too low, can't carry out postorder work, forms insoluble inclusion body, can not correctly be folded into activated protein, poisonous or do not match with the host and can not express fully to expressive host.The host system that is used for scorpion venom gene successful expression is varied, as intestinal bacteria, baculovirus, yeast, eukaryotic cell and tobacco, willow etc.These expression systems respectively have quality, and look different genes and different.Recently, (Zhang Shoutao, Guo Aiguang, Xiao Leyi such as Zhang Shoutao, Dong Jianjun, Zhao Tianzeng, Xue Qigeng, Kong Tianhan, 2002 30 6 phases of volume of Guo Wei " Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology's journal ": 30-33) obtain the cDNA of buthus martensii Karscs neurotoxin BmKCT by the amplification of reverse transcription polymerase chain reaction (RT-PCR) method and be cloned into the pTrcHisA expression vector changing expression production scorpion BmKCT peptide in the e. coli bl21 (DE3) over to, yet its output is not high, and most of inclusion body that forms, and is soluble.And do not contain the proteolytic enzyme restriction enzyme site that a plurality of Histidines in the expression vector and BmKCT peptide are separated in the PCR primer of design, make that the BmKCT peptide of expressing generation must be a fusion recombinant protein that contains a plurality of additional set propylhomoserins (His) and other aminoacid sequence.These extra Histidines can influence the structure of recombinant peptide so that its relevant function with other aminoacid sequence.
Chinese scholar Li Wen is prosperous, Ceng Xianchun, Zhu Shunyi etc. have made up high-quality buthus martensii Karscs (Buthusmartensii Karsch) poison gland cell cdna library, from the storehouse, filter out a kind of gene (the GenBank accession number is AF 135821) (Xian-Chun Zeng of chloride ion channel modulators, Wen-Xin Li, Shun-YiZhu, Fang Peng, Zhi-Hui Zhu, Kai-Lang Wu, Fu-Hua Yiang.Cloning andcharacterization of a cDNA sequence encoding the precursor of aChlorotoxin-like peptide from the Chinese scorpion Buthus martensiiKarsch.Toxicon 38:1009～1014,2000), its amino acid sequence coded with have 68% homology from the isolating catilan of African scorpion (chlorotoxin), called after buthus martensii Karscs catilan (Buthusmartensii Karsch ChloroToxin, BmKCT) gene.We have made up the genetic engineering bacterium of this gene and have efficiently expressed and purifying, studies show that, reorganization BmKCT albumen can effectively suppress the chlorine electric current (Yang Rui on the star-like glioma cell U251 cytolemma, Peng Fang, Liu Hui, Cao Zhi is low-priced, Li Wenxin, hair is admired Jiang Dahe, expression and the Function Identification of buthus martensii Karscs catilan bmkct, " Chinese biological chemistry and molecular biosciences journal " 21 1 phases of volume in 2005: 19～23), and the activity of tangible anti-glioma arranged, so we claim that this cDNA sequence is the anti-glioma peptide of scorpion gene, the synthetic polypeptide is an anti-glioma peptide of scorpion under this cDNA instructs.
Summary of the invention
The objective of the invention is to be to provide a kind of anti-glioma peptide of scorpion recombination bacillus coli, can express and have active anti-glioma peptide, solvable, the output height.
Another object of the present invention also is to provide a kind of construction process of anti-glioma peptide of scorpion recombination bacillus coli, and method is easy, and is safe, low production cost, and purifying is simple and product purity is high.
A further object of the present invention provides a kind of application of anti-glioma peptide of scorpion in the medicine of preparation treatment or prevention anti-glioma that produces with gene engineering method.
To achieve these goals, made up a kind of recombination bacillus coli (Escherichia coli) DE3 (containing recombinant plasmid rBmKCT/pGEX-5X) that can express anti-glioma peptide of scorpion, depositary institution: Chinese typical culture collection center, address: China. Wuhan. Wuhan University, preservation date on January 20th, 2006, deposit number: CCTCC NO:M206015, classification name: Escherichia coli (DE3)/rBmKCT/pGEX-5X, this Pseudomonas is in Gram-negative bacteria, can be at LB (Luria-Bertani) substratum (tryptone 10 gram/L, yeast extract 5 gram/L, sodium-chlor 10 gram/L, pH7.0) growth fast in, it contains recombinant plasmid rBmKCT/pGEX-5X recombination bacillus coli, and this plasmid is promotor with tac, has amicillin resistance, carry the Thiadiazolidine isomerase gene, and connected the anti-glioma peptide of scorpion gene in this gene downstream, the gene order of small intestine kinases restriction enzyme site is contained in the junction.Can be used for producing and have the active anti-glioma peptide of scorpion of anti-glioma.
