CN104610443B - A kind of high stability restructuring Procalcitonin, Preparation method and use - Google Patents
A kind of high stability restructuring Procalcitonin, Preparation method and use Download PDFInfo
- Publication number
- CN104610443B CN104610443B CN201510085322.5A CN201510085322A CN104610443B CN 104610443 B CN104610443 B CN 104610443B CN 201510085322 A CN201510085322 A CN 201510085322A CN 104610443 B CN104610443 B CN 104610443B
- Authority
- CN
- China
- Prior art keywords
- procalcitonin
- pct
- high stability
- gly
- linking arm
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/585—Calcitonins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Endocrinology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Procalcitonin is recombinated the invention provides a kind of high stability, the structure of the albumen is：The katacalein of 1 calcitonin linking arm of PCT amino polypeptide linking arms 2, amino acid sequence such as SEQ ID NO:Shown in 1.The present invention substituted for being easy in PCT 58 59aa of degraded with flexible peptide chain linking arm, 92 95aa two parts peptide chains, using the amino acid sequence of the above-mentioned antigen of Escherichia coli optimal codon reverse translation, obtain the high stability PCT antigen genes being made up of Escherichia coli optimal codon, then clonal expression is carried out, high stability PCT antigens are obtained.Compared to wild type PCT, the antigen is except with more high stability, also maintaining wild PCT antigenicity.The high stability PCT GFPs that the present invention is provided are that, using Escherichia coli optimal codon composition, have higher expression quantity in Escherichia coli, be adapted to a large amount of prepare.PCT is recombinated simultaneously and can be used for the preparation of immune detection Plays product and the drafting of standard curve, so as to realize the accurate quantitative analysis to PCT contents.
Description
Technical field
The present invention relates to the transformation of Procalcitonin (Procalcitonin, PCT) high stability, codon optimised genes, its is heavy
Histone, further relates to the evaluation and the application in standard items Drawing of Curve of the protein stability.
Background technology
Procalcitonin (Procalcitonin, PCT) is human calcitonin (Calcitonin, CT) precursor substance, by 116
Individual amino acid composition, relative molecular mass 13KD.PCT is that C cells are produced and secreted by thyroid follicle, and by intracellular special
Proteases for decomposing is active calcitonin.PCT contents are very low in normal human serum, but in bacterial infection or septicemia, septicopyemia
Situations such as mass formed by blood stasis is to steeply rise, same to leucocyte, interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and C is anti-
Albumen (CRP) these inflammatory factors are answered to compare, its sensitivity and specificity etc. are higher.Therefore it can be made whether according to PCT concentration
There is the tentative diagnosis of bacterial infection, and choose whether to use antibiotic.Usual clinic thinks to work as PCT in serum>0.25ng/
Antibiotic can be used during ml；PCT>5ng/ml must use antibiotic.Therefore, PCT levels can be thin as identification in serum
Bacterium gradient of infection and antibiotic instruct the clinical indices used, prompting bacterium infection and its gradient of infection, judge therapeutic effect,
Instruct to played an important role in terms of antibacterial drug therapy.
There are a variety of methods, such as radioimmunology currently used for what PCT was detected, enzyme exempts from method, immunochemiluminescence method, Gold standard
Deng, and these methods must prepare the standard items of gradient concentration with PCT antigens, calculate the content of PCT in sample.Because PCT is anti-
Original contains degradation site, therefore, extremely unstable under normal condition, easily degraded, greatly influences the accuracy of testing result.
PCT is the inactive preceding peptide material of calcitonin (Calcitonin, CT), by positioned at No. 11 chromosomes
Calc-I gene codes on (11p1514), generate Calc-ImRNA, normally by alternative splicing (1~4 extron of coding)
Under the conditions of, PCT mRNA translate into the preceding PCT containing 141 amino acid residues in parafollicular cell rough surfaced endoplasmic reticulum (RER),
Molecular mass is 16kD.Preceding PCT enters endoplasmic reticulum, cuts off the signal peptide of N- ends with enzyme-specific through glycosylating, and generation contains
The PCT of 116 amino acid residues, in golgiosome, PCT amino polypeptides (1- is ultimately formed through series hydrolysis enzyme effect
57aa), calcitonin (60-91aa) and katacalein (96-116aa).It can be seen that 58-59aa, 92-95aa are respectively water
Site is solved, the presence of these hydrolases is probably to cause the unstable key factor of PCT reference materials.
