CN100383250C - Method of expressing bird catching arachinid toxin I and its special carrier - Google Patents
Method of expressing bird catching arachinid toxin I and its special carrier Download PDFInfo
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- CN100383250C CN100383250C CNB2004100966583A CN200410096658A CN100383250C CN 100383250 C CN100383250 C CN 100383250C CN B2004100966583 A CNB2004100966583 A CN B2004100966583A CN 200410096658 A CN200410096658 A CN 200410096658A CN 100383250 C CN100383250 C CN 100383250C
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Abstract
The present invention discloses a method of expressing tiger line fowling spider toxin-I and a special carrier thereof. The special carrier of expressing the tiger line fowling spider toxin-I is a recombinant baculovirus expression carrier containing a tiger line fowling spider toxin-I gene. The 5' end of the tiger line fowling spider toxin-I gene is connected with a histidine label coding sequence. The tiger line fowling spider toxin-I gene with the histidine label coding sequence is provided with a nucleotide sequence of a sequence 1 in a sequence table. In the method of expressing the tiger line fowling spider toxin-I, the special expression carrier can be converted to colibacillus containing baculovirus plasmids, and the recombinant baculovirus can be obtained. The recombinant baculovirus can transfect insect cells, and the tiger line fowling spider toxin-I can be expressed. The present invention has the advantages of high protein expression quantity (900 ng/ml) and high bioactivity, and has significant industrial application prospect and actual significance.
Description
Technical field
The present invention relates to a kind of method and dedicated carrier thereof of expressing bird catching arachinid toxin I.
Background technology
Bird catching arachinid toxin I (HWTX-I) is a kind of polypeptide class neurotoxin that acts on presynaptic membrane of separation and purification from the thick poison of China's rare tarantula kind Ornithotoctonus huwena, its relative molecular mass is 3.75kD, be made up of 33 amino-acid residues, wherein 6 cysteine residues are connected to form 3 disulfide linkage with 1-4,2-5,3-6 mode.It is a kind of neurotoxin that acts on presynaptic membrane, and is a kind of N type Ca
2+The blocker of ionic channel, thereby be a kind of excellent tools reagent of studying ionic channel and acceptor interaction.Find after deliberation that recently this neurotoxin also has tangible analgesia effect, compares with morphine, its habituation is low and do not have other toxic side effect, may be developed to a kind of novel analgesic, has potential clinical value and good prospects for application.But only depend on natural Ornithotoctonus huwena resource can't satisfy needs to the research and development and the clinical application thereof of this neurotoxin.
It is reported, the chemosynthesis of human fluorenylmethyloxycarbonyl solid phase method had once been arranged HWTX-I, but the cost of this synthetic method is higher, and resultant quantity is limited, can't apply (Liang Songping, Xia Zanxian, Xie Jinyun, the chemosynthesis of bird catching arachinid toxin I and structure thereof and physiologically active analysis.Chinese science (C collects), 1997,27 (5): 391-397).Also there is the people in intestinal bacteria, HWTX-I to be carried out prokaryotic expression, but 3 disulfide linkage are arranged because of HWTX-I has, cause the renaturation of this prokaryotic expression product difficult, and the lipopolysaccharides in the Bacillus coli cells wall is that strong intracellular toxin is former, thereby also be unfavorable for large-scale development and use (Li Min, Li Luyi, Liang Songping, the clone and the expression of chemosynthesis huwentoxin I gene with this expression system.Life science, 1998,2 (3): 229-232; Diao Jianbo, Liu Junliang, Liang Songping, the reorganization bird catching arachinid toxin I expression and the purifying of pET31b carrier.Chinese biological chemistry and molecular biosciences journal, 2003,19 (2): 195-200).The somebody has expressed HWTX-I in pichia pastoris, though expression amount is higher, but its biologic activity of the purified back of the recombinant protein of expressing only is about 60% (Nie Dongsong, Li Min, Xu Huiming etc., expression and the purifying of reorganization bird catching arachinid toxin I in pichia pastoris of natural protein.The biotechnology journal, 2002,18 (2): 172-177).Aforesaid method all fails to realize efficiently expressing activated HWTX-I.
