WO2013170408A1 - 一种Tat PTD—Endostatin重组蛋白及其制备方法与应用 - Google Patents
一种Tat PTD—Endostatin重组蛋白及其制备方法与应用 Download PDFInfo
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- WO2013170408A1 WO2013170408A1 PCT/CN2012/001276 CN2012001276W WO2013170408A1 WO 2013170408 A1 WO2013170408 A1 WO 2013170408A1 CN 2012001276 W CN2012001276 W CN 2012001276W WO 2013170408 A1 WO2013170408 A1 WO 2013170408A1
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- WIPO (PCT)
- Prior art keywords
- endostatin
- protein
- tat
- tat ptd
- ptd
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Classifications
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
Definitions
- the invention provides a Tat PTD-Endostatin recombinant protein, a preparation method and application thereof, and belongs to the field of protein transduction.
- Endostatin is a potent inhibitor of endogenous vascular endothelial cell proliferation found in 1997 by O'Reilly et al. The study found that ES can specifically inhibit the formation of vascular endothelial cells and inhibit the formation of vascular endothelial cells of various origins, and does not affect resting vascular endothelial cells, without drug resistance and toxic side effects. China has developed endostatin derivatives into a new class of national endostar with independent intellectual property rights (Endostar,
- ES ocular neovascular diseases in ES.
- ES still has some shortcomings. Because of its short half-life in vivo, poor accession ability, and large clinical dose, it increases the economic burden of patients. If it can improve its stability by a certain means, prolong its half-life in vivo, and increase its ability to inject into the body to increase its activity in vivo, thereby reducing the dose or prolonging the administration cycle, it will greatly improve the therapeutic effect of ES and promote its Clinical application.
- Tat protein that penetrates the cell membrane may help solve the problem of getting the drug through the eye barrier into the site of action.
- Tat egg. White is derived from the human immunodeficiency virus HIV-1 transactivator.
- Maurice and Paul found that Tat protein can be translocated into cells.
- Vives et al. demonstrated that Tat protein is rich in basic amino acids.
- the positively charged polypeptide fragment 47-57 amino acid sequence fragment of Tat protein
- Tat protein transduction domain PTD
- the research shows that Tat PTD can carry protein peptides, foreign genes, liposomes, inorganic molecules and other substances into the cells.
- Tat PTD Compared with other transport carriers, Tat PTD has its advantages: (1) Transduction efficiency is not limited by the size of the linker, and it does not affect the activity of the "cargo molecule" transported; (2) within a certain range It does not cause cell damage, and does not have obvious immunogenicity, antigenicity and inflammation. (3) It can penetrate the blood-brain barrier and is expected to solve the problem of macromolecular drugs entering the biological barrier structure.
- Tat PTD after Tat PTD is linked to macromolecular proteins, it can be understood as a chemical modification of macromolecular proteins, which prolongs the peptide chain, which is expected to increase protein stability and prolong biological half-life. Improve its efficacy in treating diseases such as cancer.
- the invention utilizes the transmembrane action of the transmembrane peptide Tat PTD and the inhibition of neovascularization by endostatin, and the two are fused together by genetic engineering to obtain the ability to penetrate the cell membrane or even the blood-brain barrier or the eye barrier. Endostatin achieves the goal of preventing ocular vascular proliferation by simple topical eye drops. This study explores large molecular eggs The new route of white eye administration and the prevention and treatment of retinal choroidal lesions are of great significance.
- the present invention provides a Tat PTD-Endostatin recombinant protein and a preparation method and application thereof.
- the present invention is achieved by the following technical scheme - a Tat PTD-Endostatin recombinant protein, which is composed of a protein transduction domain of the transactivation transduction protein Tat of human immunodeficiency virus (HIV-1) and human endostatin.
- a fusion protein wherein the amino acid sequence of the protein transduction domain (Tat PTD) of the transactivation transduction protein Tat of human immunodeficiency virus is represented by SEQ ID N0.1, and the amino acid sequence of human endostatin is SEQ. ID N0.2 is shown.
- the present invention also provides a DNA sequence capable of expressing a Tat PTD-ES fusion protein having the sequence represented by SEQ ID N0.3 (Tat PTD) and SEQ ID N0.4 (Endostatin) or encoding the same protein with the genetic code thereof Degenerate sequence.
- the Tat PTD-Endostatin recombinant protein can be expressed by yeast or E. coli system as follows:
- the method for expressing Tat PTD-Endostatin recombinant protein by yeast is as follows:
- Tat PTD-ES fusion gene comprising a gene of interest encoding Tat PTD and a gene encoding Endostatin, and amplifying;
- the above positive transformant is fermented, and the fermentation product is isolated and purified to identify the fermentation product, which is Tat PTD-Endostatin recombinant protein.
- the yeast strain in the step (4) is Pichia pastoris or Saccharomyces cerevisiae.
- the method for expressing Tat PTD-Endostatin recombinant protein by Escherichia coli is as follows:
- Recombinant plasmid pET28a and Tat PTD-ES were digested with restriction endonucleases Nde I and flwH / respectively, and the digested products were separately recovered, and then ligated with T4 DNA ligase;
- Competent E. coli Rossetta competent cells were prepared, and the expression plasmid was heat-transformed into Rossetta competent cells and cultured on LB plates containing kanamycin (5 ( ⁇ g/ml) for 16 h. Transformants, select positive transformant single colonies for colony PCR verification, and obtain positive transformants;
- the above positive transformant is fermented, and the fermentation product is isolated and purified to identify the fermentation product, which is Tat PTD-Endostatin recombinant protein.
- the step (5) is as follows:
- Inclusion body inclusion body The inclusion body is centrifuged after ultrasonication, and the inclusion body is renatured by dilution renaturation or dialysis renaturation or ultrafiltration renaturation method;
- Tat PTD-Endostatin fusion protein The renatured protein was purified by affinity chromatography or ion exchange chromatography, and the target protein with higher purity was obtained after salt removal.
- the specific method for the renaturation of the 3 is as follows: After the cell wall is broken (using high pressure dispersion, enzymatic cleavage or ultrasonic method to break the cells), 10000 g is centrifuged for 30 min, and the supernatant is discarded to obtain inclusion bodies; urea containing detergent is used.