The degradation site gene that this research is mutated in PCT by anneal extension PCR method and bridging PCR methods, on this basis
Build prokaryotic expression system high efficient expression recombination, after obtaining the higher destination protein of purity through column chromatography, stability
Experiment experiment display：The restructuring PCT albumen for knocking out protease cutting site is efficiently solved in conventional PCT antigens examination criteria product
The bottleneck problem easily degraded.The examination criteria product of reliable high stability are provided for PCT immune detection, for expanding
The clinical practice of PCT detections, preferably instructs antibiotic medication, significant.
The content of the invention
It is an object of the invention to provide a kind of restructuring PCT antigens of high stability.Another object of the present invention is provided with large intestine
The above-mentioned antigen epitope genes of bacillus optimal codon composition.The invention provides the PCT restructuring obtained on said gene basis
Antigen.Present invention also offers the experimental result in above-mentioned antigen in PCT immune detections in terms of stability, the antigen is realized
Experiment purpose with higher stability.
Specifically, the present invention provides a kind of restructuring PCT antigens of high stability, it is characterised in that:The antigen fragment is
PCT amino polypeptide-linking arm 1- calcitonins-linking arm 2- katacaleins.Described amino polypeptide, calcitonin, katacalein are equal
For the part of PCT antigens, its site is located at 1-57aa, 60-91aa and 96-116aa of wild PCT antigens respectively.It is described
Linking arm 1 is (- Gly-Gly-), and the linking arm 2 is (- Ser-Gly-Gly-Gly), the restructuring Procalcitonin amino acid sequence
Row such as SEQ ID NO:Shown in 1.
More specifically, the restructuring Procalcitonin fragment linking arm 1 and linking arm 2 substitute the 58- in natural PCT sequences
59aa, 92-95aa two parts peptide chain.
The nucleotide sequence such as SEQ ID NO of the codon optimised genes of above-mentioned high stability restructuring PCT antigens:2 institutes
Show.
A kind of high stability recombinates the preparation method of Procalcitonin, comprises the following steps：
1) high stability Procalcitonin amino acid sequence is designed：Using linking arm 1 (- Gly-Gly-) and linking arm 2 (-
Ser-Gly-Gly-Gly natural calcitonin original position) is replaced respectively in 58-59aa hydrolytic sites (- Lys-Arg-) and positioned at 92-
95aa hydrolytic sites (- Gly-Lys-Lys-Arg-), the amino acid sequence such as SEQ ID NO of the PCT albumen after replacement:Shown in 1；
2) codon optimization and acquisition of high stability Procalcitonin gene：Using Escherichia coli optimal codon by step
1) SEQ ID NO in:1 amino acid sequence reverse translation is into nucleotide sequence such as SEQ ID NO:Shown in 2, extended using annealing
Round pcr, obtains the high stability Procalcitonin gene being made up of Escherichia coli optimal codon；
3) clone of high stability Procalcitonin gene, expression：By step 2) the middle high stability calcitonin former base obtained
Because carrying out double digestion, it is connected with the expression vector pBVIL-1 for also passing through double digestion, builds corresponding expression plasmid, Ran Houzhuan
Change E.col i HB101 competent cells, obtain target gene height expression bacterial strain, and are cultivated, and express, purify acquisition height surely
Qualitative restructuring Procalcitonin.
A kind of application of high stability restructuring Procalcitonin in high stability PCT antigens are prepared.
A kind of codon optimised genes answering in high stability PCT antigens are prepared of high stability restructuring Procalcitonin
With.
A kind of application of high stability restructuring Procalcitonin in PCT detection reagent Plays product Drawing of Curve.
A kind of PCT immune detections reference material, Procalcitonin is recombinated comprising above-mentioned high stability, and wherein PCT reference materials are
Standard curve method is drawn using quadratic polynomial equation model to carry out, the drafting available for PCT concentration standard curves in sample
And detection.
