CN111621480A - Purification and detection method of exosome generated by Newcastle disease virus infected HeLa cell - Google Patents

Purification and detection method of exosome generated by Newcastle disease virus infected HeLa cell Download PDF

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CN111621480A
CN111621480A CN202010409254.4A CN202010409254A CN111621480A CN 111621480 A CN111621480 A CN 111621480A CN 202010409254 A CN202010409254 A CN 202010409254A CN 111621480 A CN111621480 A CN 111621480A
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exosome
purification
ndv
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cell
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谭磊
丁铲
仇旭升
周昌娈
孙英杰
廖瑛
孟春春
宋翠萍
刘炜玮
于圣青
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Shanghai Veteromaru Research Institute Caas China Animal Health And Epidemiology Center Shanghan Branch Center
Shanghai Veterinary Research Institute CAAS
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Abstract

The invention belongs to the field of biological detection methods, and relates to a purification and detection method of exosome (exosome) generated after Newcastle Disease Virus (NDV) infects a cervical cancer cell line HeLa cell. The exosome purification method of the invention sequentially comprises the following steps: (1) centrifuging the cell supernatant containing the exosome to be detected at 10000-12000 g for 20-35 min; (2) the supernatant was centrifuged at 135000-145000 g for 90-100 min. The detection method comprises the following steps: determining an exosome morphology by a transmission electron microscope; determination of exosome surface marker protein CD 63; exosome size, concentration analysis. The invention can be used for purifying and detecting exomes generated after the NDV infects the HeLa cells, and lays a foundation for researching the effect of the exomes in virus infection.

Description

Purification and detection method of exosome generated by Newcastle disease virus infected HeLa cell
Technical Field
The invention belongs to the field of biological detection methods, and particularly relates to an exosome (exosome) purification and detection method after NDV (Newcastle disease virus, NDV) infects HeLa cells of a cervical cancer cell line.
Background
Newcastle Disease Virus (NDV) is a ssRNA virus, enveloped. The virus particles are polymorphic, and have circular, elliptical, and long rod shapes. The diameter of the mature virus particle is 100-400 nm. The envelope is a bilayer membrane derived from the binding of lipids of the outer membrane of the host cell to viral glycoproteins. The surface of the envelope is provided with spikes with the length of 12-15 nm, and the spikes are provided with hemagglutinin, neuraminidase and hemolysin. The virus has a core of ssRNA molecules with attached protein capsomeres, wound into a helically symmetric nucleocapsid with a diameter of about 18 nm. The mature virus is released extracellularly in a budding manner.
NDV has strong resistance to external environment, and can be inactivated after being acted at 55 deg.C for 45min and under direct sunlight for 30 min. The infectivity of the virus is not affected when the virus is stored at 4 ℃ for several weeks, at-20 ℃ for several months or at-70 ℃ for several years. Viruses can still be isolated in chicken houses, egg nests, egg shells and feathers within 8 weeks after the outbreak of newcastle disease.
Humans can be infected with NDV, with conjunctivitis symptoms most commonly occurring within 24 hours post-infection. In the case of the first reported case in 1942, the patient had swollen lymph nodes in front of and around the ear and had soft hand. At the initial 48 hours of infection, the patient is generally suffering from headache, discomfort and mild chills, which may last for 24 hours. Topical treatment was used to relieve conjunctivitis symptoms and restore vision within 1 week. The virus was still isolated from tears of serologically positive infected subjects 2 weeks after infection. The symptoms reported later in the case of newcastle disease are similar and often show ocular symptoms, sometimes changes in one eye, occasionally both eyes, accompanied by eyelid edema, conjunctival congestion, lacrimation, pain, and the like. Fever, tremor, headache, pharyngitis, photophobia and the like rarely occur.
Exosomes are a class of vesicles secreted by cells with diameters of about 30-120nm, and present a typical "saucer" structure under an electron microscope. In 1983, Harding et al first discovered such vesicles in sheep reticulocyte culture supernatant. In 1986, John Stone et al named these vesicles "exosomes" and the chinese name was "exosomes" as it appeared to be capable of "trans-endocytosis". In the first few years of exosome discovery, it has been considered a "garbage truck" for cells to transport waste proteins. It is well known that major histocompatibility complex (MHC II) molecules are either essential for adaptive immunity or are essential. In 1996, Raposo et al discovered MHC II molecules in B-lymphocyte exosomes transformed by Epstein-Barr virus (EBV), thereby revealing the latent identity of another layer of exosomes, i.e., another way of intercellular communication, which affects multiple cellular processes due to the transfer of carried signal molecules to adjacent or distant cellular tissues.
