CN101434964B - Cholera toxin B subunit and lumbrokinase fusion gene, as well as recombinant vector and host cell including the same - Google Patents
Cholera toxin B subunit and lumbrokinase fusion gene, as well as recombinant vector and host cell including the same Download PDFInfo
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Abstract
The invention discloses a cholera toxin B subunit and a lumbrokinase fusion gene, a recombinant carrier and a host cell that contain the gene. The cholera toxin B subunit and lumbrokinase fusion gene is a nucleotide sequence shown in SEQ ID NO.10 of the sequence table. The invention has the advantages that the CTB and LK fusion gene has high-efficiency expression of CTB and LK fusion protein through pichia GS115, has higher expression system yield, stable pichia expression and ultra-wide application, the purified fusion protein has biological activity under inspection and can be used for preparing fusion protein medicaments with remarkable thrombus curing effect, and CTB can be used as an adjuvant for remarkably improving the orally taking absorbing and utilizing rate of LK and greatly raising the drug action of LK.
Description
Technical field
The present invention relates to the bioengineering field gene recombination technology, be specifically related to the construction process of a kind of fusion gene and genetic engineering bacterium.
Background technology
(Lumbrokinase, LK), (Earthworm plasminogen activator EPA), is a kind of proteolytic ferment with fibrinolytic that extracts in the earthworm body to Lumbrukinase to be called earthworm fibrinolysin again.It is distributed widely in the digestive tube inner chamber of earthworm, and Fibrinogen and plasminogen activation are plasmin in the blood of directly degrading.Studies show that Lumbrukinase no matter vein or oral administration in a large number, all have thrombolytic effect.Lumbrukinase is direct thrombolysis both, can activate thrombolysis again; And can reduce platelet aggregation rate (it is hyperfunction to suppress endogenous coagulation function), promote vascular endothelial cell to produce t-PA[Iannucci NB, Camperi SA, Cascone O, Purification oflumbrokinase from Eisenia fetida using aqueous two-phase systems and anion-exchange chromatography, Separation and Purification Technology, 2008,64 (1): 131-134], and inhibition PAI generates (strengthening endogenous fibrinolytic), the blood vessel function that contracts (diastole unstriated muscle, alleviating vascular spasm) that suppresses endothelin (ET), blood viscosity lowering (promotion microcirculation).As thrombolytic drug, Lumbrukinase successively in Korea S and China's listing, demonstrates tangible curative effect in cerebrovascular treatment of thrombotic disorders.
Toxins,exo-, cholera (CT) is that the molecular weight that is produced by vibrio cholerae is the enterotoxin of 84kD, covalently bound the forming of pentamer that is formed by 1 A subunit (CTA) and 5 B subunits (CTB).CTA is a toxicity subunit, and CTB is nontoxic receptors bind subunit, can with Sphingolipids,sialo (GM1) receptors bind on all karyocyte films.The effect of A subunit by B subunit enters cell and brings into play its toxic action [Sprander BD Structure and function of cholera toxin and relatedEscherichia coil heat lable enterotoxin[J] Microl, Rew.1992,56:622-643].CTB has stronger immunological adjuvant activity, particularly when its when chemical process or gene fusion technology make itself and uncorrelated antigen form coupling protein or fusion rotein, the adjuvanticity when its immunological adjuvant activity is better than itself and uncorrelated antigen mixed immunity.Since CTB can with Sphingolipids,sialo (GM1) receptors bind on all karyocyte films, based on this characteristic, discover that CTB not only can be used for immunological adjuvant, can also be delivery carrier as effective medicine.The permeability of medicine in enteron aisle becomes pharmaceutical protein and passes the major obstacle that intestinal epithelial cells enters blood circulation.Yet acceptor connects lead oral and is delivery system and provides feasible approach for the absorption of pharmaceutical protein.In order to verify whether CTB can become the effective protein proteins medicine and be delivery carrier, people such as Arati are CTB and GFP protein gene, and have realized expression in tobacco chloroplast.With this feeding mouse that grows tobacco, and made detailed physiological and biochemical analysis [Arati Limaye to taking this mouse that grows tobacco, Vijay Koya, 1 Mohtashem Samsam, and HenryDaniell.Receptor-mediated oral delivery of a bioencapsulated green fluorescent protein expressedin transgenic chloroplasts into the mouse circulatory system.The FASEB Journa l2006,20:37-46.].In this research, they find that the CTB-GFP fusion rotein can be absorbed by intestinal epithelial cell and gut associated lymphoid tissue.The pentamer fusion rotein can be attached on the GM1 acceptor of surface of cell membrane, and enters phagosome by endocytosis.This subsequently fusion rotein enters endoplasmic reticulum, and herein, the Furin enzyme that extensively exists in the born of the same parents cuts fusion rotein, and CTB and GFP albumen separate, and CTB is brought to substrate one side of cell, still with the GM1 receptors bind.The GFP molecule then is excluded the extracellular by golgi body, thereby enters lymphokinesis, further enters blood circulation.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of CTB and LK fusion gene are provided.