The present invention makes up the method for producing anti-glioma peptide of scorpion, comprise design PCR upstream and downstream primer, with the round pcr anti-glioma peptide gene fragment that from scorpion of Buthus martensii venom cDNA storehouse, increases, and be transformed into recipient bacterium intestinal bacteria DE3 (Fan Peng by expression vector, Xianchun Zeng, Xiaohua He, Jun Pu, Wenxin Li et al, Molecular cloning and functional expression of a gene encoding anantiarrhythamia peptide derived from the scorpion toxin, Eur.J.Biochem., 269:4468-4475,2002).It is characterized in that, the PCR primer of design contains small intestine kinases restriction enzyme site, anti-glioma peptide gene fragment process digestion with restriction enzyme with pcr amplification, be cloned into expression vector pGEX-5x-1 plasmid (FanPeng, Xianchun Zeng, Xiaohua He, Jun Pu, Wenxin Li et al, Molecular cloning and functionalexpression of a gene encoding an antiarrhythamia peptide derived from the scorpion toxin, Eur.J.Biochem., 269:4468-4475,2002) in, be transformed into and obtain recombination bacillus coli DE3 (containing the rBmKCT/pGEX recombinant plasmid) among the intestinal bacteria DE3, be used to produce soluble and activated reorganization anti-glioma peptide of scorpion.The anti-glioma peptide of scorpion that adopts method of the present invention to produce has the effect that suppresses neurospongioma specificity chloride channel.Its construction step is as follows:
A) from self-built scorpion venom gland cDNA library (Xian-Chun Zeng, Wen-Xin Li, Shun-Yi Zhu, Fang Peng, Zhi-Hui Zhu, Kai-Lang Wu, Fu-Hua Yiang.Cloning andcharacterization of a cDNA sequence encoding the precursor of aChlorotoxin-like peptide from the Chinese scorpion Buthus martensiiKarsch.Toxicon 38:1009～1014,2000) the cDNA sequence of screening chloride channel inhibitor B mKCT gene in, except the coded signal peptide sequence, in 35 amino acid whose mature peptide sequences of its derivation, with have 68% homology from the isolating chlorotoxin of African scorpion (catilan), claim that this cDNA sequence is the anti-glioma peptide of scorpion gene: gene order is SEQID NO:1.
gcaaaactct?attaaaaatg?aagttcctct?acggaatcgt?tttcattgca?ctttttctaa 60
ctgtaatgtt?cgcaactcaa?actgatggat?gtgggccttg?ctttacaacg?gatgctaata 120
tggcaaggaa?atgtagggaa?tgttgcggag?gtattggaaa?atgctttggc?ccacaatgtc 180
tgtgtaaccg?tatatgaata?attaaaaatg?tacacctgaa?cagatcattt?aatgaataat 240
aaatattaat?aagcattaaa?a 261
Above-mentioned is the cDNA sequence of anti-glioma peptide of scorpion, the 18-197 nucleotides sequence is classified protein coding region as, the nucleotide sequence coded signal peptide of 18-89 district wherein, remaining coding region part is a mature peptide, the 238-243 sequence is a poly adenine nucleotide tailing signal sequence.Following protein precursor is synthetic so this gene can be encoded:
MKFLYGIVFI?ALFLTVMFAT?QTDGCGPCFT?TDANMARKCR?ECCGGIGKCF?GPQCLCNRI 59
This protein precursor just can be formed with active mature protein-anti-glioma peptide of scorpion through the processing after translating:
CGPCFTTDAN?MARKCRECCG?GIGKCFGPQC?LCNRI 35
B) according to anti-glioma peptide of scorpion gene order (SEQID NO:1) design primer A1 (5 '-GCCGGATCCCCGATGACGATGACAAGTGTGGGCCTTGCTTTAC-3 ') and A2 (5 '-GCCCTCGAGTCATATACGGTTACACAGACATTG-3 '), carry out pcr amplification, just can obtain to contain the gene or the gene fragment of the anti-glioma peptide of scorpion of restriction enzyme digestion sites sequence:
gccggatccc?cgatgacgat?gacaagtgtg?ggccttgctt?tacaacggat?gctaatatgg 60
caaggaaatg?tagggaatgt?tgcggaggta?ttggaaaatg?ctttggccca?caatgtctgt 120
gtaaccgtat?atgactgctc?ccg 143
C) with restriction enzyme the PCR product that reclaims and expression vector pGEX-5x-1 plasmid being carried out enzyme respectively cuts.
D) use T
4Dna ligase, anti-glioma peptide of scorpion gene after ligase enzyme is cut and expression vector pGEX-5x-1 make up recombinant expression vector rBmKCT/pGEX.
E) preparation competence intestinal bacteria DE3, to connect product (rBmKCT/pGEX) is transformed among the intestinal bacteria DE3, clone's that obtains cut by enzyme or PCR identifies that the back goes into fragment to skewer and check order, correct clone's of sequencing result is the recombination bacillus coli DE3 (containing recombinant plasmid rBmKCT/pGEX-5X) that contains the anti-glioma peptide of scorpion gene.
A kind of method of structure anti-glioma peptide of scorpion recombination bacillus coli of optimization: it is characterized in that: PCR upstream primer (SEQ ID NO:2)
A1,5 '-GCCGGATCCCCGATGACGATGACAAGTGTGGGCCTTGCTTTAC-3 ', this primer contain BamHI restriction enzyme digestion sites and small intestine kinases restriction enzyme site;
PCR downstream primer (SEQ ID NO:3)
A2, this primer of 5 '-GCCCTCGAGTCATATACGGTTACACAGACATTG-3 ' contains the XhoI restriction enzyme digestion sites.
Cut rear clone to pGEX-5x-1 with the pcr amplification products therefrom through BamHI and XhoI enzyme, be transformed into and make up recombination bacillus coli DE3 (containing rBmKCT/pGEX-5X) among the intestinal bacteria DE3.