Currently, exosome contents are mainly divided into three major classes: proteins, RNA and lipids. Several databases such as EVPedia, Vesicledia and ExoCarta have been extensively included with exosome-related data resources. 9769 proteins, 3408 mRNAs and 1116 lipids have been found in exosomes as counted by ExoCarta (http:// www.exocarta.org /) database listings. Some common marker proteins exist on the surface of exosomes secreted by different cells, such as heat shock protein 70 (HSP 70), heat shock protein 90 (HSP 90); the four transmembrane proteins CD9, CD63, CD81 and CD82, etc. Other host proteins captured by exosomes due to their involvement in cyst membrane formation may also be used as markers to identify exosomes, such as Apoptosis-related genes (ALGs) and Tumor susceptibility genes 101 (TSG 101). However, it has been found that viral infection may result in altered levels of these proteins in the exosomes. The difference and function of exosome contents from different cell sources mainly depend on cell types and physiological and pathological states thereof, which proves that exosome capture nucleic acid and protein are regulated behaviors, and the fact also provides important biological significance for the research of exosome.
Since existing separation techniques are based on the size, structure and capture of some membrane proteins of exosomes, it is difficult to completely distinguish them from other vesicle and macromolecular protein complexes. Therefore, the separation, purification, detection and identification of exosomes are important for studying biomacromolecules and cell states in physiological processes.
Disclosure of Invention
The invention aims to provide a method for purifying exosome after NDV infects Hela cells.
Another technical problem to be solved by the present invention is to provide a method for detecting exosomes produced after NDV infects Hela cells.
In one aspect, the invention provides a method of purifying an exosome, the method comprising:
the sample of the purification method is obtained by purifying a Hela cell culture infected with NDV; the purification sequentially comprises the following steps:
(1) centrifuging the cell supernatant containing the exosome to be detected at 10000-12000 g for 20-35 min;
(2) the supernatant was centrifuged at 135000-145000 g for 90-100 min.
The centrifugation method is completed by matching an ultracentrifuge tube/bottle and an ultracentrifuge.
In a preferred embodiment of the present invention, the centrifugation conditions in step (2) are a speed of 140000g and a centrifugation time of 90 minutes.
Preferably, the cell supernatant obtained in step (2) is centrifuged again at 135000g-145000g for 90-100 min.
In a preferred embodiment of the present invention, the cell supernatant after centrifugation in step (2) is transferred into an ultracentrifuge tube, 140000g is set as a centrifugation speed, after 90min of ultracentrifugation, PBS is added to discard the supernatant for heavy suspension precipitation, the cell supernatant is centrifuged again for 90min at 140000g, and 200 μ L PBS is added to discard the supernatant for heavy suspension precipitation, so as to obtain an exosome sample.
Preferably, the NDV-infected Hela cell culture is centrifuged at 1000g to 2000g for 3 to 10min and the cell supernatant is subjected to step (1).
The product obtained by the purification method can be detected by the following method and steps:
determining an exosome morphology by a transmission electron microscope;
determination of exosome surface marker protein CD 63;
and/or
exosome size, concentration analysis.
In a preferred embodiment of the invention, the exosomes are exosomes secreted by NDV after infection of Hela cells.
Preferably, the infected NDV is found to have some particles with a diameter of about 100nm, as observed by transmission electron microscopy and nanoparticle tracking analysis, in an exosome sample of uninfected HeLa cells, well within the exosome size range, slightly larger than the diameter of an exosome produced by HIV virus infected T cells (50-90 nm).
Determination of Exosome marker proteins may be accomplished by protein identification means conventional in the art, for example using Western blotting, ELISA, or HPLC methods.
Preferably, the exosome has a diameter of 50-200nm and is contained in an amount of 4 x 106-8*106Particles/ml. Particle and content determination of exosomes may be achieved by nanoparticle tracking analyzers.
In another aspect, the purification and detection methods of the invention can be used to identify exosomes produced by NDV-infected Hela cells.
For example, morphological features are identified by ultracentrifugation purification and transmission electron microscopy; nanoparticle Tracking Analysis (NTA) to detect particle size; exosomes were identified comprehensively by methods such as Western Blot analysis of exosome-tagged proteins.
The invention adopts an optimized ultracentrifugation method to extract exosome, non-exosome particles are firstly precipitated to the bottom of a tube through low centrifugal force, and exosome is obtained by utilizing higher centrifugal force after supernatant is extracted.
