CN1766106A - Brain natriuretic peptide (BNP)preparation method - Google Patents

Abstract

The invention discloses a preparation method for BNP to overcome the shortcomings that needs high cost, has low yield, and is limited in material resource in prior art. Wherein, using PCR technique to amplify objective gene; with Pet32a as carrier plasmid, enzyme-incision orientation inserting into objective gene to form recombinant plasmid; with escherichia coli DH5 alpha and BL21(DE3) as expression bacterial strain and His pole as purification pole, separating and purifying the objective protein. This invention is convenient to detect and enhance recombinant protein stability. The diagnosis reagent both prepared with BNP by immunological method can diagnose many cardiovascular diseases rapidly, exactly and specificically.

Description

Brain natriuretic peptide (BNP) preparation method

Technical field

The invention belongs to field of biomedicine technology, relate to a kind of cardiovascular peptide hormone---the preparation method of brain natriuretic peptide (BNP).

Background technology

At present, cardiovascular disorder has become the whole world sanitarian one and has threatened greatly, and the number that cardiovascular disorder is died from the whole world every year is up to 1,700 ten thousand, and age of onset is rejuvenation trend, and the crowd's heart trouble sickness rate that is in the work age bracket is more and more higher.The at present more common central force depletion of cardiovascular disorder, coronary heart disease, rheumatic heart disease, high worry etc., and the diagnosis of some disease is wherein still lacked specific quantizating index.As to coronary heart disease diagnosis, lack the useful quantitative biochemical indicator for a long time always.The patient is early stage normal expiratory dyspnea to occur, but its symptom, sign lack specificity, and the expiratory dyspnea discrimination ratio that causes with pulmonary disorder is difficulty, and determines that quickly and accurately the dyspneic cause of disease is extremely important to effective treatment of disease.And for example also difficult usually to the diagnosis of heart failure, in early days, as expiratory dyspnea, shank swelling and motion back fatiguability etc. also is some non-specific symptoms, conventional diagnosis depends on ultrasonic cardiogram, but can not popularize because of expense is expensive, application is restricted, and the unusual change that often is later than biochemical indicator that occurs on the ultrasonic cardiogram.Thereby the specific index of seeking a kind of quick, accurate and effective diagnosis of cardiovascular diseases has become a big emphasis often to be solved.

B-Type natriuretic peptide (BNP) has another name called the brain diuretic peptide, is a kind of cardiovascular peptide hormone of being separated in the pig brain by Japanese scholar sodoh in 1988 at first, belongs to one of sharp sodium peptide family member.Mainly synthetic justacrine in ventricular muscles has vasodilation, antagonism RAAS (RAAS), inhibition sympathetic activity, promotes effects such as natruresis, minimizing water-sodium retention.BNP also regulates the contractile function and the pressure load of ventricle specifically, and antagonism coronary vasospasm and pulmonary hypertension prevent the expression of myocardial fibrosis and vascular smooth muscle cell curing and endotheliocyte plasminogen activator inhibition (PAI).In clinical application, can be used for diagnosis to (HF) in heart failure, left ventricle contractile function confusion and coronary heart disease etc., these patient's blood plasma BNP level obviously rises (the BNP level is very low in normal people's peripheral blood, only is the pg level), and the severity of its rising degree and disease is proportionate.Also can be used for assessment to numerous disease prognosis, such as (HF), acute myocardial infarction (AMI) in heart failure, primary pulmonary hypertension, pulmonary infarction, or even general population's prognosis evaluation, its BNP level is high more, its prognosis is just poor more, and its case fatality rate is also high more.Also help the differential diagnosis of some disease, as to the dyspneic discriminating of the impatient property of pulmonary and heart source, the former BNP concentration is normal, and the latter then obviously raises.Also can be used for heart failure, the treatment of outstanding acute heart failure because the arteriovenous expansion of BNP tunable increases stroke output, increases cardiac output under the situation that does not change heart rate; Because of the disorderly patients with heart failure that causes of left chamber contractile function, use the BNP treatment, can reduce pulmonary capillary wedge pressure; And the level of reduction aldosterone and catecholamine, and then the sharp sodium of diuresis.In view of its obvious curative effects, (Food and Drug Administration FDA) just ratifies the clinical application of BNP for the acute mistake compensatory patients with heart failure of treatment as far back as calendar year 2001 in FDA (Food and Drug Adminstration).