Mature peptide section according to the anti-glioma peptide of scorpion genes encoding, promptly the gene fragment except signal peptide sequence designs primer, be anti-glioma peptide of scorpion cDNA 87-101nt place design PCR upstream primer A1,5 '-GCCGGATCCCCGATGACGATGACAAGTGTGGGCCTTGCTTTAC-3 ' contains BamHI restriction enzyme digestion sites and small intestine kinases restriction enzyme site sequence; According to the cDNA174-197nt place design downstream primer A2 of anti-glioma peptide of scorpion, 5 '-GCCCTCGAGTCATATACGGTTACACAGACATTG-3 ' contains Xho I restriction enzyme digestion sites.Cut rear clone with the pcr amplification products therefrom through BamHI and Xho I enzyme and be transformed into intestinal bacteria DE3 again in the pGEX-5x-1 and obtain anti-glioma peptide of scorpion recombination bacillus coli DE3 (containing recombinant plasmid rBmKCT/pGEX-5X), in order to express the anti-glioma peptide of scorpion of biologically active.
The peptide that obtains with this anti-glioma peptide of scorpion recombination bacillus coli production has obvious anti-glioma activity, is solvable, non-inclusion body state and output height, and output is 2mg anti-glioma peptide of scorpion/L culture.
A kind of anti-glioma peptide of scorpion is characterized in that, the aminoacid sequence of this bioactive peptide following (SEQ IDNO:4):
CGPCFTTDANMARKCRECCGGIGKCFGPQCLCNRI。
Reorganization anti-glioma peptide of scorpion provided by the invention is consistent with the mature amino acid sequence of deriving from anti-glioma peptide of scorpion cDNA, and does not contain influential bioactive additional set propylhomoserin fragment and other aminoacid sequence.The reorganization anti-glioma peptide of scorpion is solvable state, does not form inclusion body, the function of promptly this Toplink actual response scorpion anti-glioma gene.Recombinant peptide provided by the invention not only has the effect that suppresses neurospongioma specificity chloride channel, and tangible anti-glioma effect is arranged.
A kind of method of producing anti-glioma peptide of scorpion comprises design PCR primer, with the PCR anti-glioma peptide of scorpion gene fragment that increases from scorpion of Buthus martensii venom cDNA storehouse, and is transformed among the intestinal bacteria DE3 by expression vector and expresses.It is characterized in that: the PCR primer of design contains small intestine kinases restriction enzyme site, anti-glioma peptide of scorpion gene fragment process digestion with restriction enzyme with pcr amplification, be cloned in the pGEX-5x-1 expression vector, be transformed into and obtain recombination bacillus coli (Escherichia coli) DE3/rBmKCT/pGEX-5X among the intestinal bacteria DE3, after IPTG induces, cut with small intestine kinases enzyme, obtain soluble anti-glioma peptide of scorpion:
CGPCFTTDANMARKCRECCGGIGKCFGPQCLCNRI
According to anti-glioma peptide of scorpion gene order designing probe gene or the gene fragment that obtains anti-glioma peptide of scorpion screened in the cDNA storehouse of scorpion, carry out pcr amplification; With restriction enzyme the PCR product that reclaims and expression vector pGEX-5x-1 plasmid are carried out enzyme and cut and use T
4Dna ligase connects, and makes up recombinant expression vector.Transformed into escherichia coli DE3 obtains recombination bacillus coli DE3/rBmKCT/pGEX-5X, induce through IPTG, express Thiadiazolidine isomerase-anti-glioma peptide of scorpion fusion rotein, behind gsh affinity chromatography column purification, cut, obtain soluble and activated reorganization anti-glioma peptide of scorpion with small intestine kinases enzyme.
A kind of method of production anti-glioma peptide of scorpion of optimization: it is characterized in that:
PCR upstream primer A1:
5 '-GCCGGATCCCCGATGACGATGACAAGTGTGGGCCTTGCTTTAC-3 ', this primer contain BamHI restriction enzyme digestion sites and small intestine kinases restriction enzyme site;
PCR downstream primer A2:
5 '-GCCCTCGAGTCATATACGGTTACACAGACATTG-3 ', this primer contains the XohI restriction enzyme digestion sites.
Cut rear clone to pGEX-5x-1 with the pcr amplification products therefrom through BamHI and XohI enzyme, behind conversion, IPTG abduction delivering, fusion rotein is carried out enzyme and cut the anti-glioma peptide of scorpion that obtains solubility with little enteropeptidase.
Gene fragment design primer according to the mature peptide section of anti-glioma peptide of scorpion gene, be anti-glioma peptide of scorpion cDNA 90-106nt place design PCR upstream primer A1,5 '-GCCGGATCCCCGATGACGATGACAAGTGTGGGCCTTGCTTTAC-3 ' contains BamHI restriction enzyme digestion sites and small intestine kinases restriction enzyme site; According to anti-glioma peptide of scorpion cDNA 174-197nt place design downstream primer A2,5 '-GCCCTCGAGTCATATACGGTTACACAGACATTG-3 ' contains the XohI restriction enzyme digestion sites.Cut rear clone with the pcr amplification products therefrom through BamHI and XohI enzyme and in pGEX-5x-1, be transformed into intestinal bacteria DE3, through IPTG abduction delivering recombination fusion protein: Thiadiazolidine isomerase-anti-glioma peptide of scorpion, utilize the Thiadiazolidine isomerase affinity column to carry out purifying, cut through small intestine kinases enzyme again, obtain the anti-glioma peptide of scorpion of biologically active.