Compared with prior art, the invention has the characteristics that：
1. invention is set up on the basis of the transformation of natural PCT antigen hydrolysis site.Natural PCT antigens are in physiological conditions
Can occur hydrolysis, cut off 58-59aa, 92-95aa two parts peptide chains form 3 kinds of albumen with difference in functionality.This research
Using technique for gene engineering and bioinformatics Antigen Epitope Prediction technology, replace natural PCT antigens with 2 peptide chain linking arms 2
Hydrolytic sites, the generation of PCT physiology hydrolysis is prevented from amino acid sequence composition, so as to obtain the restructuring with more high stability
PCT antigens.
2. invention is set up on the basis of the restructuring PCT antigens obtained using codon-optimization techniques.Because natural PCT is anti-
Original is expressed in human body cell, that is, the mammalian cell system used, and the restructuring PCT antigens that this research is obtained are to set up
On escherichia expression system, both have different optimal codons, also have different requirements to the structure of gene.For
The restructuring PCT antigens of higher expression quantity are obtained, the present invention utilizes codon-optimization techniques, obtains by Escherichia coli advantage password
Molecular high stability PCT optimization genes, realize its high efficient expression in Escherichia coli, meet PCT detection reagents pair
The demand of yield.
Beneficial effects of the present invention：In order to obtain the restructuring PCT antigens with high stability, the present invention is directed to PCT albumen
2 hydrolytic sites 58-59aa, 92-95aa in sequence have carried out amino acid substitution respectively, meanwhile, in order that the height obtained is steady
Qualitative PCT albumen has the antigenicity of wild PCT albumen, and the present invention replaces original water using the linking arm of soft peptide chain composition
Site is solved, the polypeptide near the corresponding site after replacement is had the space of abundance, preferably keeps the sky of PCT after amino acid substitution
Between conformation.
The problem of in order to overcome wild MP gene codons bias, the present invention is reversely turned over using Escherichia coli optimal codon
The amino acid sequence of above-mentioned antigen is translated, the high stability PCT antigen genes being made up of Escherichia coli optimal codon, its base is obtained
High stability PCT antigen genes are obtained because sequence is as shown in sequence 1 in sequence table, and using annealing extension PCR technology.
Clonal expression is carried out to above-mentioned high stability PCT antigen genes using gene recombination technology, high stability PCT is obtained
Antigen, the PCT antigens of the high stability obtained using the PCT detection reagents of Roche Holding Ag to the present invention are entered with commercially available PCT antigens
Row stability compares, and evaluates its meaning in PCT detections.
Brief description of the drawings
Fig. 1 is compared for transformation PCT antigens with the sequence of natural PCT antigens；
Fig. 2 is the standard items curve that the examination criteria product prepared using transformation PCT antigens are drawn.
Embodiment
The principle and feature of the present invention are described below in conjunction with accompanying drawing, the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the present invention.
To achieve the above object, the present invention is used as linking arm according to natural PCT amino acid sequences using two sections of soft peptide chains
Replace PCT 2 hydrolytic sites, design high stability PCT amino acid sequence.Reversely turned over using Escherichia coli optimal codon
It is translated into nucleotide sequence；Using annealing extension PCR technology, the PCT antigen epitope genes of high stability, gene engineering expression are obtained
Afterwards, large-scale purification prepares transformation PCT antigens.The stability of restructuring PCT antigens is obtained using breaking test evaluation.
The technical scheme is that：
Natural PCT amino acid sequences are downloaded on NCBI websites, using linking arm 1 (- Gly-Gly-) and linking arm 2 (-
Ser-Gly-Gly-Gly PCT) is replaced respectively to be located at 58-59aa hydrolytic sites (- Lys-Arg-) and hydrolyze position positioned at 92-95aa
Point (- Gly-Lys-Lys-Arg-), obtains high stability PCT amino acid sequence (1 in such as sequence table).And use large intestine bar
Gene order (2 in such as sequence table) of the bacterium optimal codon reverse translation into nucleotide sequence, i.e. high stability PCT.
