CN109187774A - A kind of quantitative detecting method of 2 porcine circovirus virus-like particle - Google Patents
A kind of quantitative detecting method of 2 porcine circovirus virus-like particle Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
Abstract
The purpose of the present invention is to provide a kind of easy to operate, high sensitivity, repeatability and the high PCV2 virus-like particle detection methods of applicability, and this method comprises the following steps: step S1, and dilution sample to be tested is simultaneously filtered clarification or centrifugal clarification；Step S2, taking protein content, determined 2 porcine circovirus virus-like particle standard items are serially diluted, and carry out HPLC detection to the serial standards after dilution, and establishing by abscissa, peak height of concentration is the regression equation of ordinate；Step S3 carries out HPLC to the sample to be tested after dilution and detects to obtain corresponding sample peak height；Step S4, the 2 porcine circovirus virus-like particle concentration in sample to be tested is calculated according to regression equation and sample peak height, wherein chromatographic column used by the HPLC in step S2 and step S3 is gel chromatographic columns, the detector used is UV detector, Detection wavelength 280nm.
Description
Technical field
The invention belongs to field of biotechnology, are related to the quantitative detecting method of virus-like particle, and in particular to boar circle
The quantitative detecting method of 2 virus-like particle of circovirus virus.
Background technique
Pig circular ring virus (porcine circovirus, PCV) belongs to circovirus section (Circoviridae) annulus disease
Poison belongs to (Circovirus), is the smallest virus found so far, particle diameter minimum only 17nm, DNA gene group leader
1.7kb, virion are icosahedral symmetry, without cyst membrane, sub-thread cyclic DNA virus.There are two serotypes by known PCV, non-
Pathogenic PCV1 and pathogenic PCV2.PCV2 is to cause pmws (Postweaning
Multisystemic Wasting Syndrome, PMWS) main pathogen, with swine fever virus (CSFV) or pig breeding and exhale
The severity of disease can be enhanced by inhaling the mixed infections such as syndrome virus (PRRSV), can be caused 10%~30% not equal dead
Rate is died, more serious pig farm death and culling rate when breaking out this disease is up to 40%, causes to seriously threaten to pig breeding industry.Currently, PCV2
Vaccine immunization is the main means of prevention and control PCV2, and antigenic content is the most important factor for influencing immune effect of vaccine.
In field in use, as without the immunizing antigen amount enough with production actual requirement, although controllable clinical onset, often because
Immunity deficiency causes pig to infect, and shows as long-term band poison and subclinical infection, seriously affects pig growth performance, cause
Larger economic loss.
PCV2 virus-like particle (Virus-Like Particles, VLPs) is that the Porcine circovirus type 2 Cap of recombinant expression passes through certainly
60 aggressiveness formed are assembled, molecular weight is about 1800kDa, and it is close with natural viral particle structure, so immunogenicity is better than
Cap protein.The morphosis of VLPs is similar to natural viral height, and immunogenicity is good, and does not contain viral gene, therefore
It is a kind of ideal vaccine form.
The VLPs structure evaluation and antigen quantitative detection of virus sample particle vaccines are the control of virus sample particle vaccines quality
Important component, but since virus-like particle is the macromolecular substances of polyprotein assembling, general biology quantitative detection side
Method is difficult to distinguish VLPs and protein monomer while quantitative, also in fubaritic solution virus-like particle stable state.Mesh
Preceding PCV2 antigen quantitative detecting method is mainly enzyme linked immunosorbent assay (ELISA) Enzyme Linked Immunosorbent
Assay (ELISA) method and SDS-PAGE method, and both methods is not suitable for the quantitative detection of PCV2VLPs: ELISA
Quantitative approach is that known antigen or antibody are adsorbed on to solid phase carrier (polystyrene micro-reaction plate) surface, marks enzyme
Antigen-antibody reaction carried out in solid phase surface, then carry out quantifying for antigen or antibody, this method cannot be distinguished
PCV2VLPs and Cap protein monomer, while it captures antibody and also limits this to the difference of the antigen affinity of different genotype
The application of method；SDS-PAGE method is a kind of semi-quantitative method, can not antigen concentration in accurate quantitative analysis sample, and equally
It cannot distinguish between PCV2VLPs and Cap protein monomer.
High performance liquid chromatography (HPLC) is a kind of easy to operate, efficient and high sensitivity analysis method, detection sensitivity
Up to ng grades, analysis speed is fast, and the relative average debiation of testing result is usually no more than 2% after same sample repetition loading, mesh
Before, HPLC is widely applied to the various fields such as biochemistry, food analysis, medical research, environmental analysis, inorganic analysis.Cause
This, the quantitative analysis of PCV2VLPs is carried out using HPLC detection technique, not only can be shortened traditional ELISA method and SDS-
The detection time of PAGE method, moreover it is possible to improve detection sensitivity and stability, be conducive to the quality for improving PCV2VLPs production of vaccine
Control improves vaccine product quality.