Obtain recombination bacillus coli DE3/rBmKCT/pGEX-5X according to the method for producing anti-glioma peptide of scorpion and send Chinese typical culture center preservation on January 20th, 2006, preserving number is CCTCCM206015.
Because the resulting different recombinant peptides of different production methods are also inevitable difference to some extent on function.In accordance with the present production process, resulting recombinant peptide has following characteristics:
1, do not contain the extra fragments that a plurality of His form in the recombinant peptide, consistent with the mature peptide of inferring by cDNA.
2, resulting recombinant peptide is soluble non-inclusion body state, and the output height, and output is 2mg anti-glioma peptide of scorpion/L culture.
3, proving that by the brain tissue of rat glioma model resulting recombinant peptide is a kind of effective neurospongioma resistant polypeptides, is 61% to the gliomatous inhibiting rate of brain tissue of rat.
The application of anti-glioma peptide of scorpion in the medicine of preparation treatment or prevention anti-glioma.
The application of anti-glioma peptide of scorpion in the medicine of preparation treatment or prevention anti-glioma that utilizes gene engineering method to produce.
Anti-glioma peptide of scorpion provided by the invention and the anti-glioma peptide of scorpion of producing by method of the present invention can be used to prepare the anti-glioma medicine.This anti-glioma peptide of scorpion can with pharmaceutically acceptable carrier, for example; Vehicle such as glucose, lactose, N.F,USP MANNITOL, glycine, water etc.; Weighting agent such as starch, sucrose etc.; Lubricant such as talcum powder, calcium stearate, polyoxyethylene glycol etc.; Absorption enhancer such as Sodium desoxycholate, ox sulphur glycocholate, N-Methyl pyrrolidone, EDTA, Tween-80, SDS etc.; Tackifier such as methylcellulose gum, polyacrylamide, hyaluronic acid sodium etc. or use with other treatment anti-glioma medicament mixed.The anti-glioma peptide of scorpion that the present invention produces can composition form by injection, eye conjunctiva absorb, snuffing is gone into, the mode of skin absorption, drop rectum with drug is used with aqua, fixing agent or other formulation.The anti-glioma peptide of scorpion that the present invention produces can mix with one or more carriers, and the conventional production method according to pharmaceutical field is made into required formulation then.This pharmaceutical composition contains weight ratio can be the anti-glioma peptide of scorpion of 0.2%-90%.
Description of drawings
Fig. 1 expression vector pGEX-5x-1 synoptic diagram.
The tac promotor can be induced with IPTG (isopropylthio-) and be expressed target protein matter; Amicillin resistance is as the selection markers of positive colony; Contain the Thiadiazolidine isomerase gene, with the goal gene amalgamation and expression, its product can carry out separation and purification with gsh part affinity chromatography.
The structure synoptic diagram of Fig. 2 recombinant expression vector pGEX-5x-1-anti-glioma peptide of scorpion (rBmKCT/pGEX).
GST represents Thiadiazolidine isomerase; A1 is a upstream primer, contains small intestine kinases restriction enzyme site and restriction enzyme Bam HI restriction enzyme site; A2 is a downstream primer, contains restriction enzyme XhoI restriction enzyme site; BmKCT is the cDNA sequence of anti-glioma peptide of scorpion.
The agarose gel electrophoresis that recombinant expression vector plasmid enzyme restriction and PCR identify in Fig. 3 anti-glioma peptide of scorpion recombination bacillus coli.
A, enzyme are cut evaluation: M1 is that (23130,9416,6557,4361,2322,2037,564,125bp), No. 1 is the plasmid pGEX-5X-1 that cuts through Bam HI enzyme to λ DNA/Hind III Markers; No. 2 recombinant plasmid rBmKCT/pGEX for cutting through Bam HI enzyme; No. 3 is the recombinant plasmid rBmKCT/pGEX through Bam HI and Xho I double digestion; Identify the anti-glioma peptide of scorpion gene fragment of gained for PCR No. 4; M2 be DNAMarkers (2000,1000,750,500,250,100bp).
B, PCR identify: M be λ DNA/Hind III Markers (23130,9416,6557,4361,2322,2037,564,125bp); No. 1 is BmKCT positive control fragment; No. 2 negative contrasts; 3-6 number is different single clone's (rBmKCT/pGEX-5X).
Fig. 4 SDS-PAGE protein electrophorese figure.
1 is protein labeling, is respectively 97,66,45,30,20.1,14.4kDa; 2 for there not being the total protein of inductive recombination bacillus coli; The total protein of 3 recombination bacillus colis of having induced for IPTG; 4 is the fusion rotein Thiadiazolidine isomerase-anti-glioma peptide of scorpion that obtains by Thiadiazolidine isomerase affinity chromatography column purification, and 5 is the fusion rotein after small intestine kinases enzyme is cut, and 6 is the anti-glioma peptide of scorpion of purifying.
The chloride channel map of current of the full cell voltage patch clamp record of Fig. 5.