- the guanidine hydrochloride solution is washed several times to remove the adhered heteroprotein (the concentration of the detergent solution is such that it can dissolve the interfering cellular proteins and membrane components without dissolving the inclusion bodies); then the inclusion bodies are dissolved by the denaturant (inclusion bodies) Dissolved under denaturing conditions, stirred at room temperature for 2 h, centrifuged at 10,000 g for 30 min, and the supernatant was an inclusion body solution; then, the denatured, denatured, bioactive protein was renatured to make it foldable to form a soluble , a biologically active conformation.
- the detergent solution is urea having a concentration of not more than 2 mol L or guanidine hydrochloride of not more than 1.5 M, and the detergent solution contains Tritonx-100 at a concentration of 0.5% (v/v)»
- the denaturing solution is cesium hydrochloride at a concentration of 5-7 M or urea at a concentration of 6-8 M.
- the dissolution of the inclusion body by the denaturant is carried out under reducing conditions.
- the reducing conditions refer to the use of a reducing agent, and the reducing agent used is dithiothreitol (DTT) or ?-mercaptoethanol.
- the renaturation is renatured by reducing the concentration of the denaturating agent (by slowly slowly or stepwise diluting the lysate to reduce the concentration of the denaturant reagent) to a level that is free of denaturation or weak denaturation.
- the renaturation buffer contains at least one thiol component in a reduced and oxidized form, and the thiol component is GSH/GSSG.
- the protein of interest is purified by affinity chromatography or cation exchange chromatography.
- Affinity chromatography was performed using Ni ion affinity chromatography, pH 5-ll.
- CM-Sepharose CM-Sepharose
- SP-Sepharose SP-Sepharose
- buffer pH 7-9 CM-Sepharose
- the desalting is: salt removal by G25-Sephadex, ultrafiltration, or dialysis.
- the Tat PTD-Endostatin fusion protein of the present invention can be used for the treatment of various diseases caused by neovascularization, including ocular proliferative diseases and various tumors such as retinopathy caused by diabetes, non-small cell lung cancer and the like.
- the invention uses the yeast and E. coli expression system to express the Tat PTD-ES fusion protein, and uses SDS-PAGE and Western Blot to identify the product, and uses Ni ion affinity chromatography to separate and purify the Tat PTD-ES with higher purity.
- the fusion protein was assayed for its activity in inhibiting the proliferation of human umbilical vein endothelial cells (EAHY926) by CCK-8 assay.
- the Tat PTD-Endostatin fusion protein of the invention retains the inhibitory effect of endostatin on the formation of new blood vessels, has strong characteristics of crossing the cell membrane, and has the potential to penetrate the physiological barrier of the body such as the blood brain or the eyeball barrier, and has the potential to It has the advantages of high guiding efficiency, easy to pass through the blood-brain barrier and eyeball barrier, overcomes the limitation of poor transmembrane effect of endostatin, and can play a better role in inhibiting the formation of new blood cells, which can be treated through the blood-brain barrier. Brain tumors, or by simple topical eye drops, achieve the goal of preventing retinal vascular proliferation.
- Figure 1 is a PCR map of the Tat PTD-ES fusion gene and Endostatin gene, wherein lane 1 is the PCR product of Endostatin, lanes 2 and 3 are PCR products of Tat PTD-Endostain gene, and M is DNA Marker I (600 bp, 500 bp, 400 bp, 300 bp, 200 bp, 100 bp).
- Figure 2 is a map of the recombinant plasmid after digestion, wherein the lane M1 is DNA Marker M 1 (600 bp, 500 bp, 400 bp, 300 bp, 200 bp, 100 bp), and lanes 1 and 3 are both pGAPZaA/Tat PTD-Endostatin recombinant plasmid digestion.
- the lane M1 is DNA Marker M 1 (600 bp, 500 bp, 400 bp, 300 bp, 200 bp, 100 bp)
- lanes 1 and 3 are both pGAPZaA/Tat PTD-Endostatin recombinant plasmid digestion.
- Lane 23 is the product of the pGAPZoA/Endostatin recombinant plasmid
- the lane M2 is the lkb DNA Ladder (10000 bp 8000 bp, 7000 bp, 6000 bp, 5000 bp 4000 bp, 3000 bp, 2000 bp, 1000 bp).
- Figure 3 is a PCR map of the recombinant plasmid, in which the PCR product of the recombinant plasmid pGAPZoA/Endostatin is used as a template, and the lanes 2 and 3 are PCR products using the recombinant plasmid pGAPZoA/Tat PTD-Endostatin as a template, and the lane M is DNA Marker I (600 bp, 500 bp, 400 bp, 300 bp, 200 bp, lOObp).
- Figure 4 shows the sequencing results of the recombinant plasmid pGAPZoA/Tat PTD-Endostatin.
- Figure 5 is a SDS-PAGE electrophoresis pattern of the supernatant of the fermentation broth, wherein lane 1 is protein Marker (94.0 KDa, 66.2 KDa, 45.0 KDa, 33.0 KDa, 26.0 KDa, 20.0 KDa, 14.4 KDa), and 2 is Pichia pastoris.
- the supernatant of the empty plasmid fermentation broth, 3, 4 is the supernatant of the positive Pichia fermentation of Tat PTD-ES, and 5 is the supernatant of the positive Pichia pastoris expressing Endostatin.
- Figure 6 shows the sequencing results of the recombinant plasmid pET28a/Tat PTD-ES.
- Figure ⁇ is the results of SDS-PAGE after E. coli ultrasound wall rupture, in which lane 1 is Es inclusion body; 2, 3. Tat PTD-ES inclusion body; 4. empty plasmid; 5. empty strain; 6. Marker; Empty plasmid supernatant; 8. Tat PTD-ES supernatant; 9. Es supernatant.
- Fig. 8 is a SDS-PAGE electrophoresis pattern of the sample obtained by refolding and purification of E. coli inclusion bodies, wherein lane 1, lane 2 is protein Marker, and lane 3 is isolated and purified sample.
- Figure 9 shows the inhibitory effect of the purified recombinant protein on the proliferation of EAHY926 cells.
- Figure 10 shows the effect of purified recombinant protein on the inhibition of angiogenesis in chick embryo chorioallantoic membrane.
- A is saline group; B is negative control; C is Tat PTD-ES group; D is ES group.
- Figure 11 is a view showing the ability of the purified recombinant protein to enter the cell, wherein A is ES and B is Tat PTD-ES.
- Example 1 Expression of a Tat PTD-ES fusion protein using an expression system
- Amplification of the Tat PTD-ES fusion gene 33 codes encoding the Tat-PTD1 amino acid and the selected restriction sites
- DH5a was prepared by routine activation, and DH5a was transformed by pGAPZ a A plasmid DNA by heat shock method. After transformation, the bacterial solution was applied to Zeocin (25 g/ml). In the low-salt LB plate, single colonies were picked the next day, amplified, and the plasmid was extracted with a kit.