It is difficult to be suitable for the quantitative detection of PCV2 VLPs however, existing HPLC detection method is applied to PCV2VLPs, it is domestic
Outer document and patent retrieval also do not retrieve relevant report, and reason includes: that 1, PCV2VLPs is by 60 aggressiveness Cap protein groups
The polymer composite dressed up, conventional reversed-phase HPLC and positive HPLC are not able to maintain the conformation of VLPs when detecting；2, ferment
The Dissolve things insides such as a large amount of albumen, nucleic acid and polysaccharide are released after mother cell is broken, some of substances can interfere PCV2VLPs to examine
Survey signal, in some instances it may even be possible to interact with PCV2VLPs, influence detection effect；3, since PCV2VLPs is purified and difficult quantitation,
At present with the standard items without commercialization of production standard curve.
Summary of the invention
To solve the above problems, providing a kind of easy to operate, high sensitivity, repeatability and the high PCV2 disease of applicability
Malicious sample particle detection technique, the present invention are designed and are detected using the chromatography of molecular exclusion chromatography according to the structure feature of PCV2VLPs
Method optimizes in conjunction with sample treatment, the preparation of standard items and testing conditions, establishes efficient HPLC detection PCV2VLPs
Purity and quantitative approach, solve the key technical problem for being difficult to fast quantification of production virus-like particle in practice.This hair
It is bright specifically to adopt the technical scheme that
The present invention provides a kind of quantitative detecting methods of 2 porcine circovirus virus-like particle, for sample to be tested
In 2 porcine circovirus virus-like particle carry out quantitative detection, which comprises the steps of: step S1, dilution
Sample to be tested is simultaneously filtered clarification or centrifugal clarification；Step S2 takes protein content determined porcine circovirus 2 type disease
Malicious sample particulate level product are serially diluted, and carry out HPLC detection to the serial standards after dilution, and foundation is with concentration
Abscissa, the regression equation that peak height is ordinate；Step S3 detects the sample to be tested progress HPLC after dilution and is corresponded to
Sample peak height；The disease of the porcine circovirus 2 type in sample to be tested is calculated according to regression equation and sample peak height in step S4
Malicious sample granule density, wherein chromatographic column used by the HPLC in step S2 and step S3 is gel chromatographic columns, the inspection of use
Survey device is UV detector, Detection wavelength 280nm.
The quantitative detecting method of above-mentioned 2 porcine circovirus virus-like particle provided by the invention, can also have in this way
Technical characteristic, wherein gel chromatographic columns be Agilent Bio SEC-5 or TSKgel G4000SWXL.
The quantitative detecting method of above-mentioned 2 porcine circovirus virus-like particle provided by the invention, can also have in this way
Technical characteristic, wherein in step S1, the dilution of sample to be tested is carried out using phosphate buffer, is contained in the phosphate buffer
150mM NaCl、2.7mM KCl、1.5mM KH2PO4And 8mM K2HPO4。
The quantitative detecting method of above-mentioned 2 porcine circovirus virus-like particle provided by the invention, can also have in this way
Technical characteristic, wherein sample to be tested be selected from the fermentation liquid of recombination engineering, cell culture fluid, isolate and purify liquid or antigen at
Any one in product.
The quantitative detecting method of above-mentioned 2 porcine circovirus virus-like particle provided by the invention, can also have in this way
Technical characteristic, wherein sample to be tested using the X-100 containing 1%Triton phosphate buffer carry out 20 times dilution.
The quantitative detecting method of above-mentioned 2 porcine circovirus virus-like particle provided by the invention, can also have in this way
Technical characteristic, wherein virus-like particle HPLC retention time be 15.5min.
The present invention also provides the quantitative detecting method of another 2 porcine circovirus virus-like particle, feature exists
In including the following steps: step S1, dilution sample to be tested is simultaneously filtered clarification or centrifugal clarification；Step S2, takes albumen to contain
Amount be serially diluted by determined 2 porcine circovirus virus-like particle standard items, and to the serial standards after dilution
HPLC detection is carried out, establishing by abscissa, peak area of concentration is the regression equation of ordinate；Step S3, after dilution
Sample to be tested carries out HPLC and detects to obtain corresponding sample peak area；Step S4, according to regression equation and sample calculated by peak area
Obtain the 2 porcine circovirus virus-like particle concentration in sample to be tested, wherein the HPLC in step S2 and step S3 is adopted
Chromatographic column is gel chromatographic columns, and the detector used is UV detector, Detection wavelength 280nm.
Invention action and effect
The quantitative detecting method of the 2 porcine circovirus virus-like particle provided according to the present invention is based on due to using
Virus-like particle is detected in the HPLC of gel chromatographic columns, therefore virus-like particle and impurity can be efficiently separated,
Achieve the purpose that detect virus-like particle.Further, can also be made using gel chromatographic columns of the invention and HPLC condition non-
The virus-like particle of polymeric form and the virus-like particle of polymeric form are separated, so as to distinguish virus-like
The different polymerized forms of grain are more suitable for product testing and the quality control of actual production process.
Detailed description of the invention
Fig. 1 is the HPLC testing result of the PCV2 virus-like particle standard items of the embodiment of the present invention；
Fig. 2 be the embodiment of the present invention in PCV2 virus-like particle standard items HPLC characteristic peak SDS-PAGE electrophoresis (A) and
Immunoblotting Western blot (B) testing result；
Fig. 3 is PCV2 virus-like particle standard items HPLC retention time 13.7min and 15.5min in the embodiment of the present invention
The electron microscope observation result of characteristic peak；
Fig. 4 is that the HPLC method of the embodiment of the present invention quantifies the standard curve of PCV2 virus-like particle；
Fig. 5 is that the yeast of the embodiment of the present invention breaks the HPLC testing result mixed after PCV2-VLPs standard items in bacteria liquid sample.
Specific embodiment
The quantitative detection side of 2 porcine circovirus virus-like particle (hereinafter referred to as virus-like particle) of the present invention
Method mainly comprises the steps that
Step S1 dilutes sample to be tested and is filtered clarification or centrifugal clarification；
Step S2, taking protein content, determined 2 porcine circovirus virus-like particle standard items progress series is dilute
It releases, and HPLC detection is carried out to the serial standards after dilution, establishing by abscissa, peak height of concentration is the recurrence of ordinate
Equation；
Step S3 carries out HPLC to the sample to be tested after dilution and detects to obtain corresponding sample peak height；
The porcine circovirus 2 type virus-like in sample to be tested is calculated according to regression equation and sample peak height by step S4
Granule density.
Illustrate a specific embodiment of the invention below in conjunction with attached drawing and embodiment.In following each embodiments, sample
Or standard items dilute in used phosphate buffer and contain 150mM NaCl, 2.7mM KCl, 1.5mM KH2PO4And 8mM
K2HPO4, other reagents obtain from general commercial sources unless otherwise specified, and the experiment condition being not specified is referring to conventional real
The condition testing condition or suggesting in accordance with supplier.
<embodiment>
1, instrument and reagent
Angilent high-efficient phase chromatogram instrument Infinity 1260 is equipped with UV detector.
The preparation of HPLC detection mobile phase: 13.4g sodium phosphate dibasic heptahydrate, 6.9g sodium dihydrogen phosphate one is taken to be hydrated
Object and 14.2g anhydrous sodium sulfate are dissolved with 800mL Milli-Q water, are settled to 1000mL, 0.22 μm of membrane filtration.
2, prepared by PCV2 virus-like particle standard items
Currently, there is no commercialized PCV2 virus-like particle standard items both at home and abroad.Therefore, the present invention is to contain PCV2
The VLPs sample of virus-like particle has been carried out pure with ion-exchange chromatography and Sephacryl S-500HR molecular sieve gel chromatography
Change, using obtained high-purity virus-like particle as standard items.
Fig. 1 is the HPLC testing result of the PCV2 virus-like particle standard items of the embodiment of the present invention.
As shown in Figure 1, being detected by HPLC, virus-like particle purity > 98% in standard items.In addition, HPLC detection is in
Existing two characteristic peaks, wherein there is an acromion, retention time 13.7min before the peak of retention time 15.5min.
Fig. 2 be the embodiment of the present invention in PCV2 virus-like particle standard items HPLC characteristic peak SDS-PAGE electrophoresis (A) and
Immunoblotting Western blot (B) testing result, in which: swimming lane 1 is the retention time 13.7min characteristic peak collected；Swimming lane
2 be the retention time 15.5min characteristic peak collected.
Fig. 3 is PCV2 virus-like particle standard items HPLC retention time 13.7min and 15.5min in the embodiment of the present invention
The electron microscope observation result of characteristic peak.
Two characteristic peaks presented in HPLC detection are collected respectively, with SDS-PAGE electrophoresis and immunoblotting Western
Blot (B) is detected, as a result as shown in Fig. 2, the component of two characteristic peaks of explanation is all Cap protein.
It is further observed with electron microscope, as a result as shown in figure 3, illustrating the two characteristic peaks all is Cap egg
The virus-like particle that white assembling is formed, wherein 13.7min characteristic peak is virus-like particle polymeric form.
3, the column of chromatographic column imitates detection
The column effect detection for first carrying out chromatographic column is needed before carrying out HPLC detection, detailed process is as follows:
Chromatography Infinity 1260 is opened, is rinsed after 20min with 0.5mL/min Milli-Q water and changes chromatography
Column mobile phase water rinses 20min；Then guard column and gel chromatographic columns are installed, 24 DEG C of column temperature, UV detector is opened, to base
Line steadily carries out zero-in afterwards.It is that standard items have carried out column effect detection with 0.01g/L p-aminobenzoic acid, testing result is such as
Under:
Retention time is 27.946min, peak height 41.3mAU, half-peak breadth 0.425min.The calculating of theoretical cam curve is public
Formula are as follows: N=5.54* (Ve/W1/2) 2, N is theoretical cam curve, and Ve is retention time, and W1/2 is half-peak breadth, therefore chromatographic column
The theoretical cam curve of TSKgel G4000SWXL is 23953, reaches factory calibration requirement >=16000, can be applied to subsequent disease
Malicious sample particle HPLC detection.
4, the detection of PCV2 virus-like particle standard items and Specification Curve of Increasing
The PCV2 virus-like particle standard items being prepared are subjected to protein quantification with BCA method, then use phosphoric acid buffer
Liquid be diluted to concentration be 4.1 μ g/mL, 8.2 μ g/mL, 16.3 μ g/mL, 32.6 μ g/mL, 43.5 μ g/mL, 65.2 μ g/mL,
The standard solution of 104.4 μ g/mL and 130.5 μ g/mL etc..100 μ L are taken to be detected, mobile phase NaH containing 50mM2PO4、
50mM Na2HPO4、100mM Na2SO4, pH 6.75, flow velocity 0.6ml/min, Detection wavelength 280nm.Recording retention time is
15.5min feature peak height, every group is repeated 3 times, and the results are shown in Table 1.
Table 1: various concentration PCV2 virus-like particle standard items testing result
The peak value that this is put due to 4.1 μ g/mL is too low, poor with other test point linear relationships, therefore marks drawing
It is removed when directrix curve.
Fig. 4 is that the HPLC method of the embodiment of the present invention quantifies the standard curve of PCV2 virus-like particle.
Make linear regression with the concentration of PCV2VLPs and retention time 15.5min feature peak height, as a result as shown in figure 4, table
It is bright when HPLC detects PCV2VLPs, the linear relationship of concentration and feature peak height is that (y is y=2.7841x+2.9523
PCV2VLPs mass concentration, unit μ g/mL；X is retention time 15.5min peak value, unit mAU)；R2 value is 0.9988,
The linear relationship of the characteristic peak peak height and concentration that illustrate PCV2VLPs is higher, it was demonstrated that the detection method of the invention can be used for
The quantitative detection of PCV2VLPs.
5, sample detection precision and the rate of recovery
PCV2 virus-like particle standard items are added in yeast cells liquid, concentration is 65.2 μ g/mL after addition, takes 100
μ L records the peak value of detection retention time 15.5min every time with HPLC Parallel testing 6 times, calculates the rate of recovery and precision, knot
Fruit is as shown in table 2 below.
Table 2: the precision and the rate of recovery of detection
6, Specimen Determination
It is quantified with the PCV2 virus-like particle amount of antigen of the strain fermentation expression recombinantly expressed to yeast, sample is used
The phosphate buffer of the X-100 containing 1%Triton takes 100 μ L to detect after diluting 20 times.
Fig. 5 is that the HPLC that the yeast of the embodiment of the present invention is broken after mixing PCV2-VLPs standard items in bacteria liquid sample detects knot
Fruit.
As shown in figure 5, having PCV2 virus-like particle at retention time 15.5min in the testing result map of above-mentioned sample
Characteristic peak.Detection 3 times is repeated, peak value is followed successively by 26.7mAU, 27.1mAU, 26.7mAU, can count by standard curve
The concentration for calculating the sample of detection is 77.66 μ g/mL, to show that the PCV2 virus-like particle antigen in fermented liquid supernatant is dense
Degree is 1553 μ g/mL.
Embodiment action and effect
According to the quantitative detecting method of 2 porcine circovirus virus-like particle provided in this embodiment, due to using base
Virus-like particle is detected in the HPLC of gel chromatographic columns, therefore virus-like particle and miscellaneous can be efficiently separated
Matter achievees the purpose that detect virus-like particle.The rate of recovery of the detection method of the present embodiment is 104%, and relative standard deviation is
1.48%, therefore the quantitative requirement of detection method can be reached, it can be used as quantifying for 2 porcine circovirus virus-like particle
Detection method application.
Further, the disease of non-polymeric form can also be made using the gel chromatographic columns of the present embodiment and HPLC condition
The virus-like particle of malicious sample particle and polymeric form is separated, so as to distinguish the different polymerization shapes of virus-like particle
Formula is more suitable for product testing and the quality control of actual production process.In addition, HPLC, which has, is easy procedure, easy to operate
The advantages that, but also the quantitative detecting method of the present embodiment is very suitable for intermediate product or product in commercial process
Detection (such as recombination engineering fermentation liquid, cell culture fluid, isolate and purify liquid or antigen finished product etc.).
The preferred embodiment of the present invention has been described in detail above.It should be appreciated that the ordinary skill of this field is without wound
The property made labour, which according to the present invention can conceive, makes many modifications and variations.Therefore, all technician in the art
Pass through the available skill of logical analysis, reasoning, or a limited experiment on the basis of existing technology under this invention's idea
Art scheme, all should be within the scope of protection determined by the claims.
Claims (7)
1. a kind of quantitative detecting method of 2 porcine circovirus virus-like particle, for the 2 porcine circovirus in sample to be tested
Virus-like particle carries out quantitative detection, which comprises the steps of:
Step S1 dilutes the sample to be tested and is filtered clarification or centrifugal clarification；
Step S2, taking protein content, determined 2 porcine circovirus virus-like particle standard items are serially diluted, and right
Serial standards after dilution carry out HPLC detection, and establishing by abscissa, peak height of concentration is the regression equation of ordinate；
Step S3 carries out HPLC to the sample to be tested after dilution and detects to obtain corresponding sample peak height；
The 2 porcine circovirus in the sample to be tested is calculated according to the regression equation and the sample peak height in step S4
Virus-like particle concentration,
Wherein, chromatographic column used by the HPLC in step S2 and step S3 is gel chromatographic columns, and the detector used is ultraviolet
Detector, Detection wavelength 280nm.
2. the quantitative detecting method of 2 porcine circovirus virus-like particle according to claim 1, it is characterised in that:
Wherein, the gel chromatographic columns are Agilent Bio SEC-5 or TSKgel G4000SWXL.
3. the quantitative detecting method of 2 porcine circovirus virus-like particle according to claim 1, it is characterised in that:
Wherein, in step S1, the sample to be tested is dilute using 20 times of the phosphate buffer progress of the X-100 containing 1%Triton
It releases.
4. the quantitative detecting method of 2 porcine circovirus virus-like particle according to claim 1, it is characterised in that:
Wherein, in step S1, the dilution of the sample to be tested is carried out using phosphate buffer, is contained in the phosphate buffer
150mM NaCl、2.7mM KCl、1.5mM KH2PO4And 8mM K2HPO4。
5. the quantitative detecting method of 2 porcine circovirus virus-like particle according to claim 1, it is characterised in that:
Wherein, fermentation liquid of the sample to be tested selected from recombination engineering, cell culture fluid, isolate and purify in liquid or antigen finished product
Any one.
6. the quantitative detecting method of 2 porcine circovirus virus-like particle according to claim 1, it is characterised in that:
Wherein, the virus-like particle is 15.5min in the retention time of HPLC.
7. a kind of quantitative detecting method of 2 porcine circovirus virus-like particle, for the 2 porcine circovirus in sample to be tested
Virus-like particle carries out quantitative detection, which comprises the steps of:
Step S1 dilutes the sample to be tested and is filtered clarification or centrifugal clarification；
Step S2, taking protein content, determined 2 porcine circovirus virus-like particle standard items are serially diluted, and right
Serial standards after dilution carry out HPLC detection, and establishing by abscissa, peak area of concentration is the regression equation of ordinate；
Step S3 carries out HPLC to the sample to be tested after dilution and detects to obtain corresponding sample peak area；
Step S4 obtains the pig circular ring virus in the sample to be tested according to the regression equation and the sample calculated by peak area
2 virus-like particle concentration,
Wherein, chromatographic column used by the HPLC in step S2 and step S3 is gel chromatographic columns, and the detector used is ultraviolet
Detector, Detection wavelength 280nm.
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