CN1594565A - Method for preparing recombinant bFGF using domestic silkworm as biological factory - Google Patents

Abstract

The invention discloses a method for producing recombinant alkali fibroblast growth factor bFGF by employing domestic silkworm as bio-factory. The invention uses baculovirus HyNPV hybridized by AcNPV and BmNPV as vector, constructs recombinant virus containing human bFGF gene, expresses human BFGF gene with biological activity while using domestic silkworm as host, solves the shortcomings that it needs complicated operations in bFGF expression in escherichia coli, that plasmid transformer is not stable in yeast expression, and that the cost is high in mammal cell expression. Recombinant human BFGFs expressed in domestic silkworm are mainly reserved in fat. Each silkworm yield amounts to 600-700 mu g, after purifying bFGF from fat using affinity chromatography. Activity analysis shows that: recombinant bFGF has bioactivity of promoting cell proliferation. Domestic silkworm can act as bio-factory to produce large amount of recombinant BFGF, which opens a broad prospect for medical and therapeutical fields.

Description

Silkworm produces recombinant human bfgf's method as " biological factory "

Affiliated technical field

The present invention relates to gene recombination technology, baculovirus gene expression system, protein purification and activation analysis, relate in particular to a kind of silkworm produces the recombinant human bfgf as " biological factory " method.

Background technology

Fibroblast growth factor (fibroblast growth factor is called for short FGF) is that the heparin Heparin that a class formation is similar, biological function is close is conjugated protein.Have report to think that there are 18 members in FGF family recently, wherein, the acidity and the Prostatropin (aFGF and bFGF) that are separated to from cow brain tissue are two members that find the earliest this family.People bFGF is made up of 146 amino acid, and molecular weight is 16.4kDa, and pI is 9.6.People bFGF all has the external short splitting action of intensive to multiple mesoblastema and the neuroderm cell that comprises vascular endothelial cell, therefore, be considered to grow in adjusting, the blood vessel in wound healing and the tumor growth process takes place and regeneration aspect have vital role.Therefore, in the clinical trial broad research bFGF actual effect on wound healing, tissue transplantation and treatment local asphyxia, demonstrate good application prospects.Cost an arm and a leg but bFGF is proteic in the market, though can produce in intestinal bacteria with engineered method, bFGF 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 be necessary to set up the method for a kind of production bFGF of efficient economy.

Baculovirus expression system is one of the most effective now eukaryotic gene expression system, at present this system comprises that mainly American-European countries is just at autographa california nuclear polyhedrosis virus (the Autographacalifornica nuclear polyhedrosis virus of widespread use, be called for short AcNPV) and be Bombyx mori nuclear polyhydrosis virus (the Bombyx mori nuclear polyhedrosis virus of representative with China, Japan, be called for short BmNPV) expression system two big classes, both respectively have advantage, but virus host scope difference.The AcNPV host range is wider; and " silkworm-recombinant baculovirus expression system " is owing to can utilize specialty economies insect one silkworm of China's number of animals raised maximum to make to express carrier; silkworm has characteristics such as individual big, easy raising; therefore have low, the advantage such as be produced on a large scale of production cost, be suitable for the requirement of bio-pharmaceuticals industrialization level.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 outstanding advantage in the host territory of expansion.Like this, can be in the host cell Sf21 of AcNPV construction of recombinant virus, low-cost in silkworm larva then, produce recombinant protein efficiently.