A is contrast, and B is the neuroglial cytoma chloride channel electric current behind the adding reorganization anti-glioma peptide of scorpion.C is through after the flushing of extracellular fluid, the GCC electric current.Shown in figure, the GCC electric current that U251 expresses behind the adding 600nM all obviously reduces, but pairing voltage of chlorine current peak and activation voltage constant (B).After then passing through the flushing of extracellular fluid, the GCC electric current is replied (C) to some extent.This shows that reorganization BmKCT can obviously suppress star-like glioma cell (U251 clone) stimulates all chlorion electric currents that produced by-105-+170mV voltage, and this restraining effect is a reversible.
Fig. 6 tumor-inhibiting action of BmKCT fusion rotein of recombinating to rat C6 cell subcutaneous transplantation knurl
The rBmKCT group is for handling the Subcutaneous tumor tissue of rat through anti-glioma peptide
Control group is a Subcutaneous tumor tissue of handling rat through stroke-physiological saline solution
Fig. 7
131I-rBmKCT is in the distribution of tumor tissues
-◆-for injecting
131The result that I-rBmKCT measures
-■-for injecting Na
131The result of I blank determination
Tumour MRI (magnetic resonance imaging) (MRI) scintigram in Fig. 8 rat brain
The MRI scintigram of A control group before stroke-physiological saline solution is handled
The B control group is handled 14 days MRI scintigram through stroke-physiological saline solution
The MRI scintigram of C treatment group before anti-glioma peptide is handled
D treatment group is handled 14 days MRI scintigram through anti-glioma peptide
Embodiment
Embodiment 1: design primer and pcr amplification anti-glioma peptide of scorpion gene
Anti-glioma peptide of scorpion gene (BmKCT gene) the sequences Design PCR primer that is provided according to SEQID NO:1, for example: A1,5 '-GCCGGATCCCCGATGACGATGACAAGTGTGGGCCTTGCTTTAC-3 ', A2:5 '-GCCCTCGAGTCATATACGGTTACACAGACATTG-3 ', to screen BmKCT gene (SEQID NO:1) clone that is obtained from scorpion venom glandular cell cDNA storehouse is template, carry out the PCR reaction, anti-glioma peptide of scorpion gene in a large number increases.The PCR reaction conditions: each 1 μ l of 1 μ l Taq polysaccharase (1U), four kinds of (VITAMIN B4, guanine, cytosine(Cyt), thymus pyrimidine) deoxymononucleotide geometric ratio mixed solutions of 0.5 μ l (10mmol/L), 16.5 μ l aseptic double-distilled waters, 10 times of PCR damping fluids of 2.5 μ l, 1.5 μ l magnesium chlorides (25mmol/L), A1 (10 μ mol/L) and A2 (10 μ mol/L) primer and BmKCT gene template, cumulative volume is 25 μ l.The PCR reaction process: 60 seconds, 55 ℃ renaturation of 300 seconds, 94 ℃ sex change of 94 ℃ of pre-sex change are extended 60 seconds, 35 times, 72 ℃ last extensions 300 seconds of circulation for 60 seconds, 72 ℃.
Embodiment 2: make up recombinant expression vector (rBmKCT/pGEX)
With restriction enzyme with gained PCR product among the embodiment 1 through phenol: chloroform: isoamyl glycol (25: 24: 1) extracting, dehydrated alcohol (2.5 times of volumes) post precipitation is with 50 μ l TE damping fluid (behind the notebook embodiment) dissolution precipitations.With restriction enzyme Bam HI and Xho I (Takara company product) the PCR product that reclaims and expression vector pGEX-5x-1 plasmid being carried out enzyme cuts.Each 1 μ l of endonuclease reaction: BamHI (14U/ μ l) and XhoI (20U/ μ l), 10 times of damping fluid 2.5 μ l, PCR product or pGEX-5x-1 plasmid 50-100ng, adding sterilized water to cumulative volume is 25 μ l, 37 ℃ of water-baths 5 hours, enzyme is cut product through phenol one chloroform extracting, dehydrated alcohol (2.5 times of volumes) post precipitation T
4Dna ligase is connected the PCR product with expression vector pGEX-5x-1, form recombinant expression vector (rBmKCT/pGEX), and building process is seen accompanying drawing 2.Ligation: T
4Dna ligase (1U/ μ l) 1 μ l, the mol ratio of PCR product and expression vector pGEX-5x-1 is 3: 1, and the DNA total amount is 0.1 μ g, 5 times of ligase enzyme reaction buffer 4 μ l, adding sterilized water to cumulative volume is 20 μ l, places 24 hours for 16 ℃.
