CN1256430C - Method for preparing recombinant vEGF using domestic silkworm as biological factory - Google Patents

Abstract

The present invention discloses a method for producing recombinant human vascular endothelial growth factor (vEGF) by using silkworm as biological factories, which is characterized in that hybridized baculovirus HyNPV of built AcNPV and BmNPV is used as a carrier to build recombinant viruses which contain human VEGF genes, and silkworms are used as the parasitifer of the recombinant baculovirus to highly express the recombinant human VEGF with high bioactivity. The method overcomes the defects that the expression of colibacillus needs complicated renaturation operation, the plasmid transformant is instable in the expression of yeast, and the production cost is high in culturing cells in mammals. The recombinant human VEGF expressed by silkworm larvae are mostly secreted in haemolymph, which is good for quickly extracting protein. The experimental results prove that the method for using recombinant baculovirus to produce recombinant human VEGF by taking silkworms as biological factories is safe, practical and feasible, and the recombinant VEGF can be produced in large scale, which opens up wide prospects for the application of the VEGF in the fields of medical science and treatment.

Description

Silkworm produces the method for recombinant human VEGF as " biological factory "

Technical field

The present invention relates to DNA recombinant technology, protein purification and activation analysis, relate in particular to a kind of silkworm produces recombinant human VEGF as " biological factory " method.

Background technology

Human vascular endothelial growth factor VEGF is the mitogen and the vasculogenesis inductor of endothelial cell specific, also is the penetrating media material of blood vessel.People VEGF is 165 amino acid whose polypeptide, and molecular weight is 19kDa, pI7.3, and it is a kind of glycoprotein, contains two identical polypeptide chains, links to each other with disulfide linkage to each other.The distinctive biologic activity of VEGF is that blood vessel endothelium is specific, comprises that powerful short cell fission effect and perviousness induce character.In addition, VEGF also with tumor-blood-vessel growth, the vascularization at wound healing and indirect stimulation obstruction of artery place is relevant.Up-to-date evidence shows that VEGF forms for embryonic blood vessel and the blood vessel generation also is very important.In addition, VEGF is essential for the blood vessel circulation propagation of female reproduction pipeline and the longitudinal growth and the endochondral ossification of bone.The VEGF induction of vascular helps treating the local asphyxia of cardiac muscle or the four limbs of several modes animal.Present research is aspect the vascular pathological that the VEGF mediation takes place to suppress for the blood vessel of will recombinate VEGF or being used for the treatment of property of VEGF transgenosis takes place two.Therefore VEGF is very extensive always in the research of the ischemic therapeutic application of blood vessel generation, wound healing and processing tissue.Application facet at medicine also has prospect very much.In these are used, need the VEGF of a large amount of biologically actives, therefore be necessary to develop a kind of efficient and economic method and produce.Can produce in intestinal bacteria with engineered method, yet VEGF need be to its folding again its biological activity of recovering with the formal representation of inclusion body in intestinal bacteria.Extra processing makes this process efficiency very low and uneconomical.In order to overcome this problem, other people once tried to express production with the mammalian cell of yeast and adenovirus infection.Yet, zymic plasmid transformant instability, Mammals culturing cell production cost is too high again.Therefore the applicant produces VEGF with baculovirus one silkworm expression system.

Baculovirus expression system is one of the most effective eukaryotic expression system.This system utilizes autographa california nuclear polyhedrosis virus AcNPV and insect cell line, as Spodoptera frugiperdaIPLB-Sf21-AE, Sf21 and Trichoplusiani, Tn-5, because Sf21, Sf9 cell and Tn-5 cell are easy to the serum-free large scale culturing, and this system is widely used in producing exogenous protein.Another baculovirus expression system utilizes Bombyx mori nuclear polyhydrosis virus BmNPV, expresses in silkworm larva and produces exogenous protein, has low, the convenient and high advantage of expression level of cost.The applicant has made up the hybridization baculovirus HyNPV (patent application is authorized, patent No. ZL01125605.2) between BmNPV and AcNPV, and it has the host territory of expansion, can infected silkworm, the greedy noctuid in meadow and the autumn mythimna separata.Like this, can be in the Sf21 cell construction of recombinant virus, low-cost in silkworm larva then, produce recombinant protein efficiently.We with silkworm as the host of recombinant baculovirus, high level expression the people VEGF of biologically active.

