CN1249225C - Preparation and application of recombined human betaine homocysteine methyl transferase - Google Patents

Abstract

The present invention discloses the preparation and the application of recombined human betaine homocysteine methyl transferase, which belongs to the field of biotechnology. The present invention relates to an efficient expression technique for recombined human betaine homocysteine methyl transferase (BHMT) which can be used as a biological preparation for curing homocysteinemia and relative angiocardiopathy thereof.

Description

A kind of preparation of recombined human betaine homocysteine methyl transferase and application

The invention belongs to biological technical field, relate to a kind of recombined human betaine homocysteine methyl transferase (BHMT) and efficiently express technology, this reorganization BHMT can be used as the biotechnological formulation of treatment hyperhomocysteinemiainjury and related cardiovascular disease thereof.

(homocysteine HCY) is metabolic intermediate in the methionine(Met) working cycle to homocysteine.Research is in recent years confirmed, the hyperhomocysteinemiainjury and the substantial connection that has as multiple cardiovascular disordeies such as human first killer's atherosclerosis, hypertension, myocardial infarctions, be of cardiovascular disorder new independently and important risk.Effective inhibition of hyperhomocysteinemiainjury has been become one of the cardiovascular disorder control very important medical approach.

There are two kinds of pathways metabolisms in vivo in HCY: the synthetic methionine(Met) that methylates again, or change halfcystine into through cystathionine by changeing the sulphur approach.Therebetween, Methylene tetrahydrofolate reductase (MTHFR), cystathionine (CBS) and methionine synthetase (MS) are necessary three key enzymes of HCY metabolism; These enzymic activitys reduce or lack as, perhaps its corresponding coenzyme is as VitB ₆, VitB ₁₂With the shortage of folic acid, will cause the HCY metabolic disturbance, plasma concentration raises.Existing research is found, the heredity disappearance of above-mentioned enzyme gene, or, the nutrition reason lacks because of causing coenzyme, can cause hyperhomocysteinemiainjury (Mayer EL, Jacobsen DW, and Robinson K.Homocysteine and Coronary Atherosclerosis.J Am Coll Cardiol, 1996,27 (3): 517-27).Yet, the high incidence that but is difficult to annotate hyperhomocysteinemiainjury with the incidence and the basic nutrition situation of present human inheritance's property defective, the research of hyperhomocysteinemiainjury patient's individuality also shows, the reason that has the patient of significant proportion not exist hereditary defect and nutrient substance to lack.The nosetiology mechanism of hyperhomocysteinemiainjury it be unclear that so far, also is difficult to reach from the approach of the blocking-up cause of disease therapeutic purpose of hyperhomocysteinemiainjury.Therefore, relevant both at home and abroad at present research focuses mostly in the cardiovascular function of damaging action and mechanism thereof cause to(for) the high-level homocysteine mass formed by blood stasis that has taken place, relevant hyperhomocysteinemiainjury animal model etc. has been failed system of systems, and the exogenous coenzyme VitB that gives is just generally adopted in the treatment measure of hyperhomocysteinemiainjury ₆, VitB ₁₂With the method for folic acid strengthening above-mentioned metabolic enzyme, but how undesirable clinical effectiveness is.

Recently it is found that methylated again another the important metabolic enzyme-betaine homocysteine methyl transferase (BHMT) of catalysis HCY.But this enzyme catalysis trimethyl-glycine general-NCH ₃Be transferred to the reaction of HCY, generate N-methylsarcosine and methionine(Met), thereby effectively reduce concentration (the Heil SG of HCY, Lievers KJA, Boers GH, et a1.Betaine-Homocysteine Methyltransferase (BHMT): Genomic Sequencing and Relevance to Hyperhomocysteinemia and Vascular Disease in Humans.MolGenet Metab, 2000,71:511-519).Biological property to people source BHMT studies show that BHMT is a kind of Zn of containing ²⁺Metalloenzyme, molecular weight 45kd, form by 406 amino acid, Chang Yiliu aggressiveness form exists, the top condition of its enzymatic action is pH7.5,37 ℃ of (Millian NS and Garrow TA.Human Betaine-Homocysteine Methyltransferase Is a ZincMetalloenzyme.Arch Biochem Biophys, 1998,356 (1): 93-98).BHMT mainly has relatively large expression in liver and renal cortex, pancreas, brain, the heart, skeletal muscle, then expression level is lower in the spleen, be body is kept the stable metaboilic level of homocysteine under physiological situation critical function protein (Sunden SLF, Renduchintala MS, Park EI, et al.Betaine-Homocysteine Methyltransferase Expression in Porcine and Human Tissues and ChromosomalLocalization of the Human Gene.Arch Biochem Biophys, 1997,345 (1): 171-174).It is than above-mentioned three kinds of metabolic enzymes, exists the homocysteine that generated transformation function more efficiently, and does not rely on the advantage of special coenzyme, can be used as the important tool enzyme that reduces body HCY.But, when body generation hyperhomocysteinemiainjury, do not find as yet that endogenous BHMT expression level has by inducibility to raise, to quicken the trend of HCY degraded.Therefore, we think, when suppressing the generation of hyperhomocysteinemiainjury by high-level BHMT, should consider the exogenous BHMT of giving.