The rhabdovirus expression vector system is a kind of eukaryotic cell expression efficiently system that grows up the eighties.With this expression system expressing protein, expression amount height not only, and it is more perfect to the post-treatment of expression product, insect cell is approaching to proteinic post-treatment system and mammalian cell, can make expression product have the characteristics of glycosylation, phosphorylation, lipaseization, amidation, cleavable signal peptide and three grades of formation or level Four higher structure, therefore the foreign protein of expressing has higher biological activity near native protein.
Summary of the invention
The dedicated expression vector therefor that the purpose of this invention is to provide a kind of bird catching arachinid toxin I.
The dedicated carrier of expression bird catching arachinid toxin I provided by the present invention is the recombination rhabdovirus expression vector that contains the bird catching arachinid toxin I gene.
For the ease of the purifying of bird catching arachinid toxin I, 5 of described bird catching arachinid toxin I gene ' end is connected with histidine-tagged (His-tag) encoding sequence.
Described bird catching arachinid toxin I gene has the nucleotide sequence of sequence 1 in the sequence table for histidine-tagged encoding sequence (wherein histidine-tagged encoding sequence is from 5 of sequence 1 ' end the 4th bit base-the 21st bit base).
Wherein, the carrier that sets out that is used for making up described dedicated expression vector therefor can be the rhabdovirus expression vector in genetically engineered field, as pFastBac1, pFastBacHTA, pFastBacHTB, pFastBaC or pFastBac Dual; Be preferably pFastBac1.
Described bird catching arachinid toxin I gene can insert between the BamHI at rhabdovirus expression vector pFastBac1 multiple clone site place and the EcoRI restriction enzyme enzyme recognition site and obtain recombination rhabdovirus expression vector pFastBac1-HWTX.
The dedicated expression vector therefor of above-mentioned bird catching arachinid toxin I all can make up according to a conventional method.
The engineering bacteria, the host cell that contain carrier of the present invention also belong to protection scope of the present invention.
Second purpose of the present invention provides a kind of method of expressing bird catching arachinid toxin I.
The method of expression bird catching arachinid toxin I provided by the present invention, be that above-mentioned dedicated expression vector therefor is transformed the intestinal bacteria that contain the baculovirus plasmid, obtain recombinant baculovirus,, express bird catching arachinid toxin I this recombinant baculovirus transfection insect cell.
Expressing the bird catching arachinid toxin I that obtains in the described method adopts nickel ion affinity chromatograph post method to carry out purifying.
The described intestinal bacteria that contain baculovirus plasmid (Bacmid) are intestinal bacteria DH10Bac;
The method of described transfection insect cell can be liposome vectors method, calcium phosphate transfection method, electroporation transfection method, gene microinjection or DEAE-dextran infection protocol; Be preferably the liposome vectors method.
Described insect cell can be Sf9 cell, Sf21 cell or Tn-5B1-4 cell, is preferably the Sf9 cell.The optimum condition of liposome vectors method transfection is: cell density is 1-2 * 10
6Individual cell/mL, infection multiplicity is 5, infection time is 72 hours.
Use the present invention and carry out the expression of catching bird spider toxin I, not only proteic expression amount height (900ng/mL), and has a higher biological activity, remedied the deficiency of existing bird catching arachinid toxin I preparation method and prokaryotic expression system phraseology, in addition, if apply the present invention to the production of industrialization bird catching arachinid toxin I, then has raw material biology growing cycle weak point, industrial scale is big, the advantage that extraction cost is low, can be used for researchs such as pharmacological effect and toxicity after expression product is purified, have important prospects for commercial application and practical significance.
Description of drawings
Fig. 1 is the structure schema of recombinant expression vector pFastBac1-HWTX
Fig. 2 is the expression schema of HWTX-I
Fig. 3 is the Western Blot analytical results of expressing protein
Fig. 4 a is the optimum result of best infection time
Fig. 4 b is the optimum result of best infection multiplicity
Fig. 5 is the purifying schema of HWTX-I expressing protein
Fig. 6 is that the biologic activity of HWTX-I expressing protein detects
Embodiment
Used experimental technique is ordinary method if no special instructions among the following embodiment.
