CN1188151A - Method for preparing recombination human basic fibroblastic growth factor and its use - Google Patents

Abstract

The present invention relates to a preparation method of recombined human basic fibroblastic growth factor. Said invention uses the principle of that genetic code possesses degeneracy to design Escherichia coli favour codon, PCR amplified human bFGFcDNA fragment and procaryotic expression carrier, for example pBV220 are assembled into high-effective expression plasmid pBbFGF, and after the escherichia coli is transformed, it uses temp. to induce bFGF gene expression to implement expression of 15% of somatic total protein. The engineering bacteria are passed through the processes of fermentation and amplification, and the somatic supernate is broken, then the two-step method of heparin affinity chromatography and gel exclusion-chromatography is used to purify product, and the protecting agent is added, the medicinal freeze-dried preparation can be made up. Said preparation can be used for curing myocardial ischemica diseases of cardiac infarction, etc., ......

Description

A kind of preparation method of recombination human basic fibroblast growth factor and application

The invention belongs to biological technical field, relate to rh-bFGF (Basicfibroblast growth factor, be called for short bFGF) gene engineering preparation method, and recombinant human bfgf itself or the preparation that contains bFGF are as the medicine or the healthcare products for the treatment of coronary heart disease and myocardial infarction.

Fibroblast growth factor is a class polypeptide growth factor that extensively is present in the multiple tissue, found from the ox pituitary gland by Gospodarwicz in 1974, because it has significantly short splitting action to inoblast, so the called after fibroblast growth factor exists with acid and two kinds of forms of alkalescence.1985, people finished the amino acid sequence analysis work of ox bFGF, and in subsequently 2 years, document has been reported ox and people's various tissue-derived bFGF aminoacid sequence successively, and has realized its gene clone and expression.There is reported in literature bFGF expression level to improve constantly (Knoerzer, Gene, 75:21-30.1989 in recent years; Squires, J Biol Chem 263:16297-16302:1988), owing to obtained a large amount of pure product, has promoted its biological function research greatly.Present studies show that, it is except having very strong short splitting action to inoblast, and from mesoblastic cell, outstanding tool is that vascular endothelial cell has very strong short splitting action to multiple, the most active short vascularization somatomedin known to being.It is one of Recent study most active fields.

Prostatropin bFGF is one of cytokine that has the important use DEVELOPMENT PROSPECT, the objective of the invention is by transform 5 ' end group because of, the bFGF gene of cloning from Chinese's peripheral blood is obtained in intestinal bacteria to be efficiently expressed, and utilize fast and convenient technology, obtain a large amount of standard compliant clinical applications, finally be used for the treatment of coronary heart disease and myocardial infarction.Estimate that bFGF has the potential clinical value to following disease:

1, wound healing and the tissue repair effect by bFGF can be used to burn, the treatment of scald, chronic ulcer, bedsore and serious soft tissue contusion;

2, by the neurotrophic effect of bFGF, can be used for maincenter and peripheral nerve injury, cerebral trauma sequela, spinal operation after, the treatment of nervous system disorders such as senile dementia, ischemic encephalopathy, neural heariing loss and optic nerve sex change;

3,, can be used for the treatment of restenosis behind chronic myocardial ischemia (coronary heart disease), acute and chronic myocardial infarction and the bypass operation of coronary artery by short vascularization of bFGF and myocardial nutrition effect.

Because the above practical value of bFGF, external existing man drugmaker of number and producer are in novel method and the novel process of development and production bFGF.

The present invention adopts following technical proposals:

(1) property of merger and intestinal bacteria are arranged to the preference of amino acid code and the difference of human body cell in view of genetic code, specialized designs of the present invention has been synthesized people bFGF gene 5 ' end and has been contained the dna fragmentation of intestinal bacteria preference codon, it can improve the expression level of bFGF gene in intestinal bacteria on amino acid composition that does not change product people bFGF and the basis that puts in order.Its step at first 5 ' and the guiding of 3 ' end primer under, in the total RNA of Chinese's peripheral blood mononuclear cell, amplify special people bFGF cDNA fragment.Pcr amplification product is inserted among the domestic efficient expression vector pBV220 that extensively adopts, be built into bFGF expression plasmid pBbFGF.It can be in intestinal bacteria HB101 and DH5 α high expression level, make bFGF account for about 15% of bacterial protein, expression rate does not reduce through going down to posterity more than 100 times.

(2) by the fermentation engineering bacterium, with M9 as basic medium, suitably increase carbon source and nitrogenous source, by optimizing processing parameters such as dissolved oxygen, stirring velocity, fermentation pH value, final cultures 0D600 value can reach about 6, biomass is every liter of culture 6～8g, and the product expression rate is between 12.8～16.5%.The product expression amount can reach every liter of culture 80～100mg.