The genetically engineered biological industry is called as the sunrise industry of 21 century.Along with the adding of China " WTO ", set up and be suitable for China's national situation and characteristic biotechnology industry and be imminent.China has very abundant silkworm resource, the silkworm individuality greatly, is very easily raised and is convenient to and manages, be fit to very much utilize its to carry out expression of recombinant proteins production as " biological factory ", have very big potential application prospect in fields such as engineered vaccine and drug manufacture, protein research, biotic pesticide.

Summary of the invention

The object of the present invention is to provide a kind of silkworm to produce the method for recombinant human VEGF as " biological factory ".Utilize HyNPV to make up and contain the recombinant virus of human VEGF gene, and utilize silkworm, express the recombinant human VEGF of producing biologically active as the host.

In order to achieve the above object, its step of the technical solution used in the present invention is as follows:

1, the method for utilizing silkworm to produce recombinant human VEGF as " biological factory " is characterized in that:

(1) clone of VEGF gene: with the extractive total RNA of people lung tissue is template, utilizes the Titan One Tube RT-PCR system one step amplification of Roche to obtain human VEGF gene; Design of primers is as follows: forward primer: 5 '-AG GGATCCCCCATGGCAGAAGGAG-3 ' contains the BamHI restriction enzyme site, reverse primer: 5 '-G AAGCTTTCACCGCCTCGGCTTGTC-3 ' contains the HindIII restriction enzyme site; The cycle number of PCR reaction, the design of temperature and time are as follows: a circulation, 94 ℃ of template sex change 2 minutes; 10 circulations, 94 ℃ of sex change 10 seconds, 57 ℃ of annealing 30 seconds, 68 ℃ were extended 1 minute; 25 circulations, 94 ℃ of sex change 10 seconds, 57 ℃ of annealing 30 seconds, 68 ℃ were extended 1 minute, and each circulation back time circulation increases by 5 seconds extension time; Last 1 circulation, 68 ℃ were extended 7 minutes; Utilize 1% agarose gel electrophoresis to carry out the PCR product analysis, and with the PCR purification kit purifying of Qiagene, subsequently purpose PCR product cloning is advanced the carrier pCR2.1 of the TA clone 3.9kb of Invitrogen company, utilized dideoxy chain termination on the nucleotide sequencing instrument, to check order and confirm clone VEGF gene order exactness;

(2) contain the structure of the recombinant baculovirus of human VEGF gene: obtain the VEGF gene fragment with restriction enzyme BamHI and HindIII digestion pCR2.1-VEGF, be cloned into baculovirus transfer vector pBlueBacHis then and obtain recombinant chou pBlueBacHis-VEGF; In insect cell, produce recombinant virus by cotransfection with the reorganization of hybridization baculovirus HyNPV dna homology.In the polystyrene tapered tube, add 1 μ gpBlueBacHis-VEGF DNA and 15 μ g HyNPV DNA then, mixing, and add 14 μ l liposome lipofectin, add sterile purified water at last to final volume 40 μ l; Sf21 culturing cell with this mixture cotransfection logarithmic phase after room temperature is cultivated 15 minutes; After 24 hours, change the fresh culture that contains 10% foetal calf serum with the TC-100 culture medium culturing of serum-free into, cultivated 5 days for 27 ℃, collect the substratum supernatant and store stoste, be used for recombinant celo virus as virus.Recombinant virus finally obtains the viral solution of high density purifying by several plaque selects of taking turns, and concentration is 10 ⁸Pfu/ml.The applicant is with this recombinant virus called after HyNPV-VEGF;