TE (Tris-EDTA) damping fluid: 10mmol/L TrisCl, 1mmol/L EDTA, pH8.0
Embodiment 3: obtain recombination bacillus coli DE3 (rBmKCT/pGEX)
Connection product among the embodiment 2 is transformed among the intestinal bacteria DE3.(37 ℃ of shaking tables are cultured to OD for tryptone 10 gram/L, inoculation intestinal bacteria DE3 among the yeast extract 5 gram/L, sodium-chlor 10 gram/L) at 3ml LB liquid nutrient medium
600Reach 0.4: 12000 and left the heart 5 minutes, remove supernatant, the 0.1MCaCl of precooling on the rocks
2The resuspended thalline of 120 μ l.Add to connect product 4 μ l (＜20ng), 10 ℃ of ice baths 30 minutes were put into 42 ℃ of heat shocks 90 seconds and were changed 10 ℃ of ice baths rapidly over to 1-2 minute.Add 1mlSOC (tryptone 10 gram/L, yeast extract 5 gram/L, sodium-chlor 10 gram/L, Repone K 2.5mmol/L, magnesium chloride 10mmol/L, glucose 20mmol/L) solution, 37 ℃ of shaking tables were cultivated 1 hour, 1200 left the heart 5 minutes, removed supernatant, added the 100 resuspended thalline of μ lSOC solution and coated on the LB flat board that contains penbritin.Picking list bacterium colony from the LB flat board that contains penbritin was cultivated 5 hours for 37 ℃ in containing the LB liquid nutrient medium of penbritin, and the method with pcr amplification detects these cultures then.Get gained culture 100 μ l and boil 60 seconds templates to detect as PCR in 100 ℃ of water-baths, the primer of PCR, reaction conditions and reaction process are with embodiment 1.Agarose gel electrophoresis by 1.2% finds the corresponding culture through the 150bp size amplified fragments of having an appointment behind the pcr amplification, sees that accompanying drawing 3. goes into fragment to the skewer in the contained plasmid of this culture and carry out dna sequencing (sequencing primer is the universal sequencing primer thing at the pGEX-5x-1 plasmid).The dna sequencing result conforms to the gene order of design, determines that this culture is the recombination bacillus coli of needed anti-glioma peptide of scorpion.Delivered Chinese typical culture collection center preservation, recombination bacillus coli Escherichia coli DE3 (rBmKCT/pGEX-5X) CCTCC NO:M206015.
Embodiment 4: the feature detection of recombination bacillus coli DE3/rBmKCT/pGEX-5X (CCTCC NO:M206015)
The recombination bacillus coli DE3/rBmKCT/pGEX-5X (CCTCC NO:M206015) that makes up is detected through China typical culture collection center, and the result shows that this bacterium is a kind of Gram-negative bacillus, each concrete feature result such as following table:
Table 1:rBmKCT genetic engineering bacterium various characteristics detected result
Embodiment 5: expression, the enzyme of reorganization anti-glioma peptide of scorpion are cut and purifying
With ratio inoculation clone's (recombination bacillus coli DE3/rBmKCT/pGEX) of 1: 100,37 ℃ were cultured to OD in containing the LB liquid nutrient medium of penbritin
6000.8 in time, add IPTG (final concentration is 1.0mM) culture induced, and add the pH value to 8.5 that NaOH transfers substratum, then with culture 28 ℃ of cultivations 4 hours to carry out the expression of goal gene.50 times of cultures after concentrated the inducing, ultrasonic wave broken cell (80HZ, 30 seconds/time, to culture become limpid till), centrifugal 5 minutes of 12000rpm, gained supernatant join in the GST affinity chromatography glue that 26 ℃ of effects made fusion rotein (GST-anti-glioma peptide of scorpion) fully combine with GST affinity chromatography glue in 1 hour behind the thorough mixing.Tris-Cl buffered soln (1.0mM with the EDTA that contains 50mM, pH8.0) wash GST affinity chromatography glue repeatedly and remove foreign protein, add small intestine kinase solution (24ug/mlGST affinity chromatography glue) then, effect is 10 minutes behind the mixing, and fusion rotein (GST-anti-glioma peptide of scorpion) is cut to separate GST and reorganization anti-glioma peptide of scorpion.Add and the isopyknic double distilled water of GST affinity chromatography glue in GST affinity chromatography glue, collection effusive liquid from GST affinity chromatography glue also detects the yield of reorganization anti-glioma peptide of scorpion by the 15%SDS-PAGE protein electrophorese.Contain reorganization anti-glioma peptide of scorpion solution through Sephadex G-50 desalination and remove the GST that may sneak on a small quantity with what collect.The mechanism of gel-filtration and condition: 100ml Sephadex G-50, use the double distilled water wash-out, flow velocity is 0.4ml/ minute.Preserved by the content of reorganization anti-glioma peptide of scorpion in the 15%SDS-PAGE protein electrophorese detection collection liquid and with its lyophilize.(electrophoresis detection the results are shown in Figure 4)
Embodiment 6: the amino acid analysis of reorganization anti-glioma peptide of scorpion
Two kinds of method hydrolysis reorganization anti-glioma peptide of scorpion: 1. 6N HCl, handled 24 hours in 110 ℃ of vacuum.2. 4N methylsulfonic acid, 0.2%3-(2-Padil) indoles, carried out 24,48,72 hours in a vacuum by 110 ℃.Sample is dissolved in the solution that 1.5ml contains 40% n-propyl alcohol and 0.1% trifluoroacetic acid, and every pipe packing 0.1ml dries up with drying nitrogen, add hydrolysing agent after, container is encapsulation process in a vacuum, collects the hydrolysis content, removes HCl.With the component of 121-MB Beckman amino acidanalyser according to the methods analyst peptide of analysis of amino acid, its result is as follows:
Embodiment 7: CGPCFTTDANMARKCRECCGGIGKCFGPQCLCNRI N-terminal determined amino acid sequence
Sample uses Applied Biosystems 476A sequenator to carry out the Edman degraded, the PTH amino acid that obtains with the analysis of AppliedBiosystems Model120APTH-analyser.The result shows that nine amino-acid sequences of N-terminal are " CGPCFTTDA ".