1, the clone of HWTX-I gene
Known amino acid sequence and the suitable restriction enzyme site of carrier pFastBac1 according to HWTX-I design a pair of primer, primer sequence is following (to contain histidine-tagged sequence in this primer sequence, the 14th bit base-the 31st bit base from primer 1 sequence), so that expressing protein is carried out purifying with embodiment 3 described methods:
Primer 1:5 '-GGATCCCCATATGCACCACCACCACCACCACTCCATG-3 '
Primer 2: 5 '-GAATTCACAGCTTCCACTTGCACCACTTGTGCTTGTCG-3 '
Genomic dna with Ornithotoctonus huwena is a template, under the guiding of primer 1 and primer 2, and pcr amplification HWTX-I gene, and add BamHI and EcoRI restriction enzyme enzyme recognition site respectively at its 5 ' and 3 ' end.Wherein, reaction system is: deionized water 39 μ l, dNTP mixture 2 μ l (final concentration is 0.8mM), upstream primer 1 μ l (final concentration is 1 μ M), downstream primer 1 μ l (final concentration is 1 μ M), template 1 μ l (1 μ g genomic dna), 10 * Taq enzyme reaction buffer solution, 5 μ l and Taq enzyme 1 μ l (1U) (Shanghai Shen You biotech company), and reaction conditions is: pre-sex change 3min under 94 ℃; 94 ℃ of following sex change 30s then, 67 ℃ of annealing 45s down, 72 ℃ are extended 2.5min down, circulate 35 times; 72 ℃ are fully extended 5min down.Amplified production carries out 2.0% agarose gel electrophoresis, and the result obtains the band about a treaty 150bp.Amplified band is cloned into the pGEM-T carrier after reclaiming, carry out gene sequencing, and the result shows that HWTX-I gene that amplification obtains has the nucleotide sequence of SEQ ID NO:1 in the sequence table.
2, carrier pFastBac1 (available from GIBCO/BRL company) is reclaimed with the low melting-point agarose gel after with BamHI and EcoRI double digestion obtain electrophoretically pure klenow fragment and cut product, use T then
4Dna ligase is cut the enzyme that reclaims product and is connected through the HWTX-I of same enzyme double digestion dna fragmentation, to connect product transformed into escherichia coli DH5 α competent cell, obtain positive recombinant through screening, to positive recombinant plasmid check order (Beijing three rich polygala root biotechnology limited liability companys), sequencing result shows clone's HWTX-I gene order correct (nucleotide sequence with sequence 1 in the sequence table), and correctly be inserted between the BamHI and EcoRI restriction enzyme site of carrier pFastBac1 the rhabdovirus expression vector called after pFastBac1-HWTX that contains the HWTX-I gene that this order-checking is correct.
The expression of embodiment 2, HWTX-I
The expression process of HWTX-I may further comprise the steps as shown in Figure 2:
1, the acquisition of recombinant baculovirus
The rhabdovirus expression vector pFastBac1-HWTX that embodiment 1 is made up transforms intestinal bacteria DH10Bac (available from the GIBCO/BRL company) competent cell that contains baculovirus plasmid (Bacmid), with blue hickie method screening reorganization Bacmid, the Bacmid that transposition takes place is because the insertion inactivation of LacZ gene, make the colony growth that contains the Bacmid that recombinates be white, the colony growth that does not contain the Bacmid that recombinates is for blue, the single colony inoculation of picking white is to the selection culture plate that contains X-gal and IPTG, carry out the white screening of indigo plant once more, the single bacterium colony of last picking white carries out liquid culture, extract the recombinant baculovirus plasmid and be used for transfection meadow three-spotted phytometra cell (Spodopterafrugiperda 9, the Sf9 cell) (American I nvitrogen company) to express HWTX-I.