We find in the experimental study for the BHMT biological function, and the outer conventional liver cell of cultivating liver cell or containing HCY at cell culture fluid of donor is with BHMT, can make in the cell and in the cell culture fluid HCY content obviously reduce; The vitro culture myocardial cell who gives excessive level HCY effect can significantly slow down myocardial cell's mortality ratio with BHMT.Show that exogenous BHMT has the effect that reduces HCY level in blood plasma and the histocyte, and the myocardial cell under the high-level HCY effect is had defencive function.And, confirmed that BHMT is a kind of Autolysosome membranin, and affinity (Ueno T is preferably arranged with cytolemma, Ishidoh K, Mineki R, et al.Autolysosomal Membrane-associated Betaine HomocysteineMethyltransferase.J Bio Chem, 1999,274 (21): 15222-15229), further point out exogenous BHMT to have and be absorbed into the potential that cell is brought into play biological function.In view of the above, we propose with recombined human betaine homocysteine methyl transferase (BHMT) as the bio-pharmaceutical that slows down the treatment of hyperhomocysteinemiainjury and related cardiovascular disease thereof.

At present, the relevant medicinal research of BHMT does not appear in the newspapers as yet, but its molecular structure and biological characteristics have been obtained quite deep understanding; And show according to the foreign literature data, people BHMT gene is cloned, expression vector establishment, genetic expression, expression product separation and purification and Analysis and Identification method are comparatively ripe, and existing BHMT Antibody Preparation is finished (Garrow TA.Purification, Kinetic Properties, and cDNA Cloning of MammalianBetaine-Homocysteine Methyltransferase.J Bio Chem, 1996,271 (37): 22831-22838).We are improved on the basis of existing research, set up the technology platform that using gene engineering technique prepares the BHMT recombinant protein, and the animal model series of hyperhomocysteinemiainjury and detect, estimate in the body of hyperhomocysteinemiainjury result of treatment and the experiment in vitro model.Based on this, BHMT is designed and tests as the development technological line of the bio-pharmaceutical of treatment hyperhomocysteinemiainjury.

The present invention adopts following technical proposals:

(1) in view of intestinal bacteria to the preference of amino acid code and the difference of human body cell, the present invention designs and has synthesized people BHMT gene 5 ' end and contain the dna fragmentation of intestinal bacteria preference codon, it can improve the expression level of BHMT gene in intestinal bacteria on amino acid composition that does not change product people BHMT and the basis that puts in order; For strengthening specific binding capacity and the transmethylase catalytic capability thereof of BHMT and HCY, BHMT N end structure domain amino acid residue sequence is transformed to 53peavrqlhrefleagadvmetftf.Its step relates under the guiding of 3 ' end primer, amplifies special people BHMT cDNA fragment in the total RNA of human liver cell; The goal gene product is inserted among the efficient expression vector pTrc99A, be built into BHMT expression plasmid pTrc-hBHMT; It can be in intestinal bacteria JM105 high expression level, make BHMT account for about 20% of bacterial protein, expression rate does not reduce through going down to posterity more than 100 times.