Embodiment 8: acute toxicity test
Select healthy Kunming small white mouse (body weight differs and is no more than 4g) 60 of 18-22g for use, be divided into 5 groups at random, every group of 12 small white mouses, male and female half and half are observed reaction of animals behind the rBmKCT fusion rotein abdominal injection, normally raise 2 days.The result shows LD
50=245.1mg/kg shows that the BmKCT fusion rotein is very low to mammalian toxicity.
Table 3
LD
50＝log
-1〔X
m-i(∑P-(3-P
m-P
n/4))〕＝245.1mg/kg
Embodiment 9: reorganization anti-glioma peptide of scorpion physiologically active detects
Detect with the method for full cell patch pincers activity recombinant peptide.The preparation of the star-like glioma cell of U251: the cover glass that will coat poly-lysine is put into 24 well culture plates, inoculate an amount of cell, with the 37 ℃ of cultivations of DEME nutrient solution that contain 10% foetal calf serum, after growth 24～36 hours, not contacting mutually with iuntercellular is principle, and slide is taken out the sample cell put into patch clamp, gives a baby a bath on the third day after its birth time with extracellular fluid, add extracellular fluid again, in order to measuring.The outer liquid (mM) of patch clamp operation: NaCl 125, and KCl 5, MgSO
41.2, CaCl
21.0, Na
2HPO
41.6, NaH
2PO
40.4 Glucose 10.5 and HEPES 32.5 transfer to pH 7.4 with 1mM NaOH.Interior liquid (mM): KCl 145, MgCl
21, CaCl
20.2 EGTA 10 and HEPES 10 transfer to pH 7.2 with 1mM KOH.Clamping down on voltage is 0mV, and test voltage is-105mV-+170mV, and the step increases and is 10mv, frequency of stimulation 0.5Hz, and 15-20 ℃ is carried out.Application EPC-9 amplifier and Pulse/Pulsefit software (HEKA elektronik, Germany).Excite nerve glioma cell and write down the chloride channel electric current of voltage adds the reorganization anti-glioma peptide of scorpion then, writes down the chloride channel electric current again, and the chloride channel electric current obviously reduces, and represents that this recombinant peptide has activity, sees accompanying drawing 5.
Embodiment 10: reorganization BmKCT fusion rotein is to the tumor-inhibiting action research of rat C6 cell subcutaneous transplantation knurl
Use the model research of rat C6 cell subcutaneous transplantation knurl reorganization BmKCT fusion rotein suppress the effect of tumour, the result shows, two treated animal Subcutaneous tumor weight in averages are respectively: physiological saline group 1.924g, rBmKCT organizes 0.735g, the actual tumour inhibiting rate of rBmKCT fusion rotein is 61.8%, statistical analysis shows that also tumour counterpoise difference is extremely remarkable between two groups, shows that the rBmKCT fusion rotein has very strong tumor killing effect.(seeing accompanying drawing 6)
Embodiment 11: reorganization BmKCT albumen is to the special target Journal of Sex Research of neuroglial cytoma
Use subcutaneous rat C6 Transplanted cells knurl animal model, tail vein injection
131The I-rBmKCT fusion rotein, control group injection Na
131I.Get respectively at different time that blood, heart, lungs, liver, kidney, pancreas, stomach are dirty, brain, muscle and tumor tissue be as sample, the radiation umber of pulse of working sample.Statistical study is found, in 180 minutes, in the tumor-bearing rat tumor tissues
131The content of I-rBmKCT in time prolongation and constantly increase (enrichment), other nonneoplastic tissue does not then have this effect, shows that reorganization BmKCT albumen is to by C
6The tumor group that cell brought out is woven with tangible special affinity interaction, in other words neuroglial cytoma (C6 cell) and transplanted tumor histocyte thereof have can with the special interacting proteins of rBmKCT (part).This special interaction of rBmKCT and neurospongioma just rBmKCT albumen as the basis of anti-glioma targeted drug.Fig. 7 shows in the tumor-bearing rat tumor tissues
131The content of I-rBmKCT in time prolongation and constantly increase (enrichment).
Embodiment 12: reorganization BmKCT protein drug is intervened the test of encephalic lotus knurl
The glioma model rat that successfully makes is divided into treatment group and control group, and will recombinate respectively BmKCT polypeptide (1mg/Kg) and isopyknic physiological saline inject in the rat brain.Check in intervening back the 1st, 2,3 week row MRI (MRI (magnetic resonance imaging) scanning), find that treatment group gross tumor volume rate of growth is slow than control group, the knurl body of its drug intervention experimental animal group obviously is less than the controlled trial animal groups, and the treatment group can extend to 30-35 days lifetime, and is all dead in the control group 28 days.The result shows that in rat brain, reorganization BmKCT albumen has the function of obvious anti-cranial glia oncocyte growth.Accompanying drawing 8 is to find by nuclear magnetic resonance image check, and the knurl body that reorganization BmKCT polypeptide drugs are intervened the experimental animal group obviously is less than the controlled trial animal groups.
Embodiment 13
Ampulla: anti-glioma peptide of scorpion 2mg
NaCl 9mg
Preparation method: anti-glioma peptide of scorpion and sodium-chlor are dissolved in 1 liter of injection water, filter gained solution, in the ampoule of under aseptic condition, packing into.