- Tat PTD-ES gene EcoR l, Not I double-cleavage containing Tat PTD-ES gene fusion gene and pGAPZ a A empty plasmid, and recovering Tat PTD-ES fusion gene and 3 kb pGAPZ after agarose gel electrophoresis a A plasmid, added to the T4 ligase buffer, and ligated overnight at room temperature.
- the ligation product was transformed into DH5a the next day and plated in a low salt LB plate containing Zeocin (25 g/ml).
- pGAPZaA without the Tat PTD-ES gene was simultaneously transformed as a blank control.
- Electrotransformation experiment Prepare competent Pichia pastoris GS115, recombinant yeast expression plasmid pGAPZ a A was linearized and introduced into competent GS115 strain by electroporation, and pGAPZ without Tat PTD-ES a A electricity was converted to a control.
- the electroporated GS115 strain was applied to a YPDS plate containing Zeocin ( ⁇ ), cultured in a 30 °C incubator for 2-3 days to grow white single colonies, and picked from a Zeocin-containing LB plate. The colonies were incubated in 2 ml of YPD medium, shake cultured in a 30 ° C shaker to logarithmic growth phase, and frozen at -80 °C.
- the fourth day of recombinant Pichia fermentation broth was centrifuged at high speed (8000r/min, 20min), and the supernatant was separated and purified from the Pichia pastoris expression supernatant by nickel ion affinity chromatography and Sephade X G25.
- -ES fusion protein the purified sample was confirmed by SDS-PAGE, which indicated that the Tat PTD-ES fusion protein could be separated from the fermentation broth by nickel ion affinity chromatography and Sephadex G25.
- Double-digested fusion gene and empty plasmid The Tat PTD-ES fusion gene (constructed by conventional methods) and the selected large intestine-expressed pET28a were double-digested with Nde I and BamH I, and the fragments were recovered separately.
- Binding of the fusion gene to the plasmid The recovered gene of interest is mixed with the plasmid in a ligation system, incubated overnight at 4'C, and the ligation product is used for the next transformation.
- the target protein was identified by SDS-PAGE and Western Blot.
- Example 3 Process flow of renaturation of inclusion body protein expressed by Escherichia coli
- Inclusion body dissolution After the cell wall is sonicated, centrifuge at 10000g for 30min, discard the supernatant to obtain inclusion bodies; wash with urea or guanidine hydrochloride solution containing detergent to remove adhering impurities; then use denaturation The solution was dissolved in the inclusion body, stirred at room temperature for 2 hours, centrifuged at 10,000 g for 30 min, and the supernatant was an inclusion body solution.
- the detergent solution was 2 mol L of urea, and the detergent solution also contained Triton x-100 at a concentration of 0.5% (v/v).
- the denaturing solution contained 20-100 mmol/L Tris/HCK 8 mol/L urea, 5 mmol/L EDTA and 10 nunol/LDTT.
- the results of SDS-PAGE are shown in Figure 7.
- the renaturation buffer was: pH 7.0-9.0, 20-100 mmol/Ltris/HCK 2 mol/L urea, 5 mmol/LEDTA, lramol/LGSSG> 0.2 mmol/L GSH.
- Tat PTD-ES fusion protein The renatured protein was purified by Ni ion exchange chromatography to obtain a higher purity target protein after removal of salt, as shown in Fig. 8.
- the frozen solution of human umbilical vein endothelial cells (EAHY926) was rapidly thawed in a 37 °C water bath, centrifuged at 1000 °C for 15 min, the supernatant was discarded, and the pellet was resuspended in culture medium and gently mixed by blowing.
- the cell suspension was counted on a cell counting plate, then inoculated into a culture flask, and placed in a C0 2 incubator (5% C0 2 , 37 ° C) for 24 h, changing the solution, and every 2 to 3 days thereafter. Change the fluid once. When the cells grow to the fused state, the cells are washed twice with PBS and then added 0.25%.
- Trypsin 1 ⁇ 2 mL observed under an inverted microscope.
- the trypsin is immediately aspirated or discarded, and the culture medium containing 15% calf serum is added to terminate the digestion.
- the head pipette repeatedly blows the cells on the bottle wall, and most of the cells are shed to form a cell suspension, which is counted under an inverted microscope, and then inoculated into a new culture flask at a density of 10 5 /mL, and the culture is continued in a constant temperature incubator.
- ES inhibited vascular endothelial cells after purification by CCK-8 method.
- Collect log phase cells adjust the cell suspension concentration to ⁇ . ⁇ 4 /well, divide into 96-well plates, 200 ⁇ per well, and incubate them in a C0 2 incubator at 37 °C, 5% CO 2 .
- the inhibitory effect of Tat PTD-ES purified by different concentrations on the proliferation of HUVEC endothelial cells is shown in Figure 9.
- the results show that the purified Tat PTD-ES fusion protein has a significant inhibitory effect on HUVEC, and the inhibitory effect increases with increasing concentration.
- concentration dependence when the concentration was increased to 200 g/mL, most of the endothelial cells were inhibited, and the inhibition rate reached 86.45%.
- the method of culturing CAM is as follows: Select a breeder farm with a fertilization rate greater than 90%, a well-grown white-skinned egg (smooth surface clean, uniform eggshell, egg-shaped specification, uniform stomatal air chamber), with warm water After washing for 3 times, immerse it in 1 ⁇ benzalkonium phenol solution for 1 min and then sterilize it in a heat-insulated electrothermal incubator at a temperature of (37 ⁇ 1) V. Place the water tray in the incubator to maintain 40%. ⁇ 60% relative air humidity, and maintain a certain ventilation and ventilation conditions.
- the egg chamber is upward, and the long axis is approximately 70°-80° to the egg tray.
- the angle of the egg should be 45° in front of the horizontal position.
- the eggs were photographed with egg lamps to select well-developed chicken embryos. Move it to a clean bench, disinfect with 75% alcohol, and dry.
- Drill a dent on the surface of the eggshell with a dental drill or a grinding wheel use a sharp needle to make a small hole in the surface of the egg chamber, drop a small amount of normal saline at the dent, and use the ear-ball to align the small hole on the surface of the chamber.
- Tat PTD-ES chick chorioallantoic membrane (CAM) production are shown in Figure 10.