In order to obtain said gene, said gene is divided into two sections of A, B, 6 primers of every section of design, through moving back twice by the present invention
Two sections of genes of A, B are obtained after fiery extension PCR, complete high stability PCT gene order is obtained after further putting up a bridge.In order to just
In gene cloning to expression vector, two restriction enzyme sites of XhoI and XbaI are introduced at the two ends of gene, to be adapted to expression vector
pBVIL-1.After double digestion in insertion vector pBVIL-1, corresponding expression plasmid is built.PCR sequencing PCR proves that each genetic fragment has been obtained
It is correctly inserted into.
After expression plasmid Transformed E .coli HB101 containing above-mentioned PCT genes, induced by IPTG, take full bacterium solution to carry out
SDS-PAGE is identified, it was demonstrated that the plasmid has obtained efficient expression, then through ion exchange column Sepharose-Q posts and gel
Purification by filtration, can obtain electrophoretically pure PCT antigens sterling.Commercialization antigen is control, using 37 DEG C of breaking tests, to obtaining
The stability of PCT antigens verified.
The testing result carried out using the PCT antigen detecting agents of Roche companies is shown：After 37 DEG C are destroyed 2 days, transformation
The coefficient of stabilization of recombinant antigen and commercialization antigen is respectively 89.99 ± 0.25%, 19.46 ± 0.73%；After destruction 7 days, both
Coefficient of stabilization be respectively 62.53 ± 0.41,5.67 ± 0.07；The stability of PCT antigens is significantly higher than control PCT antigens after transformation
(P ＜ 0.01, t=180.56；P ＜ 0.01, t=271.44).The PCT antigens that further present invention is obtained match series concentration
Examination criteria product, definite value is carried out to the PCT of various concentrations using fluorescence immune chromatography detection technique, and draw standard curve,
Statistical analysis is shown：PCT concentration can be effectively fitted with fluorescence intensity by quadratic polynomial equation, and both have obvious phase
Closing property (r=0.9913P<0.01) the high stability PCT antigens that, prompting is obtained can be used for PCT antigen concentrations as standard items
Detection.
Lower mask body four aspects of the present invention.
The design of the high stability PCT antigen amino acid sequences of embodiment 1
Natural PCT amino acid sequences GI is downloaded on NCBI websites:76880474, using linking arm 1 (- Gly-Gly-) and even
Connect arm 2 (- Ser-Gly-Gly-Gly) and replace PCT respectively positioned at 58-59aa hydrolytic sites (- Lys-Arg-) and positioned at 92-95aa
Hydrolytic sites (- Gly-Lys-Lys-Arg-), obtain high stability PCT amino acid sequence (1 in such as sequence table).After replacement
PCT amino acid sequences for compared with former sequence as shown in figure 1, wherein different amino acids with " * " show, same amino acid use
"=".
The codon optimization and acquisition of the high stability PCT antigen genes of embodiment 2
Using Escherichia coli optimal codon by the reverse translation of sequence 1 into nucleotide sequence, i.e. high stability PCT gene
Sequence (2 in such as sequence table).
In view of the accuracy of current domestic gene primer synthesis, PCT genes are designed to two sections, every section of A, B by the present invention
Gene designs 6 primers and synthesized respectively, and every prime end has the matching sequence of 16 nucleotides, the primer of every section of synthesis
Sequence is as shown in the 3-14 in sequence table.The preparation method is described for convenience, is corresponding primer sequence name, SEQ ID
NO:3 to 8 are respectively designated as AF1, AR1, AF2, AR2, AF3, AR3；SEQ ID NO:9 to 14 be respectively designated as BF1, BR1,
BF2、BR2、BF3、BR3；
Methods described is as follows：
Synthesis said gene needs 3 wheels to anneal extension PCR reaction altogether, first round PCR reaction system be hundred Imtech 2 ×
The μ l of PCR reaction solutions 25,23 μ l, XF1 and XR1 primers of distilled water each 1 μ l, interim X represent A, B, and PCR reaction conditions are 94 DEG C 1 point
Zhong Hou, 94 DEG C 1 minute, 55 DEG C 1 minute, 72 DEG C 1 point, 5 circulation；Follow-up two-wheeled PCR reaction systems be hundred Imtech 2 ×
The μ l of PCR reaction solutions 25,22 μ l, XFn and XRn primers of distilled water each 1 μ l, n-1 wheel PCR primer 1 μ l, n represent 2,3；Reaction condition
Be 94 DEG C after 3 minutes for PCR reaction conditions, 94 DEG C 1 minute, 55 DEG C 1 minute, 72 DEG C 1 minute, 72 DEG C of extensions again after 30 circulations
5 minutes, that is, obtain two kinds of genes of A, B.Take A, 1 B gene product a 1 μ l, add the μ l of 2 × PCR reaction solutions 25, the μ l of distilled water 21,
Each 1 μ l of AF3 and BR3 primers, reaction condition is that PCR reaction conditions are 94 DEG C after 3 minutes, 94 DEG C 1 minute, 55 DEG C 1 minute, 72 DEG C
1 minute, after 30 circulations again 72 DEG C extend 5 minutes, that is, obtain high stability PCT gene order.