(3) set up purifying process on this basis, made with extra care comprising fragmentation and product extracting, heparin affinity chromatography purifying, the gel exclusion chromatography of thalline.This technology is because purified product comes from the expression supernatant of solubility, so the product specific activity is higher, and structure is single.Introducing heparin affinity chromatography mainly is the highly selective of having utilized it, and further gel exclusion chromatography can be removed small amount of impurities albumen and pyrogen.Every liter of tunning can get the nearly 40mg of the pure product of people bFGF, the rate of recovery about 50%, and purity＞96%, specific activity can reach 1 * 10 ° of U/mg level, obviously exceed the reported in literature level.The a collection of bFGF preparation that obtains tens thousand of person-portions of utilization 30L fermentor tank.

(4) after pure product add stablizers such as human serum albumin, N.F,USP MANNITOL and phosphate buffered saline buffer, use the filtering with microporous membrane degerming, lyophilize powdering finished product.Use water for injection dissolving back, can be used for the treatment of wound, nerve injury and myocardial ischemia disease.

The Acute Myocardial Infarction of treatment Experimental Rabbits, give g/kg/ people bFGF of 1.4 μ 6 times, 10 days courses of treatment, can make rabbit heart stalk area and infarcted myocardium weight reduce 50.9% and 53.8% than control group, obviously be better than positive control medicine pannonit, obviously improved heart function and hemodynamic index, histology shows that also the infarct blood vessel hyperplasia is active.The result uses bFGF after showing the generation myocardial infarction in early days, can promote the cardiac muscle healing of Acute Myocardial Infarction, improves the prognosis of myocardial infarction.People bFGF can increase coronary flow after the experimental result prompting of myocardial ischemia rat is used, and the index of albumen, enzyme and the damage of other reflecting myocardium obviously reduces in the coronary artery effluent liquid.

The present invention uses recombinant DNA technology, under the prerequisite that does not change coded amino acid, changes gene 5 ' end parts Nucleotide, thus the expression that has improved product, and product exists with the high activity, soluble form.The downstream purification mode is easy, and the yield height reaches the specification of quality of clinical application.This product is used for the expeirmental myocardial ischemia treatment of diseases, can obtain significant curative effect.

The present invention describes with following example:

Embodiment 1, PCR design of primers and people bFGFF gene amplification

People bFGF protein molecule amino acid basedly constitutes by 146, wherein 8 aminoacid sequences of N-terminal are Pro-Ala-Leu-Pro-Glu-Asp-Gly-Gly, because protogene 5 ' terminal sequence GC content is too high, be unfavorable in prokaryotic system, expressing, therefore the primary thought of the design is to reduce by 5 ' end GC content, and 5 after the design ' end primer sequence is:

5?EcoRI

AGGAATTC?ATG?CCA?GCT?TTG?CCC?GAG?GAT?GGT?GGT?A

3 ' end primer sequence is:

5 BamHI are two to be stopped

ATGGATCC?TCA?TCA?GCT?CTT?AGC?AG?A?CAT?TGG

Adopt synthetic above-mentioned two oligodeoxynucleotides of solid phase tris phosphite method, the separation and purification of preparation gel electrophoresis.

Adopt micro-guanidinium isothiocyanate/phenol/chloroform single stage method total RNA of purifying in Chinese's peripheral blood mononuclear cell, make A260/A280 ratio＞1.8, get the total RNA of 10 μ l (about 4.5 μ g), add 40pmol5 ' end primer, 70 ℃ of thermally denature 5min, the room temperature cooling is lowered the temperature it naturally, after waiting to reduce to room temperature, adds following sample in 42 ℃ of water-baths successively:

Reverse transcription reaction system: 10 * reverse transcription damping fluid, 3 μ l

RNA enzyme inhibitors 20U

40nM trisodium phosphate 3 μ l

AMV reversed transcriptive enzyme 15U (1 μ l)

DNTP (each 2.5mM) 2 μ l

Add no RNA enzyme water to 30 μ l

42 ℃ of water-bath 60min of said mixture add H ₂70 ℃ of effects of O 70 μ l 5min deactivation ThermoScript II, this reactant can be used as the starting template of pcr amplification, and negate transcription product 20 μ l add following ingredients:

10 * PCR damping fluid, 10 μ l

DNTP (each 2.5mM) 16 μ l

5 ' and each 40pmol of 3 ' both sides primer

Add H ₂O to 99.5 μ l

94 ℃ of said mixtures, add 0.5 μ l Tap archaeal dna polymerase 2.5U behind the 3min, mixing, 40 seconds, 72 ℃ extensions of 40 seconds, 58 ℃ annealing of 94 ℃ of reaction of degeneration 60 seconds, carry out extending 6min behind 30 round-robin amplified reactions, get reactant and carry out electrophoresis and identify the single band observe 0.46kb, meet the length of complete people bFGF gene, and can with 5 of mark ' end primer hybridization.

Embodiment 2, expression and the sequential analysis of people bFGF gene in intestinal bacteria

With pcr amplification product and the expression vector plasmid pBV220 of restriction enzyme EeoRI and BamHI double digestion 0.46kb, separate through agarose gel electrophoresis, reclaim the dna fragmentation of 0.46kb and 3.66kb respectively.Mole such as two fragments mixes, and 12～16 ℃ of ligations of T4 dna ligase are spent the night, and connector is transformed into CaCl ₂In the E.coli bacillus coli DH 5 alpha of handling, through the penbritin screening and culturing.Select single bacterium colony, the preparation plasmid is identified with digestion with restriction enzyme, contains the correct person of people bFGFF gene fragment and direction of insertion, called after pBbFGF, corresponding engineering bacterium called after pBbFGF/DH5 α.