(3) silkworm expression in vivo and reorganization VEGF purifying: use silkworm larva to play silkworm 5 ages, inoculate above-mentioned recombinant virus and infect expression; Before the inoculation, larva is immersed in the ice-water bath anesthesia 10 minutes, injection viral suspension concentration is 10 ⁶Pfu/ml adopts hypodermic method, and every silkworm is injected 20 μ l, injects and gives mulberry after 1 hour, raises down at 23-25 ℃; In metainfective three days, can't see tangible symptom, by the 4th day, larva began appetite and weakens, and presented infection symptoms gradually; Infected ground, back five days or the 6th day, the larva of infection is most of dead, before this collector's silkworm larva.

With the hemolymph of larva parent material, use following method to collect hemolymph: above the 15ml collection tube, larva to back side bending, with a hand fixedly head and the afterbody of larva, to be allowed outside belly and abdominal foot stretch to as the recombinant human VEGF purifying.No. 25 punctures abdominal foots with the Parafilm sealing are collected blood, allow blood directly drip in the collection tube, in order to prevent the blood oxidation blackening, add the 10mmol/L dithiothreitol (DTT) DTT of 1/10 volume in advance, and making its final concentration is 1mmol/L;

Because recombinant protein N-end has 6 * His tail, can carry out protein purification under non-sex change condition with the Ni-NTA affinity column; The hemolymph of collecting from the larva that infects is with non-sex change binding buffer liquid, Na ₃PO ₄50mmol/L, NaCl 0.5mol/L, pH8.0 dilutes; Sample is incorporated into the Ni-NTA post subsequently, and is last, and recombinant protein is with the non-sex change elution buffer wash-out that contains the 250mmol/L imidazoles, by identifying recombinant protein behind the SDS-PAGE coomassie brilliant blue staining;

(4) reorganization VEGF activation analysis is identified: the biological activity of analyzing recombinant human VEGF with Human umbilical vein endothelial cells HUVEC; Cell is 37 ℃ of cultivations in the substratum 199 that contains Earle ' s salt, replenish 5% CO ₂, add 100units/ml penicillin in the substratum in addition, the 100units/ml Streptomycin sulphate, the 4.0mg/ml amphotericin B, bovine serum is added in 20% hot deactivation, 5units/ml heparin and 50mg/ml endothelial cell growth supplementation material ECGS; Cell is inoculated in 24 hole tissue culturing plates after with tryptic digestion, and cell density is approximately 1.0 * 10 ⁴Cells/well, every hole adds the same substratum of 1ml.After 24 hours, substratum is replaced by contains 10%SCS and 5units/ml heparin, but do not contain the fresh culture 199 of ECGS; The reorganization VEGF of purifying with 0.22 μ m sterilising filter filtration sterilization, joins and cultivates in the plate hole behind gradient dilution, and every pore volume is no more than 10 μ l; After 72 hours, wash twice,, be resuspended among the 200 μ l PBS, after 0.4% the blue dyeing of phenol, calculate number of cells with hemocytometer with centrifugal collecting cell behind the tryptic digestion with PBS; The result shows that reorganization VEGF can stimulate HUVEC propagation, and this shows that the VEGF that expresses in the silkworm larva has biologic activity.

The present invention compares with background technology, and the beneficial effect that has is:

The present invention has solved VEGF preferably needs in complicated renaturation manipulation, the yeast expression plasmid transformant unstable and in the too high shortcoming of Mammals culturing cell production cost at expression in escherichia coli, silkworm larva express recombinant people VEGF safety, biological activity height, and most of quilt secretion is advanced in the hemolymph, is very beneficial for proteinic rapid extraction.Reorganization VEGF can mass production be that VEGF has opened up wide prospect in application pharmaceutically.