2, recombinant baculovirus transfection Sf9 cell
Adopt the liposome vectors method to carry out transfection, concrete grammar is:
1) cultivation of Sf9 cell
The Sf9 cell (American I nvitrogen company) that will be in logarithmic phase is 9 * 10 by every hole access 1mL cell density
5The cell quantity of individual cell/mL is inoculated in Grace ' the s insect substratum that contains 10% foetal calf serum and (contains hydrolysis milk-protein, yeast extract (American I nvitrogen company) and 25 μ g/mL gentamicins, Sigma) on six orifice plates, cultivated 1 hour, and made cell attachment for 27 ℃.
2) in step 1), contain and add the recombinant baculovirus plasmid that 1 μ g (every hole) step 1 obtains in six orifice plates of adherent Sf9 cell, and at lipofectamine (Cellfectin Reagent, American I nvitrogen company) under the effect, cultivated 5 hours for 27 ℃ earlier, Grace ' the s insect substratum that more renews then continues to cultivate 72 hours.After cultivating end, collect supernatant, promptly obtain recombinant baculovirus (P1), in the Sf9 cell, obtain (P2, P3 etc.) after the amplification, measure its titre with the terminal point dilution method.
3) with the recombinate shape virus infection Sf9 cell of suitable titre, collecting cell then, after PBS washing 1 time, the lysate that is suspended from 1/20 volume of culture (contains 10mmol/L Tris-HCl pH7.4,150mmol/L NaCl), puts in the ice bath ultrasonication cell, obtain containing the lysis supernatant of total protein then through high speed centrifugation, after the packing-80 ℃ frozen standby.
3, the immunoblotting assay of expressing protein
The lysis supernatant that contains total protein that step 2 is extracted carries out polyacrylamide gel electrophoresis earlier, (with natural HWTX-I is antigen with anti-HWTX-I rabbit polyclonal antibody again, preparation according to a conventional method) be one anti-, with goat anti-rabbit igg (Promega company) is two anti-, detect the HWTX-I expression with immunoblotting (Western Blot), (swimming lane 1 is for infecting the Sf9 lysis supernatant liquor of recombinant baculovirus as shown in Figure 3 for the result, swimming lane 2 is not for infecting the Sf9 lysis supernatant liquor (negative control) of recombinant baculovirus, swimming lane 3 is for infecting the Sf9 cells and supernatant of recombinant baculovirus, swimming lane 4 is natural HWTX-I (positive control), one positive band appears in swimming lane 1 at the 4kD place, consistent with the molecular weight size of natural HWTX-I, show to have obtained to express correct HWTX-I.
4, the mensuration of HWTX-I expression amount
Adopt enzyme-linked immunosorbent assay technology (ELISA) to measure the HWTX-I expression amount.Concrete grammar may further comprise the steps: 1) the natural HWTX-I of 100 μ L (1 μ g/ml is dissolved in pH 9.6 carbonate buffer solutions) is added in the enzyme plate hole, 96 hole, hatched 48 hours for 4 ℃; 2) remove liquid in the hole, seal, 37 ℃ of incubations 2 hours with 5% skim-milk; 3) (with natural HWTX-I is antigen to prepare antigen one anti-HWTX-I rabbit polyclonal antibody in centrifuge tube, preparation according to a conventional method) solution, wherein the concentration of antibody is 2nM, and the concentration of antigen (natural HWTX-I) is respectively 10mM, 20mM, 40mM, 80mM, 160mM, 320mM, 640mM; The lysis supernatant that contains expressing protein that obtains of step 2 simultaneously behind 1: 2 serial dilution of PBST, also mixes with the antibody of 2nM.Above solution room temperature was placed 3 hours; 4) every hole adds the above-mentioned solution of 100 μ l respectively, 37 ℃ of incubations 2 hours; 3) every hole adds the goat anti-rabbit igg (Promega company) of 100 μ L horseradish peroxidase-labeled, room temperature incubation 40 minutes; 4) every hole adds 100 μ L TMB/H
2O
2Develop the color after 15 minutes, use 1N H
2SO
4Termination reaction is measured the A450nm value with microplate reader at last.The result shows that the expression amount of HWTX-I is a 900ng/mL cracking supernatant, has obtained higher expression.