The engineering bacteria that contains above expression plasmid has more a protein band through amplification cultivation, temperature-induced about 17kb.This albumen can manifest specific reaction with people bFGF monoclonal antibody.

Under the sequencing primer guiding, survey from two respectively the people bFGF gene fragment of inserting is carried out dna sequence analysis, affirmed that the PCB product that is increased meets Design Conception, its amino acid sequence coded identical with people bFGF sequence (nucleotide sequence and the amino acid sequence corresponding that record are seen Fig. 1).

The fermentative production of embodiment 3, people bFGF

After 30 ℃ of incubated overnight of engineering bacteria, be inoculated in the M9 substratum in 10% ratio, 30 ℃ are cultivated 0D600 is 1.0～1.5 o'clock, temperature is risen to 42 ℃, add casein hydrolysate (1g/L) and glucose (4g/L) simultaneously, this moment, bFGF promptly induced generation, collected thalline behind the 4h.Electrophoresis is identified the bFGF expression level, thin layer scanning show bFGF account for bacterial protein 12.8～16.5% between.

The preservation of embodiment 4, engineering bacteria and stability

Engineering bacteria adds 30% glycerine ,-20 ℃ of preservations.Short-term is preserved bacterial classification can use soft agar LB flat board.Be to check the stability of engineering bacteria, with original strain, 50 generations and 100 generation bacterium respectively the extracting plasmid carry out enzyme and cut evaluation, the three is identical as a result.The product expression level of three bacterium amplification cultivation things is suitable simultaneously, proves that the engineering bacteria among the present invention is very stable.

The separation and purification of embodiment 5, bFGF

Because people bFGF exists with soluble form in thalline, therefore supernatant at first separates through heparin affinity chromatography behind bacterial cell disruption, and then refining through gel exclusion chromatography, can obtain the pure product of bFGF more quickly.

The heparin gel post is through 20mM Tris-HCl, 1mM EDTA-Na ₂, the 2mM beta-mercaptoethanol, 0.1M NaCl goes up sample after the pH7.4 damping fluid balance, last sample after contain 0.5, behind the above-mentioned buffer solution elution foreign protein of 1M NaCl, usefulness contains the above-mentioned buffer solution elution bFGF of 1.5M NaCl again.

Above-mentioned thick pure products is done further refining behind ultrafiltration and concentration after Sephacryl S-200 gel exclusion chromatography post, collect the pure product of bFGF of molecular weight 17kd.

The evaluation of embodiment 6, bFGF product

SDS-PAGE and HPLC purity checking are all more than 98%, and Balb/C 3T3 cell strain mtt assay is measured its biological activity, and specific activity is 1 * 10 ⁶More than the U/mg.N-terminal protein sequence analysis result is identical with native protein in the body, and order is P.A.L.P.E.D.G.G.S.G.A.F.P.P.G.H.

Embodiment 7, sterile filtration, packing, freeze-drying

According to protein content and activity, add auxiliary material and stablizer in following ratio

Every 20 of rh-bFGF, 50,100 μ g

N.F,USP MANNITOL 25mg/ props up

Human serum albumin 1mg/ props up

Damping fluid is 20mM PB, pH7.0, and above raw material must meet the requirement of injecting drug use.

Sterile filtration: reach at cleanliness factor under 100 grades the condition, with 0.22 μ M filtering with microporous membrane, packing, freeze-drying, seal, labeling, vanning.Be stored in 2～8 ℃ the cold storage environment.

Claims (3)

1, a kind of preparation method of recombination human basic fibroblast growth factor comprises the gene of PCR design of primers, amplification, the structure of expression plasmid, the purifying process of expression product, and its characteristic is:

(1) 5 ' end PCR primer and amplification cDNA 5 ' terminal sequence are:

EcoRI

AGGAATTC?ATG?CCA?GCT?TTG?CCC?GAG?GAT?GGT?GGT?A

(2) above-mentioned bFGF cDNA is assembled into efficient expression plasmid pBbFGF with the prokaryotic expression carrier pBV220 that contains the PL.PR promotor;

(3) pBbFGF transformed into escherichia coli, with temperature-induced bFGF expression of gene, product exists in the engineering bacteria with soluble form;

(4) by fermentation technique amplification engineering bacteria, make basic culture solution with M9, the temperature-induced nutritive ingredients such as casein hydrolysate, glucose of adding simultaneously;

(5) the broken thalline in fermentation back is collected supernatant and is adopted heparin affinity chromatography and gel exclusion chromatography two step method purified product.

2, add stablizer such as human serum albumin according to the pure product of people bFGF of claim 1 preparation after, make finished product after sterile filtration, packing, the freeze-drying, be stored in 2～8 ℃ the cold storage environment.

3, according to claim 1,2 described pharmaceutical preparations, can be used for the treatment of myocardial ischemia diseases such as Acute Myocardial Infarction, the treatment of coronary heart disease and health care medicine.

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