(2), as basic medium, suitably increase carbon source and nitrogenous source, and mix ZnCl with 2YT by the fermentation engineering bacterium ₂By optimizing processing parameters such as dissolved oxygen, stirring velocity, fermentation pH value, final cultures OD600 value can reach about 2.0, and biomass is every liter of culture 20-25g, and the product expression rate is between 20-25%.The product expression amount can reach more than every liter of culture 200mg.

(3) set up purifying process on this basis, refining etc. comprising the fragmentation of thalline and inclusion body extracting, sephadex chromatography and fast protein liquid chromatography.

The process stabilizing of being set up, rate of recovery height, active good.Principal feature of the present invention is:

1. according to the biological characteristics of known BHMT; based on us to reducing in the histocyte about exogenous BHMT and HCY level in the cell culture fluid; and, propose with BHMT as the biotechnological formulation that slows down the treatment of hyperhomocysteinemiainjury and related cardiovascular disease thereof to the discovery that the myocardial cell under the high-level HCY effect has defencive function.

2. set up the animal model of hyperhomocysteinemiainjury.Hyperhomocysteinemiainjury nosetiology mechanism is not clear, still unformed both at home and abroad at present this method for building animal model.We adopt and deprive homocysteine metabolic enzyme associated coenzymes in the diet, or give the technological line of animal handling, and have set up good stability, and repeatability is high, and hyperhomocysteinemiainjury animal model series is in various degree arranged.

3. set up the exogenous BHMT treatment hyperhomocysteinemiainjury Evaluation on effect system of estimating.Comprise: whole animal blood plasma, cardiovascular function damage and the monitoring that recovers, the detection method of isolated cells HCY level, the detection method of cardiovascular cell injury etc.

4. set up the technological line that reorganization BHMT efficiently expresses.Comprise: the utilization of recombinant DNA technology change gene 5 ' end parts Nucleotide, thereby the expression that has improved product has strengthened specific binding capacity and the transmethylase catalytic capability thereof of BHMT and HCY.

The present invention describes with following example:

Embodiment 1, PCR design of primers and BHMT gene clone

Based on the people BHMT full DNA sequence of Garrow TA report,, BHMT N end structure domain amino acid sequence is transformed to 53peavrqlhrefleagadvmetftf for strengthening specific binding capacity and the transmethylase catalytic capability thereof of BHMT and HCY.The DNA of the 1st to the 80th amino acid residue sequence of coding BHMT obtains with chemical synthesis, and makes up the restriction site of a Nco I at its 5 ' end: 5 '-GCCCCCAACGGGTGCCATGGTTGTG GTGTCCAG-3 '; The DNA of the 77th to the 406th amino acid residue sequence of coding BHMT obtains with the PCR method, and makes up the restriction site of a BamH I at its 3 ' end, annexs the principle of property according to genetic codon, designs its 3 ' end primer sequence and is:

BamH?I

5’-CG? GGATCC?TCA?CTG?TGA?TTT?GAA?TTT

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

Adopt micro-guanidinium isothiocyanate/phenol/total RNA of chloroform single stage method purifying in human liver tissue, make A260/A280 ratio＞1.8, get the total RNA of 10 μ l (about 5 μ g), add 50pmol 3 ' 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: 5 * reverse transcription damping fluid, 5 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 25 μ l

42 ℃ of water-bath 60min of said mixture add H ₂70 ℃ of effects of O 75 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

3 ' end primer 40pmol

Add H ₂O to 99.5 μ l

Add 0.5 μ l Taq archaeal dna polymerase 2.5U behind 95 ℃ of said mixtures, the 2min, mixing, 95 ℃ of sex change, 61 ℃ of annealing, 72 ℃ of extensions each 60 seconds, carry out extending 8min behind 35 round-robin amplified reactions, get reactant and carry out the electrophoresis evaluation, observe the single band of about 1.0kb, meet goal gene length, and can hold primer hybridization with 3 ' of mark.

At last two segment DNAs are connected.

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

The structure of expression vector can be by the restriction enzyme site at a Nco I of first initiator codon ATG of BHMT cDNA place's design construction, goal gene product and expression vector plasmid pTrc99A with restriction enzyme Nco I and BamH I double digestion 1.2kb, separating the moles such as two dna fragmentations that obtain through agarose gel electrophoresis mixes, 12-16 ℃ of ligation of T4 dna ligase spent the night, and connector pTrc99A-hBHMT recombinant plasmid transformed is gone into CaCl ₂Among the E.Coli JM105 that handles, 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 BHMT gene fragment and direction of insertion and is the purpose engineering bacteria.

Engineering bacteria through under 37 ℃ of conditions with containing ZnCl ₂Substratum amplification cultivation, IPTG induce, about 45kd, have more a protein band.This albumen can present specific reaction with people BHMT monoclonal antibody.

Under the sequencing primer guiding, from both sides the recombinant human B HMT gene segment that inserts is carried out dna sequence analysis respectively, affirmed that the product that is obtained meets Design Conception.

The separation and purification of embodiment 3, reorganization BHMT

People BHMT exists with loose inclusion body form in thalline, recovery, purifying inclusion body behind the bacterial cell disruption, the extracting supernatant liquor at first passes through the sephadex chromatography preliminary purification, and is refining through fast protein liquid chromatography again, the pure product of the BHMT that can faster obtain recombinating.

Sephadex chromatography post Sephadex G-100 is through containing the 10mMTris-HCl of 5mM β one mercaptoethanol and 500 μ M EDTA, go up sample after the pH8.7 damping fluid balance, sample is through linear gradient elution, substep is collected the reorganization BHMT preliminary purification product of 45kd, further refining through fast protein liquid chromatography again, collecting molecular weight is the pure product of reorganization BHMT of 45kd.

The evaluation of embodiment 4, reorganization BHMT

Product SDS-PAGE method purity checking, used spacer gel is 5%, separation gel is 12%; Detected result shows that purity is all more than 98%.The N-terminal protein sequence analysis is the result show, the product that obtains really for target protein, order is M.P.P.V.G.G.K.K.A.K.K.G.I.L.E.R.

The activity calibrating of embodiment 5, reorganization BHMT

External conventional cultivate liver cell or myocardial cell add 10 in cell culture fluid ^-4M HCY, the BHMT0.5mg/ml that recombinates detects HCY change in concentration in the liver cell culture liquid, and detects myocardial cell's function and mortality ratio.The result shows that reorganization BHMT makes HCY content reduction by 20% in the liver cell culture liquid, and the cardiomyocyte cell death rate reduces by 2.1 times.Show that exogenous reorganization BHMT has the effect that reduces blood plasma HCY level, and the myocardial cell under the high-level HCY effect is had defencive function.

Claims (3)

1. bio-pharmaceutical one betaine homocysteine methyl transferase (BHMT) for the treatment of hyperhomocysteinemiainjury is characterized in that its N end structure territory 53-76 amino acids residue sequence is: 53peavrqlhrefleagadvmetftf.

2. the preparation method of the described betaine homocysteine methyl transferase of claim 1 (BHMT) comprises the steps:

(1) dna sequence dna of the 1st to the 80th amino-acid residue of chemical synthesis process preparation coding BHMT, wherein N holds 53-76 amino acids residue sequence according to claim 1;

(2) make up the restriction enzyme site of a Nco I at its 5 ' end, as 5 ' end primer, its sequence is 5 '-GCCCCCAACGGGTGCCATGGTTGTGGTGTCCAG-3 ', synthetic 3 ' the end primer of design, make it contain BamH I restriction enzyme site, sequence is 5 '-CGGGATCCTCACTGTGATTT GAATTT-3 ';

(3) extract the total RNA of purifying human liver tissue, under 3 ' end primer guiding, reverse transcription PCR, preparation comprises the people BHMT cDNA fragment of BHMT 77-406 amino acids residue encoding sequence;

(4) under 5 ' end and 3 ' end primer guiding,, obtain reorganization BHMT dna sequence dna through the PCR reaction;

(5) be connected construction of expression vector pTrc99A-hBHMT with behind above-mentioned BHMT dna sequence dna and the expression vector pTrc99A double digestion;

(6) expression vector that obtains is transformed among the E.coli JM105, makes up the BHMT recombinant strains;

(7) the BHMT expression strain of preparation is induced express recombinant BHMT;

(8) BHMT of separation and purification expression identifies and determination of activity.

3. the described betaine homocysteine methyl transferase of claim 1 (BHMT) application in the myocardial cell protection medicine under preparation hyperhomocysteinemiainjury medicine and high-level HCY effect.