- the number of blood vessels in the Tat PTD-ES group was significantly smaller than that in the negative control group, indicating that Tat PTD-ES was evident.
- Inhibition of CAM blood vessel formation was not significantly different from Es.
- EAHY926 endothelial cells were seeded in 12-well plates (lxl0 4 ceUs/well), and serum-free DMEM medium (containing FITC-Es, FITC-Tat PTD-ES, each drug group according to protein concentration 100 protein/mL) 1 mL was added. . Incubate in a 37 ° C carbon dioxide incubator, incubate for 2 h, rinse with iced PBS 3 times, observe the Tat PTD-ES and ES cells by fluorescence microscope.
- Tat PTD-ES The results of Tat PTD-ES entry are shown in Figure 11. From the results, Tat PTD-ES has better ability to enter cells and has the potential to penetrate physiological barriers.
- the Tat PTD-ES fusion protein expressed by the present invention retains the effect of Endostatin on inhibiting the proliferation of vascular endothelial cells, and is expected to be used for treating various diseases caused by neovascularization, including ocular vascular proliferative diseases and Tumors, such as retinopathy caused by diabetes, non-small cell lung cancer, etc., are expected to perform better than Endostatin.
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Abstract
本发明公开了一种TatPTD-Endostatin重组蛋白,是由人类免疫缺陷病毒的反式激活转导蛋白Tat的蛋白转导域和人内皮抑素构成的融合蛋白,其中,人类免疫缺陷病毒的反式激活转导蛋白Tat的蛋白转导域的氨基酸序列如SEQ ID NO.1所示,人内皮抑素的氨基酸序列如SEQ ID NO.2所示。本发明蛋白保留了内皮抑素抑制新生血管的生成作用,具有转导效率髙、易透过血脑屏障、眼球屏障的优点,克服了内皮抑素的穿膜效果差的局限性,可发挥更好的-抑制新生血管细胞生成的作用,能够用于治疗新生血管生成引起的各种疾病,包括眼部血管增生性疾病及各种肿瘤,如糖尿病引起的视网膜病变,非小细胞肺癌等。
Description
一种 Tat PTD-Endostatin重组蛋白及其制备方法与应用 技术领域
本发明提供了一种 Tat PTD-Endostatin重组蛋白及其制备方法与应用, 属于蛋白转导领 域。
背景技术
内皮抑素 (Endostatin, ES)是 1997年 O'Reilly等首次发现的一种内源性血管内皮细胞 增殖的强效抑制剂。研究发现, ES可特说异性抑制新生血管内皮细胞的生成, 并对多种起源的 新生血管内皮细胞生成有抑制作用, 且不影响静止的血管内皮细胞, 无耐药性, 毒副作用小。 我国已将血管内皮抑素衍生物研制成具有自主知识产权的国家一类新药 Endostar (恩度,
书
YH16), 用以治疗非小细胞肺癌。此外研宄者们在 ES防治眼部新生血管性疾病方面也取得一 些可喜的成就。 但是 ES仍旧存在一些缺点, 由于其体内半衰期短, 入胞能力差, 临床上的 使用剂量较大, 增加了患者的经济负担。 如果能够通过一定的手段提高其稳定性、 延长其体 内半衰期、 并增加其入胞能力达到提高其体内活性从而减少给药剂量或延长给药周期, 将会 大大提高 ES的治疗效果, 促进其在临床上的应用。
能够穿透细胞膜的 Tat蛋白可能帮助解决使药物透过眼球屏障进入作用部位问题。 Tat蛋. 白来源于人类免疫缺陷病毒 HIV-1的反式激活因子, 1988年 Maurice和 Paul发现 Tat蛋白能 够跨膜递送入细胞, 1997年 Vives等证明 Tat蛋白中有一段富含碱性氨基酸、 带有正电荷的 多肽片段(Tat蛋白中 47-57位氨基酸序列片段) 与其转导功能密切相关, 并称之为 Tat蛋白 转导域 (PTD), 其序列是 YGRKKRRQRRR。 到目前为止研宄表明, Tat PTD能够运载蛋白 多肽、 外源基因、 脂质体、 无机分子等多种物质有效进入细胞内。 与其他运输载体相比, Tat PTD具有其优越性: (1 ) 转导效率不受连接物大小的限制, 且对其运输的 "货物分子"的活性 不产生影响; (2)在一定范围内不会造成细胞损伤, 不具有明显的免疫原性、 抗原性和致炎 性; (3) 能穿透血脑屏障, 有望解决大分子药物进入生物屏障结构发挥疗效的问题。 此外, Tat PTD与大分子蛋白质相连以后,从结构上来看,也可以理解为对大分子蛋白质进行了一定 的化学修饰, 延长了肽链, 有望增加蛋白质的稳定性, 延长生物半衰期, 也有利于提高其在 ' 治疗肿瘤等疾病的疗效。
