CN109187774B - Quantitative detection method of porcine circovirus type 2 virus-like particles - Google Patents
Quantitative detection method of porcine circovirus type 2 virus-like particles Download PDFInfo
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Abstract
本发明的目的在于提供一种操作简单、灵敏度高、重复性和适用性高的PCV2病毒样颗粒检测方法，该方法包括如下步骤：步骤S1，稀释待测样品并进行过滤澄清或离心澄清；步骤S2，取蛋白含量已被确定的猪圆环病毒2型病毒样颗粒标准品进行系列稀释，并对稀释后的系列标准品进行HPLC检测，建立以浓度为横坐标、峰高为纵坐标的回归方程；步骤S3，对稀释后的待测样品进行HPLC检测得到对应的样品峰高；步骤S4，根据回归方程及样品峰高计算得到待测样品中的猪圆环病毒2型病毒样颗粒浓度，其中，步骤S2及步骤S3中的HPLC所采用的色谱柱为凝胶色谱柱，采用的检测器为紫外检测器，其检测波长为280nm。
The object of the present invention is to provide a PCV2 virus-like particle detection method with simple operation, high sensitivity, high repeatability and applicability, the method includes the following steps: Step S1, dilute the sample to be tested and carry out filtration clarification or centrifugal clarification; step S2, take the porcine circovirus type 2 virus-like particle standard whose protein content has been determined for serial dilution, and perform HPLC detection on the diluted serial standard, and establish a regression with the concentration as the abscissa and the peak height as the ordinate Equation; Step S3, perform HPLC detection on the diluted sample to be tested to obtain the corresponding sample peak height; Step S4, calculate the concentration of porcine circovirus type 2 virus-like particles in the sample to be tested according to the regression equation and the sample peak height, Wherein, the chromatographic column used in the HPLC in step S2 and step S3 is a gel chromatographic column, and the detector used is an ultraviolet detector with a detection wavelength of 280 nm.
Description
Technical Field
The invention belongs to the technical field of biology, relates to a quantitative detection method of virus-like particles, and particularly relates to a quantitative detection method of porcine circovirus type 2 virus-like particles.
Background
Porcine Circovirus (PCV) belongs to the genus Circovirus (Circovirus) of the family Circovirus (Circovirus), is the smallest virus discovered so far, and has a particle diameter of only 17nm at the minimum, a DNA genome length of 1.7kb, and the virion is an icosahedral symmetric, non-enveloped, single-stranded circular DNA virus. Two serotypes of PCV are known, nonpathogenic PCV1 and pathogenic PCV 2. PCV2 is the main pathogen causing Postweaning Multisystemic Wasting Syndrome (PMWS), mixed infection with Classical Swine Fever Virus (CSFV) or Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) can enhance the severity of the disease, can cause 10-30% of different mortality, and the death rate of a more serious pig farm is up to 40% when the disease breaks out, thereby causing serious threat to the pig industry. Currently, PCV2 vaccine immunization is the main means for preventing and controlling PCV2, and the antigen content of PCV2 vaccine immunization is the most important factor influencing the immune effect of vaccine. When the vaccine is used in the field, if the amount of immune antigen which is not enough and actually required by production does not exist, the clinical morbidity can be controlled, but the infection of the pigs is often caused by insufficient immunity, which is shown as long-term toxic and sub-clinical infection, the growth performance of the pigs is seriously influenced, and the great economic loss is caused.
PCV2 Virus-Like Particles (VLPs) are 60-polymer formed by self-assembly of PCV2Cap protein which is expressed recombinantly, have the molecular weight of about 1800kDa, are similar to the structure of natural virion, and have better immunogenicity than the Cap protein. VLPs have a morphological structure that is highly similar to that of the native virus, are well immunogenic, and do not contain viral genes, and are therefore an ideal vaccine format.