Summary of the invention

The object of the present invention is to provide a kind of silkworm to produce recombinant human bfgf's method as " biological factory ".Utilize HyNPV to make up and contain the recombinant virus of people bFGF gene, and utilize silkworm, express the recombinant human bfgf who produces 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) clone of bFGF gene: with human brain cDNA is template, and the pcr gene amplification technique obtains people bFGF gene, and design of primers is as follows: forward primer: 5 '-ACGT AGATCTCCCGCCTTGCCCGAG-3 ' contains the BglII restriction enzyme site; Reverse primer: 5 '-ACGT AGATCTTCAGCTCTTAGCAGA-3 ' contains the BglII 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 50 seconds; 30 circulations were annealed 30 seconds for 55 ℃, and 70 ℃ were extended 1 minute; Last 1 circulation, 70 ℃ were extended 10 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 gone into 3.9kb carrier pCR2.1, utilize dideoxy chain termination on the nucleotide sequencing instrument, to check order and confirm clone bFGF gene order exactness;

(2) contain the structure of the recombinant baculovirus of people bFGF gene: pCR2.1-bFGF obtains the bFGF gene fragment with restriction enzyme BglII digestion, is cloned into baculovirus transfer vector pAcGP67b then and obtains recombinant chou pAcGP67b-bFGF; In insect cell, produce recombinant virus by cotransfection with hybridization baculovirus HyNPVDNA homologous recombination, the method of cotransfection is: 1 μ g pAcGP67b-bFGF DNA and 15 μ g HyNPV DNA mix, 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 mixed solution cotransfection logarithmic phase after room temperature is cultivated 15 minutes.Earlier after 24 hours, change the fresh culture that contains 10% foetal calf serum into, cultivated 5 days for 27 ℃, collect the substratum supernatant, be used for recombinant celo virus as virus stock solution used with the TC-100 culture medium culturing of serum-free; Recombinant virus is taken turns plaque select by 3, finally obtains the viral solution of high density purifying, and concentration is 108pfu/ml, and the applicant is with this recombinant virus called after HyNPV-bFGF;

(3) silkworm expression in vivo and recombinant bfgf purifying: use silkworm larva to play silkworm 5 ages, inoculate above-mentioned recombinant virus and infect expression; Preceding larva is immersed in of inoculation carried out short period of time anesthesia in the ice-water bath, adopt hypodermic method injection recombinant virus suspension then, and concentration is 10 ⁶Pfu/ml, every silkworm is injected 20 μ l, injects and feeds mulberry after 1 hour, and raise down at 23-25 ℃; Infect collector's silkworm larva in back 72-96 hour, dissect the fatty body of infected silkworm, and with this parent material as purification of recombinant human bFGF; The pH 7.6 of precooling will be dissolved in after the fatty body homogenate, in the 20mM Tris-HCl damping fluid, behind high speed centrifugation, supernatant liquor directly gone up heparin Heparin affinity column, owing to bFGF and heparin have the intensive affinity bFGF is attached on the chromatography column, remove foreign protein with the above-mentioned same buffer solution elution that contains 0.4M NaCl then, the damping fluid one-step elution with 2.0M NaCl goes out bonded bFGF at last.Identify recombinant protein by the SDS-PAGE electrophoretic analysis, the result shows that the recombinant bfgf purity of acquisition is very high;

(4) the recombinant bfgf activation analysis is identified: the biological activity of analyzing the recombinant human bfgf with Human umbilical vein endothelial cells HUVEC; Cell is 37 ℃ of cultivations in substratum 199, 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 recombinant bfgf 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 with centrifugal collecting cell behind the tryptic digestion with PBS; After 0.4% the blue dyeing of phenol, calculate number of cells with hemocytometer.The result shows that recombinant bfgf can stimulate division, the propagation of HUVEC cell, shows that the recombinant human bfgf who 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 bFGF 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, and silkworm larva express recombinant people bFGF has the advantage that cost is low, output is high, biological activity is strong.The method that this patent is set up can the mass production recombinant human bfgf, has broad application prospects in the medical science and the clinical treatment field in future.