本发明利用穿膜肽 Tat PTD的穿膜作用和内皮抑素的抑制新生血管的生成作用, 将二者 通过基因工程的手段融合在一起, 以期获得能够穿透细胞膜甚至是血脑屏障或眼球屏障的内 皮抑素, 达到通过简单的局部滴眼给药预防眼部血管增生的目标。 这一研究对探索大分子蛋
白质眼部给药的新途径和视网膜脉络膜病变的防治具有重要意义。
发明内容
针对上述现有技术, 本发明提供了一种 Tat PTD-Endostatin重组蛋白及其制备方法与应 用。
本发明是通过以下技术方案实现的- 一种 Tat PTD-Endostatin重组蛋白, 是由人类免疫缺陷病毒 (HIV-1 ) 的反式激活转导蛋 白 Tat的蛋白转导域和人内皮抑素构成的融合蛋白, 其中, 人类免疫缺陷病毒的反式激活转 导蛋白 Tat 的蛋白转导域 (Tat PTD ) 的氨基酸序列如 SEQ ID N0.1 所示, 人内皮抑素 (Endostatin) 的氨基酸序列如 SEQ ID N0.2所示。
本发明还提供了能够表达 Tat PTD-ES融合蛋白的 DNA序列, 其具有 SEQ ID N0.3(Tat PTD)和 SEQ ID N0.4(Endostatin)所示的序列或编码相同蛋白质的与其具有遗传密码简并性的 序列。
所述 Tat PTD-Endostatin重组蛋白可以通过酵母或大肠杆菌体系表达, 方法如下: 采用酵母表达 Tat PTD-Endostatin重组蛋白的方法, 步骤如下:
( 1 ) 常规方法制备含有编码 Tat PTD 的目的基因和编码 Endostatin 的目的基因的 Tat PTD-ES融合基因, 并扩增;
( 2) 用限制性内切酶 Eco ? Not /分别酶切质粒 pGAPZoA和 Tat PTD-ES融合基因, 并分别回收酶切产物, 然后用 T4 DNA连接酶连接;
( 3 )上述连接产物转化大肠杆菌 JM109感受态细胞, 筛选阳性克隆并用 PCR及 DNA 测序分析鉴定重组质粒 pGAPZoA/Tat PTD-Endostatin, 最终获得重组表达质粒 pGAPZaA/Tat PTD-Endostatin;
(4)制备感受态的酵母菌 GS115 ,重组表达质粒 pGAPZoA/Tat PTD-Endostatin单酶切线 性化后经电转化法导入感受态的 GS115菌中,同时以不含 Tat PTD-Endostatin的 pGAPZoA电 转作对照, 在含 Zeociii的 LB平板上培养 2-3天筛选转化子, 挑选阳性转化子单菌落进行菌 落 PCR验证, 获得阳性转化子;
( 5 ) 上述阳性转化子发酵, 分离纯化得到发酵产物, 鉴定发酵产物, 即为 Tat PTD-Endostatin重组蛋白。
所述步骤(4) 中的酵母菌株为毕赤酵母或酿酒酵母。
采用大肠杆菌表达 Tat PTD-Endostatin重组蛋白的方法, 步骤如下:
( 1 ) 常规方法制备含有编码 Tat PTD 的目的基因和编码 Endostatin 的目的基因的 Tat
PTD-ES融合基因, 并扩增;
(2) 用限制性内切酶 Nde I、 flwH /分别酶切质粒 pET28a和 Tat PTD-ES融合基因, 并分别回收酶切产物, 然后用 T4 DNA连接酶连接;
(3 )连接产物转化大肠杆菌 JM109感受态细胞, 筛选阳性克隆并用 PCR及 DNA测序 分析鉴定重组质粒 pET28a/Tat PTD-Endostatin , 最终获得重组表达质粒 pET28a/Tat PTD-Endostatin;
(4)制备感受态的大肠杆菌 Rossetta感受态细胞,将表达质粒热激法转化入 Rossetta感 受态细胞中, 在含卡那霉素 (5(^g/ml) 的 LB平板上培养 16h, 筛选转化子, 挑选阳性转化 子单菌落进行菌落 PCR验证, 获得阳性转化子;
( 5 ) 上述阳性转化子发酵, 分离纯化得到发酵产物, 鉴定发酵产物, 即为 Tat PTD-Endostatin重组蛋白。
所述步骤 (5) 具体如下:
① 挑选阳性转化子, 过夜培养后, 按 1 : 100 (体积比)接入 LB培养基中, 震荡培 养至 OD600 为 0.8-1.0 (约 6h) , 加入 IPTG (加入后的浓度为 0.25mmol/L) , 37 °C , 200r/min, 诱导 6h, 收菌;
②将菌体超声破壁(或采用高压分散、酶裂解法破碎细胞),采用 SDS-PAGE对目的蛋白 进行鉴定;
③包涵体复性: 菌体超声后离心得包涵体, 采用稀释复性或透析复性或超滤复性法对包 涵体进行复性;
④分离纯化 Tat PTD-Endostatin融合蛋白:将复性成功的蛋白质经亲和层析或离子交换层 析进行纯化, 除盐后得纯度较高的目的蛋白。
所述③中复性的具体方法如下: 菌体破壁 (采用高压分散、 酶裂解法或超声波法破碎细 胞)后, 10000g离心 30min, 弃去上清得到包涵体; 采用含去污剂的尿素或盐酸胍溶液洗涤 多次去除粘附的杂蛋白 (去污剂溶液的浓度使其能够溶解干扰的细胞蛋白质和膜成分而不溶 解包涵体); 然后采用变性液对包涵体进行溶解(包涵体在变性条件下溶解), 在室温搅拌 2h 后, 10000g离心 30min, 上清为包涵体溶液; 然后, 将包涵体溶解的、 变性的无生物活性的 蛋白质复性, 使其能够折叠形成可溶的、 有生物活性的构象。
所述去污剂溶液为浓度不大于 2mol L的尿素或不大于 1.5M的盐酸胍, 去污剂溶液中含 有 Tritonx-100, 浓度为 0.5% (v/v)»
所述变性液为 5-7M浓度的盐酸胍或 6-8M浓度的尿素。
所述变性液对包涵体的溶解在还原条件下进行。
所述还原条件是指使用还原试剂, 所使用的还原试剂为二硫苏糖醇 (DTT)或 β-巯基乙 醇。
所述复性是通过降低变性试剂的浓度 (通过缓慢地连续或逐步稀释溶解液来降低变性试 剂的浓度)至无变性作用或弱变性作用的水平来复性。
所述复性时, 复性缓冲液中含有至少一种还原和氧化形式的硫醇成分, 硫醇成分为 GSH/GSSG。
所述④中, 复性成功后采用亲和层析或阳离子交换层析纯化目的蛋白质。
亲和层析采用 Ni离子亲和层析, 缓冲液 pH5-ll。
阳离子交换层析采用 CM-Sepharose, SP-Sepharose, 缓冲液 pH7-9。