Currently, the more common methods for exosome purification are ultracentrifugation, polymer precipitation, size exclusion, immunoaffinity, membrane affinity, and the like. Polymer precipitation methods suffer from the disadvantage that the co-precipitated proteins contain co-exosome impurities and the final sample cannot be completely removed of the polymer material; size exclusion has the disadvantages of relatively cumbersome and time-consuming operation and inability to remove vesicle impurities of similar size to exosomes; the disadvantage of the immunoaffinity method is that the amount of purified exosome is relatively low, the cost is high, and the method is not suitable for large-scale popularization and use; the membrane affinity method has the disadvantages that it may cause the exosomes to agglomerate, entrap some small cell debris, and the elution process may cause the exosomes to be destroyed.
In contrast, advantages of the present invention include:
compared with the traditional method, the operation time is reduced by at least 4-6h, and the working efficiency is improved.
And secondly, compared with other exosome non-centrifugal method experiment cost, the experiment cost is reduced by about two thirds. The cost of a common exosome purification kit per time is about four hundred yuan RMB, while the cost of the purification once by the method of the invention is about one hundred yuan RMB
Therefore, the exosome purification method provided by the invention has the characteristics of high extraction speed and low cost.
The invention uses ultracentrifugation method to separate and purify HeLa cell exosome infected with NDV and not infected with NDV. And (3) observing the exome morphology under a transmission electron microscope, analyzing the size and concentration of the exome by a nanoparticle tracking analyzer, displaying surface marker protein by Western blot and the like for experimental examination and identification.
Detection methods reported by Exosome include an immunoblotting (Western Blot) method, a mass spectrometry method, a PCR analysis method, a flow cytometry analysis method, a transmission electron microscopy analysis method, a nanoparticle analysis tracking technology and the like.
The mass spectrometry has the defects of long sample preparation period and low analysis specificity; the PCR analysis has the defect that false positive is often generated to influence the next test; flow cytometry analysis suffers from the disadvantage that the detection sensitivity is difficult to meet for particles less than 200nm in diameter, and for exosome particles to be smaller than this diameter, thus limiting the application of this method.
The invention adopts three methods of immunoblotting (Western Blot), transmission electron microscopy analysis and nanoparticle analysis tracking technology to jointly identify exosome, and the exosome is comprehensively detected and identified from a membrane surface marker (CD63) of the exosome, a typical exosome 'tea-tray' physical form, an exosome particle size, an exosome concentration and the like, and extracts of which the detection methods all accord with the characteristics of the exosome can be determined as the exosome. The invention determines the real effectiveness of an exosome sample and lays a solid theoretical and material foundation for the research of exosome in NDV infection and transmission.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is an exosome morphology under a transmission electron microscope.
FIG. 2 is a graph showing the results of identifying the exosome surface marker protein CD 63.
FIG. 3 is a graph of particle size versus concentration for NTA measurements.
Detailed Description
The invention is further illustrated below with reference to specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
Example 1 viral infection and Collection of cell supernatants
HeLa cells were cultured in 75cm of DMEM medium containing 10% Fetal Bovine Serum (FBS)2And (3) when the cell coverage rate reaches 90%, washing the culture flask with PBS for three times, then changing into a DMEM medium without FBS for culturing for 24 hours, and collecting the supernatant to obtain a control group culture medium supernatant sample.
In the NDV Herts/33 strain infected group, when the HeLa cell coverage rate reaches 90%, TCID is calculated by using FBS-free DMEM medium50The NDV is inoculated with cells according to 1MOI, after 1h, the culture medium is discarded, the cells are washed for three times by PBS, the FBSDMEM-free culture medium is added again to remove extracellular virus particles of uninfected cells, and then the cells are continuously cultured for 24h to obtain a NDV infected group cell supernatant sample.
Example 2 ultracentrifugation extraction of exosomes
When the cell supernatants of the control group and the NDV infected group are collected to 200mL, exosome derived from HeLa cells is extracted by an ultracentrifugation method.
(1) The collected cell supernatant was centrifuged at 300g for 10min at 4 ℃ in a 50mL centrifuge tube.
(2) Discarding the precipitate, transferring the cell supernatant after centrifugation in (1) to a new 50mL centrifuge tube, and centrifuging for 10min at 2000 g.
(3) Discarding the pellet, transferring the cell supernatant after centrifugation in (2) to a 250mL Beckman high speed centrifugation tube, trimming, and high speed centrifugation at 10,000 g for 30 min.