Second purpose of the present invention provides the yeast expression vector pPIC9k-CTB-Furin-LK that a kind of structure contains CTB and LK fusion gene.
The 3rd purpose of the present invention provides a kind of CTB of containing and LK fusion gene engineering bacterium.
The 4th purpose of the present invention provides a kind of CTB and LK fusion gene encoded protein.
The 5th purpose of the present invention provides CTB and the application of LK fusion gene encoded protein in the oral thrombolytic drug of preparation.
Technical scheme of the present invention is summarized as follows:
A kind of choleratoxin B subunit and Lumbrukinase fusion gene, by the nucleotide sequence shown in the SEQ ID NO.10 in the sequence table, described choleratoxin B subunit is abbreviated as CTB, and described Lumbrukinase is abbreviated as LK.
A kind of recombinant vectors pPIC9k-CTB-Furin-LK that contains CTB and LK fusion gene is formed by following step structure:
(1) make up the intermediate carrier pBluescript SK-CTB-Furin-LK that contains CTB and LK fusion gene:
1. the CTB gene shown in the SEQ ID NO.1 is connected with total synthesis method with the flexible peptide catenation sequence gene shown in the SEQ ID NO.3, places carrier pBluescript SK-CTB;
2. the LK gene shown in the SEQ ID NO.2 is synthesized with total synthesis method and place carrier pUCm-T-LK;
3. designing by the upstream primer P1 shown in the SEQ ID NO.5 with by the downstream primer P2 shown in the SEQ ID NO.6, is template with the pUCm-T-LK plasmid, carries out pcr amplification, obtains the Furin-LK fusion gene, and described Furin gene order is shown in SEQ ID NO.4;
4. with the Furin-LK fusion gene through EcoRI and HindIII double digestion, with carrier pBluescript SK-CTB through EcoRI and HindIII double digestion, the two carries out ligation, obtains containing the intermediate carrier pBluescript SK-CTB-Furin-LK of Furin gene shown in the flexible peptide catenation sequence gene shown in the CTB gene shown in the SEQ ID NO.1, the SEQ ID NO.3, the SEQ ID NO.4 and the LK gene shown in the SEQ ID NO.2;
(2) construction of expression vector pPIC9k-CTB-Furin-LK:
Design is by the upstream primer P3 shown in the SEQ ID NO.7, with by the downstream primer P4 shown in the SEQ ID NO.8, with pBluescriptSK-CTB-Furin-LK is template, carry out pcr amplification, pcr amplification product is cut through the NotI enzyme, yeast expression vector pPIC9k is cut through the NotI enzyme, and the two carries out ligation, obtains containing the recombinant vectors pPIC9k-CTB-Furin-LK of CTB and LK fusion gene.
A kind ofly contain CTB and LK fusion gene engineering bacterium, will be integrated on the genome of pichia spp GS115 after the pPIC9k-CTB-Furin-LK linearizing.
A kind of by CTB and LK fusion gene encoded protein, be aminoacid sequence shown in the SEQ ID NO.9 in the sequence table.
The application in the oral thrombolytic drug of preparation of a kind of CTB and LK fusion gene encoded protein.
The invention has the advantages that by CTB and LK and merge the fusion gene that forms, can efficiently express CTB and LK fusion rotein by pichia spp GS115, the productive rate of its expression system is higher, Pichia anomala expression is stable, use very extensive, purified fusion rotein has biologic activity through check, can prepare becomes the fusion rotein medicine, the fusion rotein medicine has obvious effect to the treatment thrombus, simultaneously CTB can obviously improve the oral absorption utilization ratio of LK, the drug action that has improved LK greatly as adjuvant.