VLPs structure evaluation and antigen quantitative detection of virus-like particle vaccines are important components for quality control of virus-like particle vaccines, but because virus-like particles are macromolecular substances assembled by multiple proteins, a common biological quantitative detection method is difficult to distinguish VLPs from protein monomers while quantifying, and cannot identify the stable state of the virus-like particles in a solution. At present, PCV2 antigen quantitative detection methods are mainly Enzyme-Linked Immunosorbent Assay Enzyme Linked Immunosorbent Assay (ELISA) method and SDS-PAGE method, and the two methods are not suitable for quantitative detection of PCV2 VLPs: the ELISA quantitative method is that known antigen or antibody is adsorbed on the surface of a solid phase carrier (polystyrene micro reaction plate), so that enzyme-labeled antigen-antibody reaction is carried out on the surface of the solid phase, and then the antigen or antibody is quantified, the method cannot distinguish PCV2VLPs and Cap protein monomers, and the difference of the antigen affinity of the capture antibody to different genotypes limits the application of the method; the SDS-PAGE method is a semi-quantitative method, and cannot accurately quantify the antigen concentration in a sample, and also cannot distinguish PCV2VLPs from Cap protein monomers.
High Performance Liquid Chromatography (HPLC) is an analysis method with simple operation, high efficiency and high sensitivity, the detection sensitivity can reach ng level, the analysis speed is high, the relative average deviation of the detection result after the same sample is repeatedly loaded is generally not more than 2 percent, and the HPLC is widely applied to various fields of biochemistry, food analysis, medicine research, environmental analysis, inorganic analysis and the like at present. Therefore, the quantitative analysis of PCV2VLPs by using the HPLC detection technology can shorten the detection time of the traditional ELISA method and the SDS-PAGE method, improve the detection sensitivity and stability, be favorable for perfecting the quality control of PCV2VLPs vaccine production and improve the quality of vaccine products.
However, the conventional HPLC detection method is difficult to apply to the quantitative detection of PCV2VLPs, and relevant reports are not searched in domestic and foreign literature and patent search, and the reasons include: 1. PCV2VLPs are macromolecular complexes assembled by 60-mer Cap proteins, and the conformation of the VLPs cannot be maintained by conventional reverse phase HPLC and normal phase HPLC during detection; 2. after the yeast cells are broken, a large amount of protein, nucleic acid, polysaccharide and other endolysates are released, wherein some substances can interfere with detection signals of PCV2VLPs and even possibly interact with PCV2VLPs to influence the detection effect; 3. due to the difficulty in purification and quantification of PCV2VLPs, no commercial standards were currently used to generate standard curves.
Disclosure of Invention
In order to solve the problems, the invention provides a PCV2 virus-like particle detection method which is simple to operate, high in sensitivity, repeatability and applicability, and the invention designs a chromatographic detection method adopting molecular exclusion chromatography according to the structural characteristics of PCV2VLPs, establishes a high-efficiency purity and quantitative method for detecting PCV2VLPs by HPLC by combining sample treatment, standard preparation and detection condition optimization, and solves the key technical problem that virus-like particles are difficult to rapidly quantify in the actual production. The invention specifically adopts the following technical scheme:
the invention provides a quantitative detection method of porcine circovirus type 2 virus-like particles, which is used for quantitatively detecting the porcine circovirus type 2 virus-like particles in a sample to be detected and is characterized by comprising the following steps: step S1, diluting the sample to be tested and carrying out filtration clarification or centrifugal clarification; step S2, taking a standard sample of the porcine circovirus type 2 virus-like particles with determined protein content for serial dilution, carrying out HPLC detection on the diluted serial standard sample, and establishing a regression equation with concentration as horizontal coordinates and peak height as vertical coordinates; step S3, carrying out HPLC detection on the diluted sample to be detected to obtain a corresponding sample peak height; and step S4, calculating to obtain the concentration of the porcine circovirus type 2 virus-like particles in the sample to be detected according to the regression equation and the peak height of the sample, wherein the chromatographic column adopted by the HPLC in the step S2 and the step S3 is a gel chromatographic column, the adopted detector is an ultraviolet detector, and the detection wavelength is 280 nm.