Embodiment

1, research material: genetically engineered operational tool enzyme and pcr amplification test kit are purchased in the living worker's biotechnology in China Shanghai service company.Human brain cDNA is available from Clotech company, and TA clone test kit, baculovirus transfer vector pAcGP67b, liposome lipofectin are Invitrogen company product.Heparin Heparin affinity chromatography uses product available from Japan and Optical Chemical Company.Dna sequencing kit is purchased the Biosystems in PEApplied.The substratum TC-100 of foetal calf serum FCS, insect cell line Sf21 and the substratum 199 that is used for cultivator huve cell HUVEC are the product of GibcoBRL.Spodoptera frugiperda cell is that the substratum ESF921 of Tn-5 purchases the systems in Expression.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:

Utilize silkworm to produce recombinant human bfgf's method as " biological factory ":

(1) clone of bFGF gene: with human brain cDNA is template, and the pcr gene amplification technique obtains people bFGF gene, and design of primers is as follows: forward primer: 5 '-ACGT AGATCTCCCGCCTTGCCCGAG-3 ' contains the BglII restriction enzyme site; Reverse primer: 5 '-ACGT AGATCTTCAGCTCTTAGCAGA-3 ' contains the BglII 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 50 seconds; 30 circulations were annealed 30 seconds for 55 ℃, and 70 ℃ were extended 1 minute; Last 1 circulation, 70 ℃ were extended 10 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 gone into 3.9kb carrier pCR2.1, utilize dideoxy chain termination on the nucleotide sequencing instrument, to check order and confirm clone bFGF gene order exactness;

(2) contain the structure of the recombinant baculovirus of people bFGF gene: pCR2.1-bFGF obtains the bFGF gene fragment with restriction enzyme BglII digestion, is cloned into baculovirus transfer vector pAcGP67b then and obtains recombinant chou pAcGP67b-bFGF; In insect cell, produce recombinant virus by cotransfection with hybridization baculovirus HyNPVDNA homologous recombination, the method of cotransfection is: 1 μ g pAcGP67b-bFGF DNA and 15 μ g HyNPV DNA mix, 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 mixed solution cotransfection logarithmic phase after room temperature is cultivated 15 minutes.Earlier after 24 hours, change the fresh culture that contains 10% foetal calf serum into, cultivated 5 days for 27 ℃, collect the substratum supernatant, be used for recombinant celo virus as virus stock solution used with the TC-100 culture medium culturing of serum-free; Recombinant virus is taken turns plaque select by 3, finally obtains the viral solution of high density purifying, and concentration is 108pfu/ml, and the applicant is with this recombinant virus called after HyNPV-bFGF;

(3) silkworm expression in vivo and recombinant bfgf purifying: use silkworm larva to play silkworm 5 ages, inoculate above-mentioned recombinant virus and infect expression; Preceding larva is immersed in of inoculation carried out short period of time anesthesia in the ice-water bath, adopt hypodermic method injection recombinant virus suspension then, and concentration is 10 ⁶Pfu/ml, every silkworm is injected 20 μ l, injects and feeds mulberry after 1 hour, and raise down at 23-25 ℃; Infect collector's silkworm larva in back 72-96 hour, dissect the fatty body of infected silkworm, and with this parent material as purification of recombinant human bFGF; The pH 7.6 of precooling will be dissolved in after the fatty body homogenate, in the 20mM Tris-HCl damping fluid, behind high speed centrifugation, supernatant liquor directly gone up heparin Heparin affinity column, owing to bFGF and heparin have the intensive affinity bFGF is attached on the chromatography column, remove foreign protein with the above-mentioned same buffer solution elution that contains 0.4M NaCl then, the damping fluid one-step elution with 2.0M NaCl goes out bonded bFGF at last.Identify recombinant protein by the SDS-PAGE electrophoretic analysis, the result shows that the recombinant bfgf purity of acquisition is very high;

(4) the recombinant bfgf activation analysis is identified: the biological activity of analyzing the recombinant human bfgf with Human umbilical vein endothelial cells HUVEC; Cell is 37 ℃ of cultivations in substratum 199, 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 recombinant bfgf 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 with centrifugal collecting cell behind the tryptic digestion with PBS; After 0.4% the blue dyeing of phenol, calculate number of cells with hemocytometer.The result shows that recombinant bfgf can stimulate division, the propagation of HUVEC cell, shows that the recombinant human bfgf who expresses in the silkworm larva has biologic activity.