5, the optimization of transfection conditions
Recombinant Protein Expression level and Sf9 cell density, virus infection plural number are relevant with the transfection time, determine its optimum parameter according to the expression amount of HWTX-I.
1) determines Sf9 cell optimum density
The recombinant baculovirus that step 2 is obtained is 5 in infection multiplicity, and infection time is that (concrete grammar is as follows: calculating infection multiplicity according to virus titer and cell quantity was 5 o'clock, and needed viral volume adds cell culture medium in 72 hours.Determining of infection time mainly is to begin to occur taking off infection symptoms in late period such as wall according to cell, and this experiment is defined as 72 hours) condition under infect the following Sf9 cell (American I nvitrogen company) of cell density that 1mL is in logarithmic phase: 1 * 10
5Individual cell/mL, 5 * 10
5Individual cell/mL, 9 * 10
5Individual cell/mL, 1 * 10
6Individual cell/mL, 2 * 10
6Individual cell/mL, 5 * 10
6Individual cell/mL, 9 * 10
5Individual cell/mL, 1 * 10
7Individual cell/mL.Measure the expression amount of HWTX-I with ELISA.The result shows that in infection multiplicity be 5, and infection time is that host cell density is 1-2 * 10 under 72 hours the condition
6The expression amount of the HWTX-I of individual cell/mL the highest (15 μ g/mg total protein).
2) determine best infection multiplicity
The recombinant baculovirus that step 2 is obtained is 1-2 * 10 at cell density
6Individual cell/mL, infection time are the Sf9 cell (American I nvitrogen company) that is in logarithmic phase under 72 hours the condition with the virus infection 1mL of different infection multiplicities, and be as follows: 1,2,5 and 10.The result shows that in host cell density be 1-2 * 10 shown in Fig. 4 b
6Individual cell/mL, infection time are under 72 hours the condition, and infection multiplicity is the expression amount the highest (15 μ g/mg total protein) of 5 HWTX-I.
3) determine best infection time
The recombinant baculovirus that step 2 is obtained is 1-2 * 10 at cell density
6Individual cell/mL, infection multiplicity is that next different time of 5 condition infects the Sf9 cell (American I nvitrogen company) that 1mL is in logarithmic phase, and is as follows: 24 hours, 48 hours, 72 hours, 96 hours and 120 hours.The result shows that in host cell density be 1-2 * 10 shown in Fig. 4 a
6Individual cell/mL, infection multiplicity is that infection time is the expression amount the highest (15 μ g/mg total protein) of 72 hours HWTX-I under 5 the condition.
The purifying of embodiment 3, HWTX-I expressing protein and the detection of biologic activity thereof
One, the purifying of HWTX-I expressing protein
Adopt and solidify Ni
2+Affinity column method purification of Recombinant HWTX-I, the purifying flow process as shown in Figure 5, concrete grammar is: after the cell pyrolysis liquid that contains the HWTX-I expressing protein that embodiment 2 is obtained filters, add Ni is housed
2+The chromatography column of resin (Ni-NTA agarose, 30210, Qiagen company), 4 ℃ of joltings are after 2 hours, with lavation buffer solution (50mMNaH
2PO
4, 300mM NaCl, 20mM imidazoles, with NaOH adjust pH to 8.0) wash post, until the A that flows through liquid
2SO＜0.01, use elution buffer (50mMNaH again
2PO
4, 300mM NaCl, 250mM imidazoles, with NaOH adjust pH to 8.0) albumen of elution of bound, every part of 1mL collects step by step, remove histidine-tagged with CNBr at last, obtain purified HWTX-I expressing protein, The sequencing results shows that the amino acid residue sequence of this expressing protein is consistent with the amino acid residue sequence of natural HWTX-I.