The quantitative detection method for the porcine circovirus type 2 virus-like particles provided by the invention can also have the technical characteristics that the gel chromatographic column is Agilent Bio SEC-5 or TSKgel G4000 SWXL.
The method for quantitatively detecting porcine circovirus type 2 virus-like particles provided by the present invention may further comprise the step of diluting the sample to be detected with a phosphate buffer solution containing 150mM NaCl, 2.7mM KCl, and 1.5mM KH in step S12PO4And 8mM K2HPO4。
The method for quantitatively detecting the porcine circovirus type 2 virus-like particles provided by the invention can also have the technical characteristics that a sample to be detected is selected from any one of fermentation liquor, cell culture solution, separation and purification solution or antigen finished product of recombinant engineering bacteria.
The method for quantitatively detecting the porcine circovirus type 2 virus-like particles provided by the invention can also have the technical characteristics that a sample to be detected is diluted by 20 times by adopting a phosphate buffer solution containing 1% Triton X-100.
The quantitative detection method for the porcine circovirus type 2 virus-like particles provided by the invention can also have the technical characteristics that the retention time of the virus-like particles in HPLC is 15.5 min.
The invention also provides another quantitative detection method of porcine circovirus type 2 virus-like particles, which is characterized by comprising the following steps: step S1, diluting the sample to be tested and carrying out filtration clarification or centrifugal clarification; step S2, taking a standard sample of porcine circovirus type 2 virus-like particles with determined protein content for serial dilution, carrying out HPLC detection on the diluted serial standard sample, and establishing a regression equation with concentration as a horizontal coordinate and peak area as a vertical coordinate; step S3, carrying out HPLC detection on the diluted sample to be detected to obtain a corresponding sample peak area; and step S4, calculating to obtain the concentration of the porcine circovirus type 2 virus-like particles in the sample to be detected according to the regression equation and the peak area of the sample, wherein the chromatographic column adopted by the HPLC in the step S2 and the step S3 is a gel chromatographic column, the adopted detector is an ultraviolet detector, and the detection wavelength is 280 nm.
Action and Effect of the invention
According to the quantitative detection method of the porcine circovirus type 2 virus-like particles, virus-like particles are detected by HPLC based on a gel chromatographic column, so that the virus-like particles and impurities can be effectively separated, and the purpose of detecting the virus-like particles is achieved. Furthermore, the gel chromatographic column and HPLC conditions can separate virus-like particles in non-multimerized form from virus-like particles in multimerized form, so that different polymeric forms of virus-like particles can be distinguished, and the gel chromatographic column and HPLC conditions are more suitable for product detection and quality control in actual production processes.
Drawings
FIG. 1 shows the HPLC detection result of PCV2 virus-like particle standard substance according to the embodiment of the present invention;
FIG. 2 shows the detection results of SDS-PAGE (A) and Western blot (B) of HPLC characteristic peaks of PCV2 virus-like particle standard in the example of the present invention;
FIG. 3 is an electron microscope observation of characteristic peaks of HPLC retention times of 13.7min and 15.5min of a standard sample of PCV2 virus-like particles in an example of the present invention;
FIG. 4 is a standard curve for quantifying PCV2 virus-like particles by an HPLC method according to an embodiment of the present invention;
FIG. 5 shows the HPLC detection results of the yeast lysate sample of the present invention after being mixed with the PCV2-VLPs standard.
Detailed Description
The quantitative detection method of the porcine circovirus type 2 virus-like particles (hereinafter referred to as virus-like particles) mainly comprises the following steps:
step S1, diluting the sample to be tested and carrying out filtration clarification or centrifugal clarification;
step S2, taking a standard sample of the porcine circovirus type 2 virus-like particles with determined protein content for serial dilution, carrying out HPLC detection on the diluted serial standard sample, and establishing a regression equation with concentration as horizontal coordinates and peak height as vertical coordinates;
step S3, carrying out HPLC detection on the diluted sample to be detected to obtain a corresponding sample peak height;
and step S4, calculating to obtain the concentration of the porcine circovirus type 2 virus-like particles in the sample to be detected according to the regression equation and the peak height of the sample.