(4) Abandoning the precipitate, transferring the cell supernatant obtained in the step (3) into a 38.5mL Beckman ultracentrifuge tube, accurately balancing, placing the tube into the ultracentrifuge tube, setting 140000g as the centrifugal speed, ultracentrifuging for 90min, discarding the supernatant as carefully as possible, adding PBS (phosphate buffer solution) for heavy suspension precipitation, combining the heavy suspensions of the same group of samples in a plurality of centrifuge tubes into a new 38.5mL Beckman ultracentrifuge tube, centrifuging for 90min at 140000g again, discarding the supernatant as far as possible, adding 200 mu L of PBS (phosphate buffer solution) for continuous blowing, completely suspending the precipitate, sucking the heavy suspensions into a brand new 1.5mL EP tube, and obtaining the HeLa cell exosome sample. The exosome samples were stored at-80 ℃.
In step (3), the centrifugation conditions were changed to 10000g × 35min, 10500g × 25 min, 11000g × 30min, 11500g × 20 min, 12000g × 35min, and the cell supernatants were all used for the next step without significant difference.
The centrifugation conditions after the supernatant from step (4) were changed to 135000g × 2 hours, 135000g × 1.5 hours, 140000g × 1.5 hours, 145000g × 2 hours, and the cell supernatants obtained were all used for the next step without significant difference.
Example 3 Transmission Electron microscopy of exosome morphology
Thawing the extracted HeLa cell exosome sample, rinsing with 0.1M PBS for 2-3min, taking 20 mu Lexosome suspension, dripping the suspension into a carrying net with the diameter of 2nm, standing for 1-2min, sucking excess liquid on the carrying net by using filter paper, adding 2% Phosphotungstic acid (PTA) staining solution for 1min, baking by using an incandescent lamp, airing, and observing the form of the HeLa cell exosome by using a projection electron microscope (TecnaG 2 Spirottin, FEI, Holland).
Example 4Western blot detection of expression of exosome surface protein CD63
mu.L of HeLa cell exosome sample was taken, and 5. mu.L of SDS-PAGE protein loading buffer (2X) was added to lyse the protein, which was then incubated in a water bath at 100 ℃ for 10 min. After 4 ℃ centrifugation at 12000 rpm for 2min, 10. mu.L of WB sample was prepared. And (3) loading the sample, adjusting the constant voltage of the sample to 80V by an electrophoresis apparatus for 90min, adjusting the voltage to 120V after the separation of each strip of the marker from the separation gel is obvious, and continuing electrophoresis. And (3) taking out the protein gel after the bromophenol blue migrates to the bottom of the separation gel, arranging a sandwich layer according to the sequence of 'positive membrane gel and negative membrane gel', changing the constant pressure into the constant current of 250mA, and transferring for 90 min. After the transfer printing is finished, sealing a Nitrocellulose membrane (NC) with prepared 5% skim milk at room temperature for 2h, discarding the skim milk, adding a proper amount of TBST buffer solution until the NC membrane is sufficiently covered, placing on a shaking bed for 5min, discarding the TBST buffer solution, and repeating the step twice. Mixing the raw materials in a ratio of 1: the CD63 protein monoclonal antibody was diluted at a rate of 3000 and incubated overnight at 4 ℃. The following day, the NC membrane was washed with TBST buffer for 10min each time to remove excess primary antibody. Mixing the raw materials in a ratio of 1: diluting the secondary antibody at 8000, incubating for 1h at room temperature, taking out an NC membrane, throwing off redundant TBST buffer solution, adding an appropriate amount of A, B solution in an ECL chemiluminescence kit in an equal volume, and detecting signals on a full-automatic chemiluminescence image analysis system.
Example 5 nanoparticle tracking Analyzer for detection of HeLa cell exosome particle size and concentration
After diluting 2mL of HeLa cell exosome sample with 1 × PBS, it was added to a new reaction chamber, and 11 different sites in the whole reaction chamber were examined with nanoparticle tracking analyzer zetaview PMX 110(Particle metric, germany), and abnormal sites therein were analyzed and removed, whereas the average, median and concentration of the measured Particle sizes were calculated using optimized position data. The optimized measurement conditions are as follows: the temperature was set to 25 deg.C, the sensitivity was set to 70 deg.C, the shutter (shutter) speed was also set to 70 deg.C, and the conductivity was set to 15000. mu.s/cm.
Example 6 Electron microscopy of exosome morphology derived from HeLa cells
Due to the fine structure of exosome, it can only be observed by electron microscopy. Thus, we isolated exosome vesicles from infected NDV and uninfected virus HeLa cells, and observed by transmission electron microscopy that some particles with a diameter of about 100nm were found in the exosome samples of infected NDV and uninfected HeLa cells, which were well within the exosome size range, and these particles had a two-layer outer membrane structure with an obvious crescent morphology, which is highly consistent with the classical "teacup holder" morphology of exosomes (as shown in fig. 1). Furthermore, the exosome particles in these two states are not significantly different in morphology, but upon visual inspection it was found that NDV infected HeLa cells may secrete a greater amount of exosomes than uninfected HeLa cells.