Clone, expression and the mark of the high stability PCT antigen genes of embodiment 3
1. the structure of high stability PCT antigen presentation plasmids
1.1 PCR primers and expression vector pBVIL-1 plasmid double digestions
Take above gene outcome and each 30 μ l of pBVIL-1 expression vectors to be put in respectively in Eeppendorf centrifuge tubes, respectively add
Enter 10 × buffer (D) 4 μ l, XhoI (10u/ μ l) and XbaI (12u/ μ l) each 1 μ l, plus sterile purified water puts 37 DEG C to 40 μ l
Water-bath digestion is stayed overnight.
The agarose gel electrophoresis purifying and recovery of digestion products：PCR primer and carrier pBVIL-1 are used after double digestion
1.2% Ago-Gel is purified, and specific method is pressed《Molecular cloning》The method of (Science Press, the second edition) is carried out.It is pure
Change a small amount of glue reclaim kits recovery that gene is produced with Shanghai Hua Shun bioengineering Co., Ltd again：I.e. under uviol lamp respectively
The agarose containing plasmid and target gene is cut, is put in Eeppendorf centrifuge tubes, it is each to add S1 liquid, put 55 DEG C of 10 points of water-baths
Clock makes peptization solution, adds equivalent isopropanol, mixes, 55 DEG C of warm bath 1 minute, then moves into after adsorption column, is said by kit respectively
Bright book is purified.
1.2. connection：In sterilizing Eeppendorf centrifuge tubes in add each 1 μ l of carrier and target gene after above-mentioned digestion,
The μ l of 10 × T4DNA Ligase buffer 1, the μ l of T4DNA Ligase (12u/ul) 1, plus sterile purified water put 16 DEG C to 10 μ l
Overnight.
1.3 conversion：In superclean bench, 100 μ l competent cells are taken with sterile pipette tip, and (competent cell is pressed《Molecule
Clone》The method of (Science Press, the second edition) is carried out) suspension is in Eppendorf, and the above-mentioned μ l of attachment 5 of addition gently revolve
Turn to mix, 30 points of ice bath.It is immediately transferred in 42 DEG C of water-baths place 2 minutes, often pipe addition 0.5ml LB culture mediums (are not added with antibiosis
Element), after 30 DEG C of shaking bath cultures 60 minutes, respectively take 0.2ml to be applied to respectively on LB agar culture plates and (contain antibiotic), room
After temperature is dried, 37 DEG C of constant temperature carton upside down overnight incubations are put.Several bacterium colonies are selected, (5ml/ pipes) is inoculated in LB respectively, was cultivated
Night, next day respectively takes 0.1ml to be transferred in LB (2ml/ pipes), and 32 DEG C are cultivated 3 hours, and 1mM IPTG Fiber differentiation 8h receive bacterium, use
SDS-PAGE identifies that selection target gene obtains high expression bacterial strain, and sequencing identification.
2. the expression and purification of antigen
The culture of 2.1 expression bacterial strains：The μ l of expression bacterial strain 20 of -70 DEG C of preservations are taken to be inoculated in (100ml in LB culture mediums
LB/500ml triangular flasks), 30 DEG C of air table overnight incubations, next day is in 5% ratio transferred species in LB culture mediums (ibid), 30 DEG C
Air table culture about 3 hours, when OD600 values reach 0.7,42 DEG C of Fiber differentiation 8h merge bacterium solution, 6000rpm centrifugations
20 minutes, supernatant is abandoned, sediment fraction is collected.