所述除盐为: 采用 G25-Sephadex, 超滤, 或透析的方法除盐。
本发明的 Tat PTD-Endostatin融合蛋白, 能够用于治疗新生血管生成引起的各种疾病,包 括眼部血管增生性疾病及各种肿瘤, 如糖尿病引起的视网膜病变, 非小细胞肺癌等。
本发明采用酵母和大肠杆菌表达体系表达了 Tat PTD-ES融合蛋白, 采用 SDS-PAGE和 Western Blot进行产物鉴定, 并采用 Ni离子亲和层析分离纯化后得到了纯度比较高的 Tat PTD-ES融合蛋白, 并通过 CCK-8法验证了其抑制人脐静脉内皮细胞(EAHY926)增殖的活 性。
本发明的 Tat PTD-Endostatin融合蛋白,保留了内皮抑素抑制新生血管的生成作用, 具有 较强的穿过细胞膜的特性, 具有穿透体内生理屏障如血脑或眼球屏障的潜力用, 具有转导效 率高、 易透过血脑屏障、 眼球屏障的优点, 克服了内皮抑素的穿膜效果差的局限性, 可发挥 更好的抑制新生血管细胞生成的作用, 可透过血脑屏障治疗脑部肿瘤, 或通过简单的局部滴 眼给药达到预防视网膜血管增生的目标。
附图说明
图 1为 Tat PTD-ES融合基因和 Endostatin基因的 PCR图谱, 其中泳道 1为 Endostatin的 PCR产物, 泳道 2、 3均为 Tat PTD-Endostain基因的 PCR产物, M为 DNA Marker I (600bp、 500bp、 400bp、 300bp、 200bp、 100bp)。
图 2 为 重 组质 粒 的 酶切 后 图 谱 , 其 中 泳道 Ml 为 DNA Marker M 1 (600bp,500bp,400bp,300bp,200bp, 1 OObp) , 泳道 1、 3均为 pGAPZaA/Tat PTD-Endostatin重 组质粒酶切后产物,泳道 23为 pGAPZoA/Endostatin重组质粒酶切后产物,泳道 M2为 lkb DNA Ladder(10000bp8000bp、 7000bp、 6000bp、 5000bp 4000bp、 3000bp、 2000bp、 lOOObp) 。
图 3为验证重组质粒时的 PCR图谱, 其中泳道 1以重组质粒 pGAPZoA/ Endostatin为模 板的 PCR产物,泳道 2、 3均为以重组质粒 pGAPZoA/Tat PTD-Endostatin为模板的 PCR产物, 泳道 M为 DNA Marker I (600bp、 500bp、 400bp、 300bp、 200bp、 lOObp) 。
图 4为重组质粒 pGAPZoA/Tat PTD-Endostatin的测序结果。
图 5为发酵液上清的 SDS-PAGE电泳图谱, 其中泳道 1为蛋白质 Marker(94.0KDa、 66.2 KDa、 45.0 KDa、 33.0 KDa、 26.0 KDa、 20.0 KDa、 14.4 KDa)、 2为毕赤酵母菌含空质粒发酵 液的上清液, 3、 4为表达 Tat PTD-ES的阳性毕赤酵母菌发酵的上清液, 5为表达 Endostatin 的阳性毕赤酵母菌发酵的上清液。
图 6为重组质粒 pET28a/Tat PTD-ES的测序结果。
图 Ί为大肠杆菌超声破壁后的 SDS-PAGE结果, 其中泳道 1为 Es包涵体; 2、 3. Tat PTD-ES包涵体; 4.空质粒; 5.空菌株; 6.Marker; 7.空质粒上清; 8.Tat PTD-ES上清; 9.Es 上清。
图 8为大肠杆菌包涵体经复性分离纯化后所得样品的 SDS-PAGE电泳图谱,其中泳道 1, 泳道 2为蛋白质 Marker, 泳道 3为经分离纯化后的样品。
图 9为纯化后的重组蛋白对 EAHY926细胞增殖的抑制作用。
图 10为纯化后的重组蛋白对鸡胚尿囊膜血管生成抑制作用的影响, A为生理盐水组; B 为阴性对照; C为 Tat PTD-ES组; D为 ES组。
图 11为纯化后的重组蛋白入胞能力的考察, 其中, A为 ES, B为 Tat PTD-ES。
具体实施方式
下面以具体实施例方式对本发明作进一步描述, 实施例中的实验方法, 如无特别说明, 均为常规方法。
实施例 1 采用 表达体系表达 Tat PTD-ES融合蛋白
a. Tat PTD-ES融合基因的扩增: 将编码 Tat-PTDl l个氨基酸的 33个密码及选定的酶切位点
(EcoR I )设计到上游引物的 5'端, 用 PCR技术从含有 Endostatin的 pGAPZaA质粒(本 发明人的实验室拥有, 常规方法构建得到的) 中扩增 Tat PTD-ES融合基因, 扩增得到的 融合基因片段为 600左右, 其 PCR结果见图 1。
b. 构建含有 Tat PTD-ES融合基因的表达质粒:
©pGAPZQA酵母表达质粒的扩增、 提取: DH5a常规活化后制备感受态, 取 pGAPZaA 质粒 DNA热激法转化 DH5a, 转化后将菌液涂布于含 Zeocin (25 g/ml) 的低盐 LB平板中, 次日挑取单菌落, 扩增, 并用试剂盒抽提质粒。
② Tat PTD-ES 基因的亚克隆: EcoR l、 Not I双酶切含 Tat PTD-ES 基因融合基因及 pGAPZaA空质粒,琼脂糖凝胶电泳后回收 Tat PTD-ES融合基因与 3kb的 pGAPZaA质粒,加 入 T4连接酶 buffer,室温连接过夜。次日取连接产物转化 DH5a,并涂布于含 Zeocin (25 g/ml ) 的低盐 LB平板中。 同时转化不含 Tat PTD-ES基因的 pGAPZaA作为空白对照。
③ Tat PTD-ES亚克隆的筛选及鉴定: 从 Zeocin低盐 LB平板中挑选单克隆, 接种于含 Zeocin的低盐 LB培养基中, 37Ό避光培养, 抽提质粒 DNA, 分别以酶切、 PCR法筛选含有 Tat PTD-ES的重组表达载体工程菌。 酶切结果见图 2, PCR结果见图 3, 筛选的阳性克隆质 粒由大连宝生物公司测序, 测序结果见图 4, 结果表明序列正确。
c Tat PTD-ES蛋白的表达:
①电转化实验: 制备感受态的毕赤酵母菌 GS115, 重组酵母表达质粒 pGAPZaA单酶切 线性化后经电转化法导入感受态的 GS115菌中, 同时以不含 Tat PTD-ES的 pGAPZaA电转作 对照。将电转后的 GS115菌株涂布于含 Zeocin ( ΙΟΟμ^πιΙ)的 YPDS平板中, 在 30'C培养箱 中培养 2-3天至长出白色单菌落,从含 Zeocin的 LB平板上挑取单菌落至 2mlYPD培养基中, 30°C摇床中振荡培养至对数生长期, -80°C冻存。
② PCR及 SDS-PAGE筛选阳性表达菌: 电转后的单克隆冻存菌 ΙΟμΙ经过过夜活化后, 以 1: 100接种于 lOOmlYPD培养液中, 30Ό振荡培养, 培养至第 4天时, 取发酵液, 分别进行 PCR及 SDS-PAGE验证, SDS-PAGE结果见图 5, 由结果可得筛选的阳性菌落能表达 Tat PTD-ES融合蛋白, 其分子量在 20KD左右。
d. Tat PTD-ES融合蛋白的分离纯化
取第四天的重组毕赤酵母菌发酵液高速离心(8000r/min, 20min), 取上清液采用镍离子 亲和层析和 SephadeXG25从毕赤酵母表达上清液中分离纯化 Tat PTD-ES融合蛋白,纯化后的 样品经 SDS-PAGE验证, 表明采用镍离子亲和层析和 SephadexG25可以将 Tat PTD-ES融合 蛋白从发酵液中分离出来。
实施例 2 采用大肠杆菌表达 Tat PTD-ES融合蛋白
a. 双酶切融合基因及空质粒: 采用 Nde I、 BamH I对 Tat PTD-ES融合基因(常规方法构建 得到) 及选用的大肠表达的 pET28a进行双酶切, 并分别进行片段回收。
b. 融合基因与质粒的连接: 在连接体系中将回收的目的基因与质粒混匀, 4'C孵育过夜, 连 接产物用于下一步转化。
c 重组质粒的转化: E.coli JM109感受态细胞 200μ1加入上述连接反应液, 混匀, 冰浴
30min, 42°C热激 90s, 冰浴 2min, 加入 0.8ml LB培养基, 37°C孵育 lh, 将孵育液涂
布于 LK平板上, 37°C培养 12~16h。
d. 阳性重组子的鉴定: 随机挑选数个具有 Kan抗性的转化子菌落, 经 LB液体培养基培 养、 小量扩增后, 抽提重组质粒为模板, 进行 PCR验证, 并进行测序, 测序结果见图 6, 结果表明序列正确。
e. 重组质粒转入工程菌株中:将测序正确的重组质粒,釆用热激法转入 i?0^etta菌株中,涂 布 LB平板 (Kan 50 g ml), 平板于 37'C培养 16-24 h;
f. 待平板上长出菌落后进行阳性转化子的筛选鉴定。
g. 挑选阳性转化子, 过夜培养后, 按 1:100接入 LB培养基中, 震荡培养至 OD6。。 约为 0·8-1·0(约 6h), 加入 IPTG (加入后的浓度为 0.25mmol/L) , 37°C , 200r/min, 诱导 6h, 收菌。
h. 将菌体超声破壁后, 采用 SDS-PAGE和 Western Blot对目的蛋白进行鉴定。
实施例 3 大肠杆菌表达的包涵体蛋白复性的工艺流程
a. 包涵体溶解: 菌体破壁超声后, 10000g离心 30min, 弃去上清得到包涵体; 采用含去污剂 的尿素或盐酸胍溶液洗涤多次以去除粘附的杂蛋白; 然后采用变性液对包涵体进行溶解, 在室温搅拌 2h后, 10000g离心 30min,上清为包涵体溶液。去污剂溶液为浓度 2mol L的 尿素, 去污剂溶液中还含有 Triton x-100, 浓度为 0.5%(v/v)。 变性液含有 20-100mmol/L Tris/HCK 8mol/L尿素、 5mmol/L EDTA及 10nunol/LDTT。 SDS-PAGE结果如图 7所示。 b. 蛋白复性: 复性缓冲液经过滤后, 将包涵体裂解液在搅拌得同时缓慢滴入复性缓冲液中, 复性体系的蛋白终浓度为 0.01-0.2mg/ml,然后将混合物置于 4'C下复性 10-50h。复性缓冲 液为: pH7.0-9.0, 20-100mmol/Ltris/HCK 2mol/L尿素、 5mmol/LEDTA、 lramol/LGSSG> 0.2mmol/L GSH。
c 分离纯化 Tat PTD-ES融合蛋白: 将复性成功的蛋白质经 Ni离子交换层析进行纯化, 除 盐后得纯度较高的目的蛋白, 如图 8所示。
实施例 4 Tat PTD-ES融合蛋白的生物活性研宄
考察 Tat PTD-ES抑制内皮细胞的活性: 以 Es为阴性对照, 采用 CCK-8法考察纯化后的 Tat PTD-ES抑制血管内皮细胞的活性, 具体实施步骤如下:
将人脐静脉内皮细胞(EAHY926)的冻存液在 37 °C水浴锅内快速融化, lOOO r/min离心 15 min, 弃上清, 沉淀细胞用培养液重悬并轻轻吹打混匀, 形成细胞悬液, 于细胞计数板上 进行细胞计数, 然后接种于培养瓶中, 置于 C02恒温培养箱 (5% C02, 37 °C ) 培养 24 h, 换液,以后每 2~3天换液一次。待细胞长至融合状态时,细胞用 PBS清洗两遍,然后加入 0.25%
胰蛋白酶 l~2 mL, 在倒置显微镜下观察, 待细胞间隙增大, 细胞回缩变圆时, 立即将胰蛋白 酶吸出或倒掉,加入含 15%小牛血清的培养液终止消化,用弯头吸管反复吹打瓶壁上的细胞, 使大部分细胞脱落形成细胞悬液, 于倒置显微镜下计数, 然后以 105/mL密度接种于新的培 养瓶中, 恒温培养箱继续培养。
采用 CCK-8法测定纯化后 ES抑制血管内皮细胞的活性。 收集对数期细胞, 调整细胞悬 液浓度为 Ι.ΟχΙΟ4/孔, 分于 96孔板, 每孔 200 μί, 置于 37 °C、 5%C02的 C02恒温培养箱中 培养使细胞贴壁; 加入药物 (纯化后的 Tat PTD-ES设 6个浓度梯度: 5 g/mL、 20 g/mL、 75 g/mL、 100 μ§/πΛ、 200 ^mh, 用培养基稀释,每个浓度设 5个平行孔), 继续培养 48 h; 小心吸取上清, PBS洗涤, 再次离心, 弃上清,加入 200μί新鲜培养基; 每孔加入 CCK-8溶 液 10μί, 37 °C继续培养 2 h后终止培养, 用酶联免疫检测仪于波长 490 nm处测定各孔吸光 度 (A49G) 值, 并计算其抑制率: 抑制率 =[1- (实验组 A49Q /对照组 A49Q ) ]><100%。 重复实验 五次, 取平均值。