Embodiment

1, material: RNA extraction agent box is purchased in Qiagene.DNA handles and the pcr amplification test kit purchase in Takara Biomedicals (Kyoto, Japan).TA clone test kit, baculovirus transfer vector pBlueBacHis, lipofectin and Ni-NTA resin are Invitrogen company product.Dna sequencing kit is purchased the Biosystems in PE Applied.Hybrid virus HyNPV between BmNPV and AcNPV is by our own up-to-date structure.Foetal calf serum FCS, the greedy frugiperda cell in meadow are the product that the substratum TC-100 of Sf21 and the substratum 199 that contains Earle ' s salt that is used for cultivator huve cell HUVEC are GibcoBRL.Spodoptera frugiperda cell is that the substratum ESF921 of Tn-5 purchases in Expressionsystems.Human umbilical vein endothelial cells HUVEC, SCS and ECGS are the product of Technoclone GmbH.The greedy frugiperda cell Sf21 in meadow uses the TC-100 substratum that adds 10% (v/v) FCS and 0.26% bacto-tryptone 27 ℃ of cultivations.Tn-5 utilizes the ESF921 of serum-free to cultivate based on cultivating in 27 ℃ of bottles.Silkworm hybrid is used in this test, and the silkworm larva mulberry leaf are raised, and temperature is 23～25 ℃.

2, workflow:

1, the method for utilizing silkworm to produce recombinant human VEGF as " biological factory " is characterized in that:

(1) clone of VEGF gene: utilize the Titan One Tube RT-PCR system one step amplification of Roche to obtain human VEGF gene; Design of primers is as follows: forward primer: 5 '-AG GGATCCCCCATGGCAGAAGGAG-3 ' contains the BamHI restriction enzyme site, reverse primer: 5 '-G AAGCTTTCACCGCCTCGGCTTGTC-3 ' contains the HindIII restriction enzyme site; The cycle number of PCR reaction, the design of temperature and time are as follows: a circulation, 94 ℃ of template sex change 2 minutes; 10 circulations, 94 ℃ of sex change 10 seconds, 57 ℃ of annealing 30 seconds, 68 ℃ were extended 1 minute; 25 circulations, 94 ℃ of sex change 10 seconds, 57 ℃ of annealing 30 seconds, 68 ℃ were extended 1 minute, and each circulation back time circulation increases by 5 seconds extension time; Last 1 circulation, 68 ℃ were extended 7 minutes; Utilize 1% agarose gel electrophoresis to carry out the PCR product analysis, and with the PCR purification kit purifying of Qiagene, subsequently purpose PCR product cloning is advanced the carrier pCR2.1 of the TA clone 3.9kb of Invitrogen company, utilized dideoxy chain termination on the nucleotide sequencing instrument, to check order and confirm clone VEGF gene order exactness;

(2) contain the structure of the recombinant baculovirus of human VEGF gene: obtain the VEGF gene fragment with restriction enzyme BamHI and HindIII digestion pCR2.1-VEGF, be cloned into baculovirus transfer vector pBlueBacHis then and obtain recombinant chou pBlueBacHis-VEGF; In insect cell, produce recombinant virus by cotransfection with the reorganization of hybridization baculovirus HyNPV dna homology.In the polystyrene tapered tube, add 1 μ gpBlueBacHis-VEGF DNA and 15 μ g HyNPV DNA then, mixing, and add 14 μ l liposome lipofectin, add sterile purified water at last to final volume 40 μ l; Sf21 culturing cell with this mixture cotransfection logarithmic phase after room temperature is cultivated 15 minutes; After 24 hours, change the fresh culture that contains 10% foetal calf serum with the TC-100 culture medium culturing of serum-free into, cultivated 5 days for 27 ℃, collect the substratum supernatant and store stoste, be used for recombinant celo virus as virus.Recombinant virus finally obtains the viral solution of high density purifying by several plaque selects of taking turns, and concentration is 10 ⁸Pfu/ml.Application is gone into this recombinant virus called after HyNPV-VEGF;