Description of drawings
Fig. 1 pPIC9k-CTB-Furin-LK enzyme is cut and PCR checking result.
Fig. 2 pichia spp bacterium colony PCR product electrophoresis result.
Fig. 3 crude extract SDS-PAGE protein electrophorese result of fermenting.
Fig. 4 mouse tail thrombus linear measure result.
Embodiment
The structure of intermediate carrier pBluescript SK-CTB-Furin-LK
At first, the CTB gene shown in the SEQ ID NO.1 is connected with total synthesis method with the flexible peptide catenation sequence gene shown in the SEQ ID NO.3, places carrier pBluescript SK-CTB; With the LK gene shown in the SEQ ID NO.2 with the synthetic carrier pUCm-T-LK that places of total synthesis method.Secondly, be masterplate with pUCm-T-LK, P1 and P2 are respectively upstream and downstream primer amplification LK fragment, and its reaction conditions is 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 90s circulates 30 times.In P1, introduce EcoRI restriction enzyme site (GAATTC), in P2, introduce HindIII restriction enzyme site (AAGCTT).Then, PCR product and pBluescript SK-CTB plasmid are cut product with the two enzyme and are connected: 16 ℃ respectively through EcoRI and HindIII double digestion, connection (2 μ l, 10 * T4 buffer, 0.5 μ l T4 dna ligase, 5 μ l carrier DNAs spend the night, 7.5 μ l foreign DNA, 5 μ lddH
2O).Connect product Transformed E-Coli.DH5 α, coat the LB flat board that contains the ammonia benzyl.Be that primer carries out PCR and obtains the 850bp product with the goal gene, enzyme is cut and is identified and obtain the purpose band, and last sequence verification result shows that vector construction is correct.
Expression vector pPIC9k-CTB-LK building process
At first, be masterplate with pBluescript SK-CTB-Furin-LK, P3 and P4 are respectively upstream and downstream primer amplification CTB-Furin-LK fragment, and its reaction conditions is 94 ℃, 30s, 54 ℃, 30s, 72 ℃, 120s circulates 30 times.In P3, introduce NotI restriction enzyme site (GCGGCCGC), in P4, introduce NotI restriction enzyme site (GCGGCCGC).Then, PCR product and pPIC9k plasmid are cut product with the two enzyme and are connected respectively through the NotI single endonuclease digestion: 16 ℃, and the connection of spending the night (2 μ l, 10 * T4buffer, 0.5 μ l T4 dna ligase, 5 μ l carrier DNAs, 7.5 μ l foreign DNAs, 5 μ l ddH
2O).Connect product Transformed E-Coli.DH5 α, coat the LB flat board that contains the ammonia benzyl.Be that primer carries out PCR and obtains 1200bp left and right sides product with the goal gene, enzyme is cut and is identified and obtain the purpose band, and last sequence verification result shows that vector construction is correct.See Fig. 1, among Fig. 1, M is Marker DNA DL10000, and the right side electrophorogram is that each band of Marker is represented the clip size synoptic diagram; 1 carries out single endonuclease digestion for carrier uses NotI, produces about 1200bp and 9000bp left and right sides segment, has illustrated that the disconnection of CTB-LK big or small slice receives on the pPIC9k carrier; 2 for using EcoRI that carrier is carried out single endonuclease digestion, produces about 500bp and 9000bp left and right sides segment, illustrates that the target segment has been connected on the pPIC9k carrier by forward; 3 is pcr amplification CTB-LK product, has obtained the purpose band about 1200bp very clearly, further specifies goal gene CTB-LK and has been connected on the pPIC9k carrier.