不同浓度纯化后的 Tat PTD-ES对 HUVEC内皮细胞增殖的抑制作用见图 9, 结果显示纯 化后的 Tat PTD-ES融合蛋白对 HUVEC有明显的抑制作用, 随着浓度的增大抑制作用增强, 具有浓度依赖性,当浓度增大到 200 g/mL时, 内皮细胞大部分被抑制,抑制率达到 86.45%。
实施例 5 Tat PTD-ES鸡胚绒毛尿囊膜血管 (CAM)生成的作用
a. 孵育 CAM的方法, 方法如下: 选择来源于受精率大于 90%的种鸡厂、 生长良好的白 皮种蛋(表面清洁光滑、 蛋壳均匀、 蛋形规范、 气孔气室均匀), 用温水清洗 3次后, 在 1 ο 的苯扎溴酚溶液中浸泡 lmin消毒后置于隔热式电热恒温培养箱中培养, 温度 (37 ± 1 ) V, 培养箱内放入水盘以保持 40%~60%相对空气湿度, 并保持一定的通风换气条件。鸡蛋气室向 上, 长轴与蛋托约呈 70°-80°角。 每天转蛋 3次, 转蛋角度以水平位置前俯后仰各 45°为宜。 鸡胚孵育 3天后, 用照蛋灯照蛋, 挑选出发育良好的鸡胚。 将其移至超净台内, 用 75%酒精 消毒后晾干。 用牙科钻或砂轮在蛋壳表面划刻出凹痕, 用尖针在种蛋气室表面扎一小孔, 在 凹痕处滴少量生理盐水, 用吸耳球对准气室表面的小孔轻轻吸气, 此时可看到凹陷处的生理 盐水下陷, 即该处 CAM下陷, 形成假气室(区别于蛋自身的气室)。 用灭菌透明胶带封闭假 气室, 制备假气室后稳定 48 h。
b. 给药方法: 将鸡胚按重量随机分组, 生理盐水组, 阴性对照组: bFGF 20 nL (50AU) /只 Es组: Es (5(^g/mL)100 μL+ bFGF 20 (50 AU) /只; Tat PTD-ES(50 g mL) + bFGF 20 |iL ( 50 AU) /只。 将稳定 48 h的种蛋假气室上的透明胶带揭开, 按上述剂量将供试液直接加到 鸡胚绒毛尿囊膜上, 注意小心加药, 尽量使药物集中于一点上。加药后用无菌透明胶带封口,
标记后放入温度(37 ± 1 ) Ό的培养箱中, 保持 40%-60%的相对空气湿度, 继续培养 48 h后 观察结果。
Tat PTD-ES鸡胚绒毛尿囊膜血管 (CAM) 生成的作用结果见图 10, 与阴性对照组相比, Tat PTD-ES组的血管数目明显小于阴性对照组,表明 Tat PTD-ES能明显抑制 CAM血管的生 成, 且与 Es没有显著性差异。
实施例 6 荧光显微镜考察重组蛋白进入细胞的能力
将 EAHY926内皮细胞接种于 12孔板中 (lxl04ceUs/well), 加入无血清 DMEM培养基(内 含 FITC-Es、 FITC-Tat PTD-ES,各用药组按照蛋白浓度 100 protein/mL) lmL。 置于 37°C 二氧化碳培养箱中孵育, 孵育 2 h后用冰冻的 PBS漂洗 3次, 荧光显微镜观察 Tat PTD-ES及 ES的入胞情况。
Tat PTD-ES的入胞结果见图 11, 由结果可得, Tat PTD-ES进入细胞的能力较好, 具有能 够穿透生理屏障的潜力。
上述活性实验研究证明本发明表达的 Tat PTD-ES融合蛋白保留了 Endostatin的抑制血管 内皮细胞增殖的作用, 有望用于治疗由新生血管生成引起的各种疾病, 包括眼部血管增生性 疾病及各种肿瘤, 如糖尿病引起的视网膜病变, 非小细胞肺癌等, 甚至有望比 Endostatin更 好的发挥疗效。
Claims
( 1 ) 常规方法制备含有编码 Tat PTD的目的基因和编码 Endostatin的目的基因的 Tat-ES 融合基因, 并扩增;
(2)用限制性内切酶 M e l、 BamH l分别酶切质粒 pET28a和 Tat PTD-ES融合基因, 并 分别回收酶切产物, 然后用 T4 DNA连接酶连接;
(3 )连接产物转化大肠杆菌 JM109感受态细胞, 筛选阳性克隆并用 PCR及 DNA测序分 析鉴定重组质粒 pET28a/Tat PTD-Endostatin , 最终获得重组表达质粒 pET28a/Tat PTD-Endostatin;
(4 )制备感受态的大肠杆菌 Rossetta感受态细胞, 将表达质粒热激法转化入 Rossetta感
受态细胞中, 在含卡那霉素的 LB平板上培养 16h, 筛选转化子, 挑选阳性转化子单菌落 进行菌落 PCR验证, 获得阳性转化子;
(5)上述阳性转化子发酵,分离纯化得到发酵产物,鉴定发酵产物,即为 Tat PTD-Endostatin 重组蛋白。
、 根据权利要求 5所述的方法, 其特征在于: 所述步骤(5)具体如下:
①挑选阳性转化子, 过夜培养后, 按 1 : 100接入 LB培养基中, 震荡培养至 OD6()() 为 0.8-1.0, 加入 IPTG, 37°C, 200r/min, 诱导 6h, 收菌;
②将菌体破壁, 采用 SDS-PAGE对目的蛋白进行鉴定;
③包涵体复性: 菌体超声后离心得包涵体,采用稀释复性或透析复性或超滤复性法对包涵 体进行复性;
④分离纯化 Tat PTD-Endostatin融合蛋白: 将复性成功的蛋白质经亲和层析或离子交换层 析进行纯化, 除盐后得目的蛋白。
、 根据权利要求 6所述的方法, 其特征在于: 所述③中复性的具体方法如下: 菌体破壁后, 10000g离心 30min,弃去上清得到包涵体;采用含去污剂的尿素或盐酸胍溶液洗涤多次去 除粘附的杂蛋白; 采用变性液对包涵体进行溶解, 在室温搅拌 2h后, 10000g离心 30min, 上清为包涵体溶液; 然后, 将包涵体溶解的、 变性的无生物活性的蛋白质复性, 使其能够 折叠形成可溶的、 有生物活性的构象。
、 根据权利要求 6所述的方法, 其特征在于: 所述④中, 采用 G25-SephadeX、超滤或透析 的方法除盐。
、 权利要求 1所述的 Tat PTD-Endostatin融合蛋白在治疗癌症或新生血管生成引起的疾病中 的应用。
0、 根据权利要求 9所述的应用, 其特征在于: 所述新生血管生成引起的疾病为非小细胞肺 癌、 糖尿病引起的视网膜病变、 老年性黄斑盘状变性引起的脉络膜血管增生。
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