Example 7WB detection of HeLa cell-derived exosome surface marker protein CD63
exosome contents are extremely abundant and from different sources, it is possible that exosome contents secreted by the same type of cell under different conditions vary. Fortunately, different exosomes all have a common set of exosome marker proteins on their membranes, with the most representative marker protein being CD63, so the publication selects CD63 to identify whether a sample is an exosome.
Isolated exosome samples extracted from HeLa cells infected with NDV and not infected with virus were found to express CD63 by WB analysis, identifying the samples as likely exosomes at the protein level (as shown in figure 2). In this process, it was found that the whole cell lysate was also partially expressed in CD63, and thus CD63 was considered to be highly expressed in exosomes.
Example 8 nanoparticle tracking Analyzer for detection of HeLa cell-derived exosome size and distribution
The average particle size in the HeLa cell exosome samples not infected with NDV was 127.9nm with particles of size 125.9nm accounting for 99.3% of the sample, and 129.2nm with particles of size 125.9nm also accounting for 99.3% of the sample, as measured with the nanoparticle tracking analyzer zetaview PMX 110 (as shown in figure 3). These results are consistent with the particle size observed by electron microscopy, which further demonstrates that the sample presented is an exosome. Meanwhile, the concentration of particles in the HeLa cell exosome samples infected with NDV and not infected with NDV is 1.0E respectively according to the detection of a nanoparticle tracking analyzer+11particles/mL and 2.5E+11particles/mL (as shown in FIG. 3), the latter concentration was about 4-fold higher than the former, probably due to the enhanced ability of HeLa cells to secrete exosomes upon NDV infection.
The invention utilizes an ultracentrifugation method to separate and purify HeLa cell exosome infected with NDV and not infected with NDV, and three types of tests are carried out: the detection method comprises experimental examination and identification of an exosome form (a transmission electron microscope), an exosome size concentration (a nanoparticle tracking analyzer), a surface marker protein (Western blot) and the like. The real effectiveness of the exosome sample is determined by combining the results, and a solid foundation is laid for researching the effect of the exosome in virus infection and transmission.
The above description is only for the specific embodiments of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present disclosure should be covered within the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.

Claims (10)

1. A method for purifying exosomes produced by Hela cells infected with newcastle disease;
the purification method sequentially comprises the following steps:
(1) centrifuging the cell supernatant containing the exosome to be detected at 10000-12000 g for 20-35 min;
(2) the supernatant was centrifuged at 135000-145000 g for 90-100 min.
2. The purification process according to claim 1, wherein the centrifugation conditions in step (2) are a speed of 140000g and a centrifugation time of 90 minutes.
3. The purification process according to claim 1, wherein the cell supernatant obtained in step (2) is centrifuged again at 135000g-145000g for 90-100 min.
4. The purification method according to claim 1, wherein the cell supernatant after centrifugation in step (2) is transferred into an ultracentrifuge tube and centrifuged at 140000g for 90 min;
then, abandoning the supernatant, adding PBS for resuspension and precipitation, and centrifuging again at 140000g for 90 min;
and finally, removing the supernatant, adding PBS, and carrying out heavy suspension precipitation to obtain an exosome sample.
5. The purification method according to claim 1, wherein step (1) is performed by centrifuging the NDV-infected Hela cell culture at a speed of 1000g to 2000g for 3 to 10min and taking the cell supernatant.
6. The purification method of claim 1, further comprising the step of detecting exosomes;
determining an exosome morphology by a transmission electron microscope;
exosome surface marker protein determination;
and/or
exosome size, concentration analysis.
7. A method of detecting an exosome, the method comprising:
determining an exosome morphology by a transmission electron microscope;
exosome surface marker protein determination;
and/or
Analyzing the size and concentration of exosome;
the exosome is secreted by NDV infected Hela cells.
8. The assay of claim 7 wherein the surface marker protein is CD63 protein.
9. The detection method of claim 7, wherein said exosomes have a diameter of 50-200 nm.
10. Use of the purification process according to any one of claims 1 to 6, for the isolation and purification of exosomes produced by NDV-infected Hela cells.
CN202010409254.4A 2020-05-14 2020-05-14 Purification and detection method of exosome generated by Newcastle disease virus infected HeLa cell Pending CN111621480A (en)

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