Urine sodium peptide is made up of three kinds of peptides: atrial natriuretic peptide (atrialnatriureticpeptide, ANP), brain natriuretic peptide (brainnatriuretic peptide, BNP) and C-type urine sodium peptide (c-type natriuretic peptide, CNP), by the three kinds of hormone precursors of encoding respectively of gene independently, the three has its unique tissue distribution and regulation mechanism.Wherein BNP mainly expresses at human brain and ventricle.Human BNP precursors (Pro-BNP) are made up of 108 amino acid, sophisticated 32 amino acid whose compositions of cracking generation again, i.e. and BNP and a N-terminal fragment, the two all is present in the blood plasma.The BNP molecular weight is 3500, and the 7th of its aminoterminal and the 23rd amino acids residue (half dirty propylhomoserin) form a ring type structure by disulfide linkage, and this is the functional domain of BNP.BNP plays a role in many pathophysiological processes by its corresponding receptors bind of this conformation.Compare with atrial natriuretic peptide, brain natriuretic peptide in vivo action time longer, drug effect is better, content is higher in biological tissue, and is easy to extract, thereby has the market potential and be worth and application prospects.

Extract BNP method cost height in order to overcome tradition, yield is low, the easy sex change of target protein, many defectives such as material source is limited, the present invention provides feasible technical route for the mass preparation of BNP.The BNP that makes by this law is mainly used in the usefulness of preparation diagnostic kit for clinical disease diagnosis.

Summary of the invention

The preparation method of brain natriuretic peptide of the present invention (BNP) may further comprise the steps:

(1) adopt round pcr will express the goal gene amplification of BNP, and its product of purifying;

(2) with PCR product directed pet32a that inserts through same double digestion behind BamH I and Hind III double digestion of purifying, obtain recombinant plasmid Pet32a+BNP;

(3) recombinant plasmid Pet32a+BNP is converted into escherichia coli DH5a, increases through cultivating, extracting and purifying, sequencing analysis obtains the correct recombinant plasmid of order-checking;

(4) will check order correct recombinant plasmid transformed to the BL21DE30 bacterium, at 30 ℃, express recombinant protein under the 0.1mM IPTG condition;

(5) the activation inoculation with express recombinant protein arrives 2-YT substratum constant-temperature shaking culture, carries out shake flat experiment, and engineering bacteria is planted daughter bacteria and inoculated in 5% ratio in the fermentation of fermentor tank middle-high density, induces under low dissolved oxygen condition, puts jar, the centrifugal thalline that gets;

(6) the centrifugal bacterial sediment that obtains is carried out lysis, wash out inclusion body, the dissolving inclusion body, the His post is crossed column purification.

The brain natriuretic peptide (BNP) that uses the invention described above method to obtain can be used as antigen and makes diagnostic kit, is applied to the specific diagnosis of cardiovascular disorder.

Embodiment

The invention will be further described in conjunction with the embodiments.

The embodiment technological line is as follows:

1, BNP goal gene amplification, construction of recombinant plasmid and Recombinant Protein Expression: the BNP target gene sequences is according to the gene pool in the U.S. state-run health Library.

To express the goal gene amplification of BNP with round pcr, and its product of purifying.Again the PCR product of purifying is given the directed pet32a that inserts through same double digestion behind BamH I and the Hind III double digestion, obtain recombinant plasmid.Then above-mentioned plasmid is converted into bacillus coli DH 5 alpha, through cultivating amplification, extracting and purifying, and carry out sequential analysis with full-automatic sequenator.The recombinant plasmid transformed that order-checking is correct, is cultivated under the condition of 0.1mM IPTG at 30 ℃ to the BL21DE3 bacterium.Cultivate back cracking bacterium liquid, separate each protein band with SDS-PAGE, and detect with Western Blot instrument.

2, the fermentation of expression strain:

The activatory bacterial strain is linked in the 2-YT substratum (containing ampicillin100 μ g/ml) by 5% inoculum size, 37 ℃, 300r/min, constant-temperature shaking culture is carried out shake flat experiment.The engineering bacteria high density fermentation carries out in B.BROUN (Germany) 15L fermentor tank.Plant daughter bacteria and inoculate in 5% ratio, 37 ℃, 300r/min, ampicillin100 μ g/ml.High dissolved oxygen was cultivated 4 hours.At 1mmol/L IPTG, induced 4 hours under the low dissolved oxygen condition, put jar, the centrifugal thalline that obtains.

3, the protein purification of BNP:

3.1 the cracking of cell: the centrifugal intestinal bacteria precipitation that obtains is added lysis buffer carry out lysis, lysis buffer: 50mmol/L TrisCl (pH8.0), 1mmol/L EDTA, 100mmol/L NaCl.

3.2 the washing of inclusion body: with 12000g cell pyrolysis liquid is carried out centrifugation repeatedly in 4 ℃ with Eppendorf centrifuge, and precipitate resuspended liquid to determine whether that most of target proteins are all in precipitation with the sds polyacrylamide gel electrophoresis analysis.

3.3 the dissolving of inclusion body:

Washed precipitate is suspended in the lysis buffer that contains 0.1mmol/L PMSF (now adding existing usefulness), 8mol/L (deionized) urea, under room temperature and certain pH value, adds the sds gel sample loading buffer again it is dissolved, and survey its solubleness.

3.4 cross column purification: the His post is collected target peak, collects target peak, vacuum lyophilization after reversed-phase column.SDS-PAGE identifies purity of protein.

In preparation BNP process, use the round pcr amplifying target genes, with Pet32a as vector plasmid, enzyme forms recombinant plasmid after cutting the directed BNP of insertion goal gene, with bacillus coli DH 5 alpha, BL21 (DE3) is as expression strain, with the His post as purifying pillar separation and purification target protein.This preparation method both can conveniently detect, and can strengthen the stability of recombinant protein again.

The BNP that uses this invention technology to obtain makes diagnostic kit by immunological method, can be fast, accurately, many cardiovascular disordeies of diagnosis and differential diagnosis specifically, have bigger social benefit and economic benefit.

Need not further to elaborate, believe and adopt the disclosed content in front, those skilled in the art can use the present invention to greatest extent.Therefore, the preferred specific embodiments of front is interpreted as only illustrating, but not limits the scope of the invention by any way.

Claims (2)

1. the preparation method of brain natriuretic peptide (BNP) is characterized in that following steps:

(1) adopt round pcr will express the goal gene amplification of BNP, and its product of purifying;

(2) with PCR product directed pet32a that inserts through same double digestion behind BamH I and Hind III double digestion of purifying, obtain recombinant plasmid Pet32a+BNP;

(3) recombinant plasmid Pet32a+BNP is converted into escherichia coli DH5a, increases through cultivating, extracting and purifying, sequencing analysis obtains the correct recombinant plasmid of order-checking;

(4) will check order correct recombinant plasmid transformed to the BL21DE30 bacterium, at 30 ℃, express recombinant protein under the 0.1mM IPTG condition;

(5) the activation inoculation with express recombinant protein arrives 2-YT substratum constant-temperature shaking culture, carries out shake flat experiment, and engineering bacteria is planted daughter bacteria and inoculated in 5% ratio in the fermentation of fermentor tank middle-high density, induces under low dissolved oxygen condition, puts jar, the centrifugal thalline that gets;

(6) the centrifugal bacterial sediment that obtains is carried out lysis, wash out inclusion body, the dissolving inclusion body, the His post is crossed column purification.

2, by the preparation method of the described brain natriuretic peptide of claim 1 (BNP), it is characterized in that: a kind of brain natriuretic peptide (BNP) that uses aforesaid method to obtain is applied to the specific diagnosis test kit of cardiovascular disorder as antigen prepd.