The conversion of recombination microzyme, screening and evaluation
The recombinant plasmid pPIC9k-CTB-Furin-LK that structure is correct changes among the GS115 with electric shocking method after using the SacI linearizing, coats on the MD flat board, cultivate after 2-3 days, for 30 ℃ with containing different concns (0,0.50,1.00,1.50,2.00,2.5 and 3.00mg/mL) transformant of G418 YPD plate screening high copy number, extract the transformant genome, be primer PCR with the goal gene after nucleic acid electrophoresis is identified, obtain expecting showing that goal gene is incorporated on the yeast chromosomal by the 1200bp band.As Fig. 2,10 His of picking
+(by the MD plate screening) and 3.00mg/mL G418 resistance clone carry out bacterium colony PCR, the positive clone of 1,3,4,5,7,9 and 10 swimming lanes, and 2,6 and 8 swimming lanes do not have obvious band.M is Marker III, and the right side electrophorogram is that each band of Marker is represented the clip size synoptic diagram.
The abduction delivering of recombinant protein
The recombination microzyme transformant that screening is obtained is inoculated in the BMGY substratum, be cultured to OD value about 6, centrifugal (4000r/min, 5min), thalline is forwarded among the inducing culture BMMY expresses, every 24h sampling and add methyl alcohol to 1%, cultivate after 5 days, it is centrifugal that (4000r/min 5min), collects culture supernatant, the nutrient solution supernatant of getting different amounts carries out the SDS-PAGE electrophoresis result and shows, goal gene obtains to efficiently express, and obtains the target protein band, and sequence is aminoacid sequence shown in sequence table SEQ ID NO.9.See Fig. 3, Fig. 3 is fermentation crude extract SDS-PAGE protein electrophorese result, and swimming lane M is albumen Marker, and the right side electrophorogram is that each band of Marker is represented the clip size synoptic diagram; Swimming lane 1 is extract SDS-PAGE electrophorogram in the supernatant liquor of expressing LK; Swimming lane 2 is for expressing extract SDS-PAGE electrophorogram in CTB and the LK fusion gene encoded protein supernatant liquor; Swimming lane 3 is a blank.As can be seen from Figure 3 the LK molecular weight is about 30kD, and CTB and LK fusion gene encoded protein are about 45kD, and molecular weight conforms to expection.
Proteic extraction in the fermented supernatant fluid
1. the pichia spp supernatant liquor is concentrated:
(1) a large amount of fermented supernatant fluid at first carries out the suction filtration of suction filter pump, places three layers of qualitative filter paper on the suction funnel, removes a large amount of yeast cell and the impurity in the fermented liquid;
(2) fermented supernatant fluid that obtains of suction filtration is further removed the impurity in the fermented liquid after the filter membrane of 0.22 μ m;
(3) it is concentrated that clarifying fermented supernatant fluid carries out ammonium sulfate, protein precipitation, in fermented supernatant fluid, slowly add the anhydrous slufuric acid ammonium, and evenly stir with magnetic stirring apparatus, avoid excessive velocities to cause protein denaturation, reach 55% concentration up to ammonium sulfate, this moment, most albumen were all separated out with precipitation forms;
(4) 12000r/min, 4 ℃ of centrifugal 20min remove supernatant, precipitation is dissolved (pH7.2) with an amount of PBS damping fluid, be stored in 4 ℃ standby.
2. dialysis tubing dialysis:
(1),,, uses the NaHCO of 10mmol/L usually in order to remove these impurity because dialysis tubing that sell in market contains impurity atom to pre-treatment such as dialysis tubing soak, boils
3Boil dialysis tubing 30min with the EDTA solution of 1mmol/L, after boiling,, wash completely, be stored in the 1mmol/EDTA solution in 4 ℃, in case microbial contamination with distilled water thorough washing dialysis tubing.
(2) end of dialysis tubing is made a call to two fast knots, the sedimentary protein solution of sulfuric acid money is moved in dialysis tubing with transfer pipet or funnel.Make a call to two fast knots at the other end of dialysis tubing, and put it into dialysed overnight among the Tris-HCl (pH7.0) of 20mmol/L, and during exchange buffering liquid, ammonium sulfate is fully dialysed, this process protein solution surpass bag long-pending 1/3, otherwise dialysis tubing rises brokenly easily in the dialysis procedure.
3. lyophilize
(1) protein solution that will dialyse places triangular flask, and-20 ℃ rapid freezing, and masking foil seals, and will wear some duck eyes with pin on the masking foil simultaneously;
(2) the refrigerated protein solution places freeze drier to carry out lyophilize 48h;
(3) with Tris-HCl (pH7.0) dissolving of cryodesiccated protein powder with 1mL 20mmol/L, be stored in 4 ℃ standby.