Embodiment 14
Nasal spray: anti-glioma peptide of scorpion 80mg
NaCl 8mg
EDTA 1mg
Borate buffer (pH6.5) 10mg
Spheron MD 30/70 10mg
Double distilled water is settled to 2ml
The preparation method: a kind of composition of each adding in the double distilled water of proper volume until dissolving fully, and then adds a kind of composition down.After being settled to 2ml, this solution is filtered on sterilizing filter, in the bottle of packing into and according to the packing of 1ml/ bottle.
Suppository: anti-glioma peptide of scorpion 10mg
Semi-synthetic fatty acid ester 1.5g
Double distilled water 10%
The preparation method: recombinant peptide after cholesterol absorption, with the semi-synthetic fatty acid ester 1.5g of fused below 70 ℃ mixing, is made suppository with an amount of dissolved in distilled water.
Embodiment 16
Eye drops: anti-glioma peptide of scorpion 4mg
EDTA 0.5mg
Sodium desoxycholate 1mg
Hyaluronic acid sodium 1mg
Nipagin A 0.03mg
Borate buffer solution (pH7.4) 5mg
Preparation method: dissolve anti-glioma peptide of scorpion with borate buffer, add dissolved EDTA and Sodium desoxycholate, constantly stir adding hyaluronic acid sodium and nipagin A down, fully mixing.
Embodiment 17
Liposomal formulation: anti-glioma peptide of scorpion 10mg
Yelkin TTS 240mg
Cholesterol 120mg
Double hexadecyl acid ester 18mg
The preparation method: with Z ether dissolving cholesterol, Yelkin TTS, and add double hexadecyl acid ester, add reorganization BmKCT albumen again, supersound process (strength of current is 0.1-0.5A) makes it to become stable w/o type emulsion.Remove Z ether at 20 ℃ of-25 ℃ of following reduction vaporizations.Add 5ml distilled water and continue the ether that reduction vaporization is removed remnants.Promptly make electronegative liposome turbid liquor.
SEQUENCE?LISTING
＜110〉Wuhan University
＜120〉a kind of anti-glioma peptide of scorpion and its production and application
＜130〉a kind of anti-glioma peptide of scorpion and its production and application
<160>4
<170>PatentIn?version?3.3
<210>1
<211>261
<212>DNA
<213>Buthus?martensii?Karsch
<400>1
gcaaaactct?attaaaaatg?aagttcctct?acggaatcgt?tttcattgca?ctttttctaa 60
ctgtaatgtt?cgcaactcaa?actgatggat?gtgggccttg?ctttacaacg?gatgctaata 120
tggcaaggaa?atgtagggaa?tgttgcggag?gtattggaaa?atgctttggc?ccacaatgtc 180
tgtgtaaccg?tatatgaata?attaaaaatg?tacacctgaa?cagatcattt?aatgaataat 240
aaatattaat?aagcattaaa?a 261
<210>2
<211>43
<212>DNA
＜213〉synthetic
<400>2
gccggatccc?cgatgacgat?gacaagtgtg?ggccttgctt?tac 43
<210>3
<211>33
<212>DNA
＜213〉synthetic
<400>3
gccctcgagt?catatacggt?tacacagaca?ttg 33
<210>4
<211>35
<212>PRT
<213>Escherichia?coli
<400>4
Cys?Gly?Pro?Cys?Phe?Thr?Thr?Asp?Ala?Asn?Met?Ala?Arg?Lys?Cys?Arg
1 5 10 15
Glu?Cys?Cys?Gly?Gly?Ile?Gly?Lys?Cys?Phe?Gly?Pro?Gln?Cys?Leu?Cys
20 25 30
Asn?Arg?Ile
35
Claims (2)
1, a kind of anti-glioma peptide of scorpion recombination bacillus coli is characterized in that: it is anti-glioma peptide of scorpion recombination bacillus coli Escherichia coli (DE3)/rBmKCT/pGEX-5X, and its deposit number is CCTCC No:M206015.
2, the application of a kind of anti-glioma peptide of scorpion in the medicine of preparation treatment or prevention anti-glioma, wherein the aminoacid sequence of anti-glioma peptide of scorpion is shown in SEQID NO.4.
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| CN101270158B (en) * | 2008-04-30 | 2010-12-22 | 武汉大学 | Target glioma resistant protein, preparation method and application |
| CN101270157B (en) * | 2008-04-30 | 2011-04-20 | 武汉大学 | Genetic engineering target glioma Acp-W1 resistant protein, preparation method and application |
| US8227439B2 (en) | 2008-05-15 | 2012-07-24 | Morphotek, Inc. | Treatment of metastatic tumors |
| KR101923235B1 (en) | 2010-02-04 | 2018-11-28 | 에이자이 아이엔씨. | Chlorotoxin polypeptides and conjugates and uses thereof |
| AU2011253424B2 (en) | 2010-05-11 | 2016-02-04 | Fred Hutchinson Cancer Center | Chlorotoxin variants, conjugates, and methods for their use |
| CA2913127A1 (en) | 2012-12-10 | 2014-06-19 | Fred Hutchinson Cancer Research Center | Methods for screening |
| US11559580B1 (en) | 2013-09-17 | 2023-01-24 | Blaze Bioscience, Inc. | Tissue-homing peptide conjugates and methods of use thereof |
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| . GenBank AF135821.;. GenPept AAG01185.;东亚钳蝎氯毒素BmKCT的表达和功能鉴定. 杨锐等.中国生物化学与分子生物学报，第21卷第1期. 2005 * |
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