(3) silkworm expression in vivo and reorganization VEGF purifying: use silkworm larva to play silkworm 5 ages, inoculate above-mentioned recombinant virus and infect expression; Before the inoculation, larva is immersed in the ice-water bath anesthesia 10 minutes, injection viral suspension concentration is 10 ⁶Pfu/ml adopts hypodermic method, and every silkworm is injected 20 μ l, injects and gives mulberry after 1 hour, raises down at 23-25 ℃; In metainfective three days, can't see tangible symptom, by the 4th day, larva began appetite and weakens, and presented infection symptoms gradually; Infected ground, back five days or the 6th day, the larva of infection is most of dead, before this collector's silkworm larva.

With the hemolymph of larva parent material, use following method to collect hemolymph: above the 15ml collection tube, larva to back side bending, with a hand fixedly head and the afterbody of larva, to be allowed outside belly and abdominal foot stretch to as the recombinant human VEGF purifying.No. 25 punctures abdominal foots with the Parafilm sealing are collected blood, allow blood directly drip in the collection tube, in order to prevent the blood oxidation blackening, add the 10mmol/L dithiothreitol (DTT) DTT of 1/10 volume in advance, and making its final concentration is 1mmol/L;

Because recombinant protein N-end has 6 * His tail, can carry out protein purification under non-sex change condition with the Ni-NTA affinity column; The hemolymph of collecting from the larva that infects is with non-sex change binding buffer liquid, Na ₃PO ₄50mmol/L, NaCl 0.5mol/L, pH8.0 dilutes; Sample is incorporated into the Ni-NTA post subsequently, and is last, and recombinant protein is with the non-sex change elution buffer wash-out that contains the 250mmol/L imidazoles, by identifying recombinant protein behind the SDS-PAGE coomassie brilliant blue staining;

(4) reorganization VEGF activation analysis is identified: the biological activity of analyzing recombinant human VEGF with Human umbilical vein endothelial cells HUVEC; Cell is 37 ℃ of cultivations in the substratum 199 that contains Earle ' s salt, replenish 5% CO ₂, add 100units/ml penicillin in the substratum in addition, the 100units/ml Streptomycin sulphate, the 4.0mg/ml amphotericin B, bovine serum is added in 20% hot deactivation, 5units/ml heparin and 50mg/ml endothelial cell growth supplementation material ECGS; Cell with tryptic digestion after the inoculation sub 24 hole tissue culturing plates, cell density is approximately 1.0 * 10 ⁴Cells/well, every hole adds the same substratum of 1ml.After 24 hours, substratum is replaced by contains 10%SCS and 5units/ml heparin, but do not contain the fresh culture 199 of ECGS; The reorganization VEGF of purifying with 0.22 μ m sterilising filter filtration sterilization, joins and cultivates in the plate hole behind gradient dilution, and every pore volume is no more than 10 μ l; After 72 hours, wash twice,, be resuspended among the 200 μ l PBS, after 0.4% the blue dyeing of phenol, calculate number of cells with hemocytometer with centrifugal collecting cell behind the tryptic digestion with PBS; The result shows that reorganization VEGF can stimulate HUVEC propagation, and this shows that the VEGF that expresses in the silkworm larva has biologic activity.