Claims (1)

1, a kind of silkworm is characterized in that as " biological factory " production recombinant human bfgf's method:

(1) clone of bFGF gene: with human brain cDNA is template, and the pcr gene amplification technique obtains people bFGF gene, and design of primers is as follows: forward primer: 5 '-ACGT AGATCTCCCGCCTTGCCCGAG-3 ' contains the BglII restriction enzyme site; Reverse primer: 5 '-ACGT AGATCTTCAGCTCTTAGCAGA-3 ' contains the BglII 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 50 seconds; 30 circulations were annealed 30 seconds for 55 ℃, and 70 ℃ were extended 1 minute; Last 1 circulation, 70 ℃ were extended 10 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 gone into 3.9kb carrier pCR2.1, utilize dideoxy chain termination on the nucleotide sequencing instrument, to check order and confirm clone bFGF gene order exactness;

(2) contain the structure of the recombinant baculovirus of people bFGF gene: pCR2.1-bFGF obtains the bFGF gene fragment with restriction enzyme BglII digestion, is cloned into baculovirus transfer vector pAcGP67b then and obtains recombinant chou pAcGP67b-bFGF; In insect cell, produce recombinant virus by cotransfection with hybridization baculovirus HyNPVDNA homologous recombination, the method of cotransfection is: 1 μ g pAcGP67b-bFGF DNA and 15 μ g HyNPV DNA mix, 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 mixed solution cotransfection logarithmic phase after room temperature is cultivated 15 minutes.Earlier after 24 hours, change the fresh culture that contains 10% foetal calf serum into, cultivated 5 days for 27 ℃, collect the substratum supernatant, be used for recombinant celo virus as virus stock solution used with the TC-100 culture medium culturing of serum-free; Recombinant virus is taken turns plaque select by 3, finally obtains the viral solution of high density purifying, and concentration is 10 ⁸Pfu/ml, the applicant is with this recombinant virus called after HyNPV-bFGF;

(3) silkworm expression in vivo and recombinant bfgf purifying: use silkworm larva to play silkworm 5 ages, inoculate above-mentioned recombinant virus and infect expression; Preceding larva is immersed in of inoculation carried out short period of time anesthesia in the ice-water bath, adopt hypodermic method injection recombinant virus suspension then, and concentration is 10 ⁶Pfu/ml, every silkworm is injected 20 μ l, injects and feeds mulberry after 1 hour, and raise down at 23-25 ℃; Infect collector's silkworm larva in back 72-96 hour, dissect the fatty body of infected silkworm, and with this parent material as purification of recombinant human bFGF; The pH7.6 of precooling will be dissolved in after the fatty body homogenate, in the 20mM Tris-HCl damping fluid, behind high speed centrifugation, supernatant liquor directly gone up heparin Heparin affinity column, owing to bFGF and heparin have the intensive affinity bFGF is attached on the chromatography column, remove foreign protein with the above-mentioned same buffer solution elution that contains 0.4M NaCl then, the damping fluid one-step elution with 2.0M NaCl goes out bonded bFGF at last.Identify recombinant protein by the SDS-PAGE electrophoretic analysis, the result shows that the recombinant bfgf purity of acquisition is very high;

(4) the recombinant bfgf activation analysis is identified: the biological activity of analyzing the recombinant human bfgf with Human umbilical vein endothelial cells HUVEC; Cell is 37 ℃ of cultivations in substratum 199, 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 recombinant bfgf 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 with centrifugal collecting cell behind the tryptic digestion with PBS; After 0.4% the blue dyeing of phenol, calculate number of cells with hemocytometer.The result shows that recombinant bfgf can stimulate division, the propagation of HUVEC cell, shows that the recombinant human bfgf who expresses in the silkworm larva has biologic activity.