2.2 extract inclusion body：Claim weight in wet base by precipitation, will be precipitated with the 20mmol/L pH8.0TE buffer solutions of 10 times of volumes
Hang, add lysozyme (1mg/ml suspensions), at room temperature magnetic agitation 10 minutes.The ultrasonic disruption bacterium in ice bath, every time
Surpass 30 seconds, be spaced 30 seconds, surpass altogether 10 times.8 DEG C, 1,2000rpm is centrifuged 20 minutes, abandons supernatant, and precipitation uses 1mol/L
NaCl (being prepared with TE) is washed once, then is washed 2 times with TE, collects precipitation.Precipitation is dissolved with 8M ureas (being prepared with PH8.0TE), plus 1%
Beta -mercaptoethanol.Then at 20 DEG C, 1,2000rpm, centrifuge 10 minutes, go precipitation to take supernatant.
2.3 purifying：The liquid solution of forgiving of above-mentioned dissolving is crossed into Q-Sepharose FF anion-exchange columns, equilibrium liquid is used
After (pH8.0,20mmol/L TE ureas containing 6mol/L, 0.1% beta -mercaptoethanol) cleaning, (equilibrium liquid is used with the NaCl of various concentrations
Prepare) elution, each eluting peak is collected, is identified through SDS-PAGE, 0.05mol/L NaCl eluting peaks are collected.After Sephardex
G-50 solvent resistant columns, collect the first eluting peak, the purity of antigen are identified using with SDS-PAGE.
Embodiment 4 recombinates the measure of PCT Antigen Stabilities
The Elecsys BRAHMS PCT reagents produced using Roche Holding Ag are to above-mentioned restructuring PCT antigens and commercialization PCT
The stability of antigen is detected.Operation carry out to specifications, 30 μ l samples, biotinylated monoclonal PCT antibody and
The monoclonal PCT antibody that ruthenium is combined substance markers carries out first time incubation, forms antigen-antibody sandwich complex, then add coating chain
The bead particulates of mould Avidin carry out second and are incubated, and complex is combined by the effect of biotin and streptomysin with magnetic bead, adopted
Detected with the analyzers of Elecsys 2010, calculated automatically by calibration curve and obtain testing result.For the ease of statistical
Present invention restructuring PCT and commercialization PCT is respectively using 4 samples in analysis, stability experiment, as a result respectively as shown in table 1,2：
The restructuring PCT estimation of stabilitys that the present invention of table 1 is obtained
The commercialization PCT estimation of stabilitys of table 2
Statistical result showed：After 37 DEG C of destructions 2 days, the restructuring PCT antigens that obtain of the present invention and commercialization PCT antigens it is steady
It is respectively 89.99 ± 0.25%, 19.46 ± 0.73% to determine rate；After destruction 7 days, both coefficient of stabilizations are respectively 62.53 ±
0.41%th, 5.67 ± 0.07%；The stability for the PCT antigens that the present invention is obtained is significantly higher than control PCT antigens (P ＜ 0.01, t
=180.56；P ＜ 0.01, t=271.44).
Embodiment 5 recombinates the drafting of PCT standard curves
1.0mg/ml PCT-C section monoclonal antibodies and sheep anti-mouse igg coating detection line and nature controlling line is respectively adopted, using containing 1%
BSA, 5% sucrose, 1%Tween 20,0.01M pH 7.4 PB are with 1：100 dilution proportion Alexa(AF)647
Fluorescein-labeled PCT monoclonal antibodies, it is lyophilized that release pad is made, and it is assembled into reagent card with detection line and nature controlling line.The present invention is obtained
The restructuring PCT obtained is diluted to 0.25ng/ml, 0.5ng/ml, 1ng/ successively using the PB containing 1%BSA, 0.01M pH 7.4
PCT examination criteria product are made in ml, 2ng/ml, 4ng/ml.The reagent card prepared is taken, 100ulPCT examination criterias are separately added into
Product, at ambient temperature, are detected, and use mELISA 1.0.1.0_x64 softwares after reaction in 3 minutes with Immunofluorescence test instrument
Standard curve is drawn, as a result as shown in Figure 2.Statistical analysis is shown：PCT concentration can pass through quadratic polynomial equation with fluorescence intensity
Effectively it is fitted, its fit equation is：
Y=A+B*X+C*X^2
Wherein A=-0.460406249999998
B=11.7704926075269
C=-1.56944892473118
Detect both PCT concentration and fluorescence intensity in sample with obvious correlation (r=0.9913P<0.01), point out
The restructuring PCT antigens that the present invention is obtained can be used for the detection of PCT antigen concentrations in sample as examination criteria product.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc. should be included in the scope of the protection.