Claims (1)

1, a kind of silkworm is characterized in that as the method for " biological factory " production recombinant human VEGF:

(1) contains the structure of the recombinant baculovirus of human VEGF gene: baculovirus transfer vector pBlueBacHis is gone in the VEGF gene clone obtain recombinant chou pBlueBacHis-VEGF; In insect cell, produce recombinant virus by cotransfection with the reorganization of hybridization baculovirus HyNPV dna homology, in the polystyrene tapered tube, add 1 μ g pBlueBacHis-VEGF DNA and 15 μ g HyNPV DNA then, mixing, and add 14 μ l liposome lipofectin, add sterile purified water at last to final volume 40 μ l; Sf 21 culturing cells with this mixture cotransfection logarithmic phase after room temperature is cultivated 15 minutes; With the TC-100 culture medium culturing of serum-free after 24 hours, change the fresh culture that contains 10% foetal calf serum into, cultivated 5 days for 27 ℃, collect the substratum supernatant and store stoste as virus, be used for recombinant celo virus, recombinant virus finally obtains the viral solution of high density purifying by several plaque selects of taking turns, and concentration is 10 ⁸Pfu/ml, the applicant is with this recombinant virus called after HyNPV-VEGF;

(2) silkworm expression in vivo and reorganization VEGF purifying: use silkworm larva to play silkworm 5 ages, inoculate above-mentioned recombinant virus and infect expression; Before the inoculation, larva is immersed in the ice-water bath anesthesia 10 minutes, injection viral suspension concentration is 10 ⁶Pfu/ml adopts hypodermic method, and every silkworm is injected 20 μ l, injects and gives mulberry after 1 hour, raises down at 23-25 ℃; In metainfective three days, can't see tangible symptom, by the 4th day, larva began appetite and weakens, and presented infection symptoms gradually; Infected ground, back five days or the 6th day, the larva of infection is most of dead, before this collector's silkworm larva;

With the hemolymph of larva parent material, use following method to collect hemolymph: above the 15ml collection tube, larva to back side bending, with a hand fixedly head and the afterbody of larva, to be allowed outside belly and abdominal foot stretch to as the recombinant human VEGF purifying; No. 25 punctures abdominal foots with the Parafilm sealing are collected blood, allow blood directly drip in the collection tube, in order to prevent the blood oxidation blackening, add the 10mmol/L dithiothreitol (DTT) DTT of 1/10 volume in advance, and making its final concentration is 1mmol/L;

Because recombinant protein N-end has 6 * His tail, can carry out protein purification under non-sex change condition with the Ni-NTA affinity column; The hemolymph of collecting from the larva that infects is with non-sex change binding buffer liquid, Na ₃PO ₄50mmol/L, NaCl 0.5mol/L, pH8.0 dilutes; Sample is incorporated into the Ni-NTA post subsequently, and is last, and recombinant protein is with the non-sex change elution buffer wash-out that contains the 250mmol/L imidazoles, by identifying recombinant protein behind the SDS-PAGE coomassie brilliant blue staining;

(3) reorganization VEGF activation analysis is identified: the biological activity of analyzing recombinant human VEGF with Human umbilical vein endothelial cells HUVEC; Cell is 37 ℃ of cultivations in the substratum 199 that contains Earle ' s salt, replenish 5% CO ₂, add 100units/ml penicillin in the substratum in addition, the 100units/ml Streptomycin sulphate, the 4.0mg/ml amphotericin B, bovine serum is added in 20% hot deactivation, 5units/ml heparin and 50mg/ml endothelial cell growth supplementation material ECGS; Cell is inoculated in 24 hole tissue culturing plates after with tryptic digestion, and cell density is approximately 1.0 * 10 ⁴Cells/well, every hole adds the same substratum of 1ml, after 24 hours, substratum is replaced by contains 10%SCS and 5units/ml heparin, but do not contain the fresh culture 199 of ECGS; The reorganization VEGF of purifying with 0.22 μ m sterilising filter filtration sterilization, joins and cultivates in the plate hole behind gradient dilution, and every pore volume is no more than 10 μ l; After 72 hours, wash twice,, be resuspended among the 200 μ l PBS, after 0.4% the blue dyeing of phenol, calculate number of cells with hemocytometer with centrifugal collecting cell behind the tryptic digestion with PBS; The result shows that reorganization VEGF can stimulate HUVEC propagation, and this shows that the VEGF that expresses in the silkworm larva has biologic activity.