Two, the biologic activity of HWTX-I expressing protein detects
It is 1 * 10 that the HWTX-I expressing protein that step 1 is purified is diluted to concentration with tyrode's solution
-5The solution of g/mL soaks mouse diaphram-phrenic nerve isolated preparation with this diluted protein solution again, gives electricity irritation then, writes down its blocking-up time simultaneously.The result shows that the HWTX-I expressing protein has the effect of blocking-up isolated mouse phrenic nerve identical with natural HWTX-I and the transmission of diaphram joint as shown in Figure 6, and biological activity is identical with natural HWTX-I.
Sequence table
<160>1
<210>1
<211>129
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
atgcaccacc?accaccacca?ctccatggcc?tgcaagggtg?tgttcgacgc?ctgcaccccc 60
ggtaagaacg?aatgctgccc?caaccgggtg?tgctccgaca?agcacaagtg?gtgcaagtgg 120
aagctgtga 129
Claims (13)
1. the dedicated expression vector therefor of a bird catching arachinid toxin I is the recombination rhabdovirus expression vector that contains the bird catching arachinid toxin I gene.
2. dedicated expression vector therefor according to claim 1 is characterized in that: 5 of described bird catching arachinid toxin I gene ' end is connected with histidine-tagged encoding sequence; The nucleotide sequence of the described bird catching arachinid toxin I gene that has a histidine-tagged encoding sequence is shown in sequence in the sequence table 1.
3. dedicated expression vector therefor according to claim 1 and 2 is characterized in that: the carrier that sets out that is used to make up described dedicated expression vector therefor is pFastBac1, pFastBacHTA, pFastBacHTB, pFastBaC or pFastBacDual.
4. dedicated expression vector therefor according to claim 3 is characterized in that: the described carrier that sets out is pFastBacl, and described dedicated expression vector therefor is pFastBacl-HWTX as shown in Figure 1.
5. the engineering bacteria and the host cell that contain claim 1 or 2 or 3 or 4 described dedicated expression vector therefors.
6. the expression method of a bird catching arachinid toxin I, be that claim 1 or 2 or 3 or 4 described dedicated expression vector therefors are transformed the intestinal bacteria that contain the baculovirus plasmid, obtain recombinant baculovirus,, express bird catching arachinid toxin I this recombinant baculovirus transfection insect cell.
7. expression method according to claim 6 is characterized in that: express the bird catching arachinid toxin I that obtains in the described method and adopt nickel ion affinity chromatograph post method to carry out purifying.
8. according to claim 6 or 7 described expression methods, it is characterized in that: the described intestinal bacteria that contain the baculovirus plasmid are intestinal bacteria DH10Bac.
9. according to claim 6 or 7 described expression methods, it is characterized in that: the method for described transfection insect cell is liposome vectors method, calcium phosphate transfection method, electroporation transfection method, gene microinjection or DEAE-dextran infection protocol.
10. expression method according to claim 9 is characterized in that: the method for described transfection insect cell is the liposome vectors method.
11. according to claim 6 or 7 described expression methods, it is characterized in that: described insect cell is Sf9 cell, Sf21 cell or Tn-5B1-4 cell;
12. expression method according to claim 11 is characterized in that: described insect cell is the Sf9 cell.
13. expression method according to claim 10 is characterized in that: the condition of described liposome vectors method transfection is: cell density is 1-2 * 10
6Individual cell/mL, infection multiplicity is 5, infection time is 72 hours.
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| CN1079475A (en) * | 1993-05-10 | 1993-12-15 | 湖南师范大学 | The extraction of toxin of tiger veins bird-catching spider and application |
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| CN1079475A (en) * | 1993-05-10 | 1993-12-15 | 湖南师范大学 | The extraction of toxin of tiger veins bird-catching spider and application |
Non-Patent Citations (2)
| Title |
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| 重组虎纹捕鸟蛛毒素_Ⅰ在巴氏毕赤酵母中的表达及纯化. 聂东宋等人.生物工程学报，第18卷第2期. 2002 * |
| 重组虎纹捕鸟蛛毒素Ⅰ用pET31 b 载体的表达和纯化. 刁建波等人.中国生物化学与分子生物学报，第19卷第2期. 2003 * |
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