Claims (9)
1. a kind of high stability recombinates Procalcitonin, it is characterised in that:The restructuring Procalcitonin fragment structure is Procalcitonin
Amino polypeptide-linking arm 1- calcitonins-linking arm 2- katacaleins, wherein amino polypeptide, calcitonin, katacalein site are located at
1-57aa, 60-91aa and 96-116aa, linking arm 1 are-Gly-Gly-, and linking arm 2 is-Ser-Gly-Gly-Gly-, described heavy
Group Procalcitonin amino acid sequence such as SEQ ID NO:Shown in 1.
2. a kind of high stability restructuring Procalcitonin according to claim 1, it is characterised in that the restructuring Procalcitonin
In fragment the 58-59aa in the former sequence of natural calcitonin, 92-95aa two parts peptide chains are substituted with linking arm 1 and linking arm 2.
3. the high stability described in a kind of claim 1 recombinates the codon optimised genes of Procalcitonin, it is characterised in that described
The nucleotide sequence of codon optimised genes such as SEQ ID NO:Shown in 2.
4. a kind of high stability recombinates the preparation method of Procalcitonin, it is characterised in that comprise the following steps：
1) high stability Procalcitonin amino acid sequence is designed：Using linking arm 1-Gly-Gly-, and linking arm 2-Ser-Gly-
Gly-Gly-, replaces natural calcitonin in situ in 58-59aa hydrolytic sites-Lys-Arg- and positioned at 92-95aa hydrolysis position respectively
Point-Gly-Lys-Lys-Arg-, the amino acid sequence such as SEQ ID NO of the Procalcitonin albumen after replacement:Shown in 1；
2) codon optimization and acquisition of high stability Procalcitonin gene：Using Escherichia coli optimal codon by step 1) in
SEQ ID NO:1 amino acid sequence reverse translation is into nucleotide sequence such as SEQ ID NO:Shown in 2, using annealing extension PCR skill
Art, obtains the high stability Procalcitonin gene being made up of Escherichia coli optimal codon；
3) clone of high stability Procalcitonin gene, expression：By step 2) in obtain high stability Procalcitonin gene enter
Row double digestion, is connected with the expression vector pBVIL-1 for also passing through double digestion, builds corresponding expression plasmid, then converts
E.coli HB101 competent cells, obtain target gene height expression bacterial strain, and are cultivated, and express, purify acquisition high stable
Property restructuring Procalcitonin.
5. high stability restructuring Procalcitonin the answering in high stability Procalcitonin antigen is prepared described in a kind of claim 1
With.
6. a kind of application of the codon optimised genes described in claim 3 in high stability Procalcitonin antigen is prepared.
7. the high stability restructuring Procalcitonin described in a kind of claim 1 is painted in Procalcitonin detection reagent Plays product curve
Application in system.
8. a kind of Procalcitonin immune detection reference material, it is characterised in that recombinate drop comprising the high stability described in claim 1
Calcium element is former.