The following description of the embodiments of the present invention will be made with reference to the accompanying drawings. In each of the following examples, the phosphate buffer used for diluting the sample or standard contained 150mM NaCl, 2.7mM KCl, and 1.5mM KH2PO4And 8mM K2HPO4Other reagents were obtained commercially in general, unless otherwise specified, and the conditions of the experiment were determined by reference to the conventional experimental conditions or according to the conditions recommended by the supplier.
< example >
1. Instruments and reagents
An angiolent high performance phase chromatograph Infinity 1260 equipped with an ultraviolet detector.
Preparation of HPLC detection mobile phase: 13.4g of disodium hydrogenphosphate heptahydrate, 6.9g of sodium dihydrogenphosphate monohydrate and 14.2g of anhydrous sodium sulfate were dissolved in 800mL of Milli-Q water to a volume of 1000mL, and the solution was filtered through a 0.22 μm filter.
2. Preparation of PCV2 virus-like particle standard
At present, no commercial PCV2 virus-like particle standard exists at home and abroad. Therefore, the present invention uses a VLPs sample containing PCV2 virus-like particles, which is purified by ion exchange chromatography and Sephacryl S-500HR molecular sieve gel chromatography, and the obtained high-purity virus-like particles are used as a standard.
FIG. 1 shows the HPLC detection result of PCV2 virus-like particle standard substance in the embodiment of the invention.
As shown in fig. 1, the virus-like particles in the standard were > 98% pure by HPLC. In addition, HPLC detection showed two characteristic peaks, with a shoulder preceding the peak with retention time of 15.5min, with a retention time of 13.7 min.
FIG. 2 shows the detection results of SDS-PAGE (A) and Western blot (B) of HPLC characteristic peaks of PCV2 virus-like particle standard in the example of the present invention, wherein: lane 1 is the collected retention time 13.7min signature peak; lane 2 is the characteristic peak collected with a retention time of 15.5 min.
FIG. 3 is the electron microscope observation result of characteristic peaks of HPLC retention time of 13.7min and 15.5min of PCV2 virus-like particle standard in the embodiment of the invention.
Two characteristic peaks shown in HPLC detection are respectively collected and detected by SDS-PAGE electrophoresis and immunoblotting Western blot (B), and the results are shown in FIG. 2, which indicates that the components of the two characteristic peaks are both Cap proteins.
Further observation by electron microscope showed that the two characteristic peaks are virus-like particles formed by assembling Cap proteins, and the characteristic peak at 13.7min is a multimerized form of virus-like particles, as shown in FIG. 3.
3. Column efficiency detection for chromatography columns
The method comprises the following specific steps of firstly carrying out column efficiency detection on a chromatographic column before carrying out HPLC detection:
turning on the chromatograph Infinity 1260, washing with 0.5mL/min Milli-Q water for 20min, and washing with mobile phase water of chromatographic column for 20 min; then a protective column and a gel chromatographic column are installed, the column temperature is 24 ℃, an ultraviolet detector is opened, and zero correction is carried out after the base line is stable. The column effect detection is carried out by using 0.01g/L para aminobenzoic acid as a standard substance, and the detection results are as follows:
the retention time is 27.946min, the peak height is 41.3mAU, and the half-peak width is 0.425 min. The theoretical plate number is calculated by the formula: n is 5.54 (Ve/W1/2)2, N is the theoretical plate number, Ve is retention time, W1/2 is half-peak width, so the theoretical plate number of the chromatographic column TSKgel G4000SWXL is 23953, the requirement of factory standard is not less than 16000, and the method can be applied to subsequent virus-like particle HPLC detection.
4. PCV2 virus-like particle standard substance detection and standard curve drawing
The prepared PCV2 virus-like particle standard is subjected to protein quantification by the BCA method, and then diluted with phosphate buffer to standard solutions with concentrations of 4.1. mu.g/mL, 8.2. mu.g/mL, 16.3. mu.g/mL, 32.6. mu.g/mL, 43.5. mu.g/mL, 65.2. mu.g/mL, 104.4. mu.g/mL, 130.5. mu.g/mL, and the like. Taking 100 mu L for detection, and the mobile phase contains 50mM NaH2PO4、50mM Na2HPO4、100mM Na2SO4The pH was 6.75, the flow rate was 0.6ml/min, and the detection wavelength was 280 nm. Retention time was recorded as 15.5min characteristic peak height and was repeated 3 times for each group, and the results are shown in table 1.
Table 1: detection results of different concentrations of PCV2 virus-like particle standard
Since the peak height at 4.1. mu.g/mL was too low to be linear with other detection points, it was removed when the calibration curve was plotted.
FIG. 4 is a standard curve for quantifying PCV2 virus-like particles by an HPLC method according to an embodiment of the present invention.
The results of linear regression of the concentration of PCV2VLPs and the characteristic peak height of retention time of 15.5min are shown in FIG. 4, and show that when PCV2VLPs are detected by HPLC, the linear relation between the concentration and the characteristic peak height is that y is 2.7841x +2.9523(y is the mass concentration of PCV2VLPs, unit mu g/mL; x is the peak height value of retention time of 15.5min, unit mAU); the R2 value is 0.9988, which shows that the linear relation between the characteristic peak height and the concentration of the PCV2VLPs is high, and the detection method provided by the invention is proved to be applicable to quantitative detection of the PCV2 VLPs.
5. Sample detection precision and recovery rate
PCV2 virus-like particle standard is added into yeast cell sap, the concentration after the addition is 65.2 mug/mL, 100 muL is taken to be detected for 6 times in parallel by HPLC, the peak height value of the retention time of each detection of 15.5min is recorded, and the recovery rate and the precision are calculated, and the results are shown in the following table 2.
Table 2: precision and recovery rate of detection
6. Sample measurement
The amount of PCV2 virus-like particle antigen expressed by fermentation of a strain expressed by recombinant yeast is quantified, and 100 mu L of sample is taken for detection after being diluted by 20 times by using a phosphate buffer solution containing 1% Triton X-100.
FIG. 5 shows the HPLC detection results of the yeast lysate sample of the present invention after being mixed with the PCV2-VLPs standard.
As shown in FIG. 5, in the detection result map of the sample, a characteristic peak of PCV2 virus-like particles exists at the retention time of 15.5 min. The detection is repeated for 3 times, the peak heights are 26.7mAU, 27.1mAU and 26.7mAU in sequence, the concentration of the detected sample can be calculated to be 77.66 mu g/mL through a standard curve, and therefore the antigen concentration of the PCV2 virus-like particles in the supernatant of the fermentation liquid is 1553 mu g/mL.
Examples effects and effects
According to the quantitative detection method for porcine circovirus type 2 virus-like particles provided by the embodiment, the virus-like particles are detected by HPLC based on a gel chromatographic column, so that the virus-like particles and impurities can be effectively separated, and the purpose of detecting the virus-like particles is achieved. The recovery rate of the detection method in the embodiment is 104%, and the relative standard deviation is 1.48%, so that the quantitative requirement of the detection method can be met, and the method can be applied as a quantitative detection method of the porcine circovirus type 2 virus-like particles.
Furthermore, the gel chromatographic column and the HPLC conditions of the embodiment can separate the virus-like particles in non-multimerized form from the virus-like particles in multimerized form, so that different polymerization forms of the virus-like particles can be distinguished, and the method is more suitable for product detection and quality control in the actual production process. In addition, HPLC has the advantages of easy process, simple operation and the like, and the quantitative detection method of the embodiment is also very suitable for detecting intermediate products or products in the industrial production process (such as fermentation liquid, cell culture liquid, separation and purification liquid or antigen finished products of recombinant engineering bacteria).
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
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