The raising of mouse and the foundation of thrombus model checking CTB and LK fusion gene encoded protein activity
According to bibliographical information, form thrombus model by can bring out mouse tail to mouse back subcutaneous injection carrageenin, the whole process that this model can reflect the thrombosis development from the scope and the degree of body surface measurement afterbody conversion.The Kunming male mice is adopted in this experiment, the SPF level, and the animal credit number is: SCXK-(army) 2002001, about body weight 20g, bring out thrombosis medicine carrageenin and be configured to 0.5% solution with physiological saline, the subcutaneous injection of mouse the small of the back.See Fig. 4, Fig. 4 is mouse tail thrombus linear measure result, 1: irritate and feed the water control group; 2: irritate and feed the pichia spp extract control group that empty carrier transforms; 3: irritate and feed LK protein extract group; 4: irritate and feed CTB and LK fusion gene encoded protein extract group.It is more similar with the pichia spp extract control group result who irritates the conversion of hello empty carrier to irritate hello water control group, mouse tail thrombus length is about to account for puts in order tail length about 35%, be about and account for whole tail length about 28% and irritate to feed LK protein extract group mouse tail thrombus length, filling hello CTB and LK fusion gene encoded protein extract group mouse tail thrombus length are about to account for puts in order tail length about 17%, the two all has significant difference with respect to the water control group, explanation resulting CTB and LK fusion gene encoded protein in experiment not only have the thrombotic effect of inhibition, and adjuvant CTB can bring into play it and strengthens the effect that LK absorbs.
＜110〉University Of Tianjin
＜120〉choleratoxin B subunit and Lumbrukinase fusion gene and comprise the recombinant vectors and the host cell of this gene
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gacatggaac?ttcctcccgg?aacaaaaatt?gtcggaggaa?ttgaagctag?accatacgag?600
ttcccatggc?aggtgtccgt?cagaaggaag?tcttccgatt?cccatttctg?cggaggtagc?660
atcatcaacg?ataggtgggt?tgtctgcgct?gctcactgca?tgcagggaga?ggcccccgct?720
ctggtttcat?tggtcgtggg?tgagcacgac?aggagtgcag?ctagtgcagt?aaggcagact?780
catgacgttg?atagcatctt?cgttcacgag?gactacaaca?caaataccct?agagaacgac?840
gtttctgtca?tcaagacatc?tgttgccatc?actttcgaca?tcaacgttgg?tccaatctgt?900
gccccagatc?cagctcaaca?gtacgtctac?agaaagagcc?agtgctccgg?atggggaact?960
atcaattcag?gtggaatctg?ctgtcccaac?attctgagat?acgtaactct?gaatgtcaca?1020
accaaccaat?tctgcgaaga?tgtataccca?ctaaattcaa?tcttcgacga?tatgatttgc?1080
gcttcagaca?acactggggg?taacgacaga?gactcctgcc?agggtgactc?cggcggccct?1140
ctgagcgtca?aggatggcag?tggaatcttc?agcctgattg?gtattgtgtc?ttggggaatt?1200
ggttgcgctt?ctggctatcc?aggagtctac?tccagggtcg?gatttcacac?agcatggatc?1260
accgacatca?tcaccaacaa?ctaa 1284
Claims (3)
1. choleratoxin B subunit and Lumbrukinase fusion gene is characterized in that described choleratoxin B subunit is abbreviated as CTB by the nucleotide sequence shown in the SEQ ID NO.10 in the sequence table, and described Lumbrukinase is abbreviated as LK.
2. one kind by described CTB of claim 1 and LK fusion gene encoded protein, it is characterized in that aminoacid sequence shown in the SEQ ID NO.9 in the sequence table.
3. CTB and the LK fusion gene encoded protein application in the oral thrombolytic drug of preparation, described CTB and LK fusion gene encoded protein are aminoacid sequences shown in the SEQ ID NO.9 in the sequence table.
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Non-Patent Citations (2)
| Title |
|---|
| Choi,E..登录号:AAA96503.GenBank数据库.1996, * |
| Hu,R.和Zhang,S.登录号:AY327442.GenBank数据库.2004, * |
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