9. Procalcitonin immune detection reference material according to claim 8, it is characterised in that the Procalcitonin reference material
It is to draw standard curve method using quadratic polynomial equation model to carry out.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510085322.5A CN104610443B (en) | 2015-02-16 | 2015-02-16 | A kind of high stability restructuring Procalcitonin, Preparation method and use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510085322.5A CN104610443B (en) | 2015-02-16 | 2015-02-16 | A kind of high stability restructuring Procalcitonin, Preparation method and use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104610443A CN104610443A (en) | 2015-05-13 |
| CN104610443B true CN104610443B (en) | 2017-08-29 |
Family
ID=53145118
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510085322.5A Active CN104610443B (en) | 2015-02-16 | 2015-02-16 | A kind of high stability restructuring Procalcitonin, Preparation method and use |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104610443B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108084256A (en) * | 2017-12-28 | 2018-05-29 | 北京市华信行生物科技有限公司 | Procalcitonin mutant and its preparation method and application |
| CN111996195A (en) * | 2020-04-26 | 2020-11-27 | 润方(长春)生物科技有限公司 | Prokaryotic recombinant expression and purification method of procalcitonin mutant protein |
| CN114478745B (en) * | 2022-03-30 | 2023-12-15 | 南京京达生物技术有限公司 | Separation and purification method of recombinant procalcitonin PCT |
| CN114437199B (en) * | 2022-04-08 | 2022-06-07 | 广州市雷德生物科技有限公司 | High-stability recombinant procalcitonin, and expression vector and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103275223B (en) * | 2013-06-04 | 2015-06-17 | 福建省洪诚生物药业有限公司 | Method for preparing procalcitonin antibody |
-
2015
- 2015-02-16 CN CN201510085322.5A patent/CN104610443B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN104610443A (en) | 2015-05-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104610443B (en) | A kind of high stability restructuring Procalcitonin, Preparation method and use | |
| CN104059133A (en) | Mutant protein A with high alkali-resisting characteristic and application thereof | |
| CN101556287B (en) | Novel protein molecular weight standard and preparation method thereof | |
| CN108912221A (en) | For producing auxilin, encoding gene, recombination fusion protein, recombinant expression carrier and the preparation method of recombination fusion protein | |
| CN104198741A (en) | Tilapia streptococcus agalactiae IgM antibody capture ELISA detection kit | |
| CN103059109B (en) | Mycoplasma pneumonia antigen, preparation method and immunodetection kit | |
| CN110746499A (en) | Serum amyloid protein A mutant and application and preparation method thereof | |
| CN111253478B (en) | Mycoplasma pneumoniae antigen and preparation method and application thereof | |
| CN111647055B (en) | N protein for detecting novel coronavirus, preparation and application thereof | |
| CN112028975A (en) | 2019 method for preparing novel coronavirus spike protein receptor binding domain protein | |
| CN114276445B (en) | Rotavirus recombinant protein specific antibody, plasmid vector and method | |
| CN113588946B (en) | Recombinant protein and method for detecting mycoplasma hyopneumoniae antibody by indirect ELISA (enzyme-linked immunosorbent assay) | |
| CN111087453A (en) | Preparation method and application method of chlamydia pneumoniae recombinant antigen | |
| CN109239351B (en) | Lotus root latent virus double-antibody sandwich enzyme-linked immunosorbent assay kit and preparation and detection methods thereof | |
| CN102221616A (en) | Indirect ELISA (Enzyme-Linked Immuno Sorbent Assay) diagnostic kit of mycoplasma gallisepticum | |
| CN102702324B (en) | Application of human procalcitonin B cell epitope peptide fragment and monoclonal antibody thereof | |
| CN113004380B (en) | Treponema pallidum recombinant antigen, preparation and application | |
| CN103834667A (en) | Chemosynthetic extracellular region gene fragment of streptococcus pneumonia PspA protein, and expression and application thereof | |
| US20190016754A1 (en) | Methods of producing and purifying matrix-binding fusion proteins by ion-exchange chromatography | |
| CN100406472C (en) | Fusion and solubility expression of cobratoxin and acidolysis release and purification of recombinant toxin | |
| CN104945488B (en) | Polypeptide with immunoglobulin binding capacity | |
| CN114317504B (en) | Limulus factor C truncated protein and preparation method and application thereof | |
| CN108530521A (en) | Recombinate the preparation and application of hepatitis C antigen | |
| CN114891079B (en) | French phoenix tree pollen allergen and application thereof | |
| CN108084256A (en) | Procalcitonin mutant and its preparation method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |