CN111254219B - Method for detecting virus packaging efficiency - Google Patents
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- 238000001514 detection method Methods 0.000 claims description 20
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- 238000001415 gene therapy Methods 0.000 claims description 9
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- 238000005516 engineering process Methods 0.000 description 8
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Abstract
The invention provides a method for detecting virus packaging efficiency, which is based on that surface charge changes after the virus successfully packages nucleic acid, and the number of virus particles for successfully packaging the nucleic acid is judged by detecting the relative condition of the surface charge of the virus, so that the virus particle packaging efficiency is calculated.
Description
Technical Field
The invention relates to a novel method for detecting virus packaging efficiency, in particular to a method for judging the packaging efficiency of virus packaging exogenous genes in the fields of transgenosis and gene therapy.
Background
Gene therapy (gene therapy) refers to the introduction of exogenous normal genes into target cells to correct or compensate for diseases caused by defective and abnormal genes for therapeutic purposes. Technical applications in terms of transgenesis are also included. That is, the exogenous gene is inserted into the appropriate recipient cell of the patient by gene transfer technology, so that the product produced by the exogenous gene can treat a disease. Compared with the traditional medical means, the gene therapy means based on the gene technology has more pertinence, can obtain more ideal therapeutic effect, can greatly relieve the pain of patients, and is widely seen in the industry. From 2012, 754 cases of gene therapy clinical trials were added worldwide. The most attractive of these is immunotherapy of tumors that utilize CAR-T cells to target tumor-associated cell surface antigens (Chimeric Antigen Receptor T-Cell Immunotherapy, chimeric antigen receptor T cell immunotherapy). CAR-T treatment was reviewed by the journal of Science in 2013 as the first of "ten major technological advances". The CAR-T technology is mainly used for treating blood tumor at the current stage, and mainly represents two products, namely Kymriah (Norhua) and Yescarta (Kite) approved by the FDA in 2017.
The flow of gene therapy is generally to insert a normal gene into the DNA of a viral vector by means of genetic engineering; (2) Packaging the recombined virus DNA in vitro to generate complete engineering virus with infection capability; (3) Injecting the recombined virus into the patient directly, infecting the pathological cells by the virus and bringing the normal genes into target cells to treat the disease; or (1) inserting a normal gene into the DNA of a viral vector; (2) Packaging the recombined virus DNA in vitro to generate complete engineering virus with infection capability; (3) Obtaining somatic cells of a patient, such as hematopoietic stem cells and the like, and culturing and amplifying in vitro; (4) Infecting the obtained patient cells with the recombinant virus, which introduces the normal gene into the target cells; (5) Carrying out in vitro culture amplification on recombinant cells carrying normal genes; (6) The recombinant cells carrying the normal genes are returned to the patient, so that the treatment of the diseases is realized.
The above methods all use vectors, and the main vectors used at present include eukaryotic vectors, viral vectors and the like, wherein the research on the viral vectors is more popular, and the viral vectors are adenovirus, lentivirus, retrovirus and the like. Lentiviral (Lentivirus) vectors are gene therapy vectors developed based on HIV-1 (human immunodeficiency virus type I) and have infectivity to both dividing cells and non-dividing cells, can be loaded with DNA fragments up to 5kb in capacity, and after entering host cells, the target genes are integrated into the genome through reverse transcription to form stable genetic material, so that the target genes can be stably expressed in the cells for a long period of time. Adenovirus (adenoviruses) particles are composed of 252 capsomers in icosahedral arrangement, without enveloped particles with a diameter of 70-90 nm. The diameter of each shell particle is 7-9 nm. Within the capsid is a linear double stranded DNA molecule of about 35000bp with inverted repeats of about 100bp at each end. The structural proteins of adenovirus are aggregated in the nucleus to form a viral capsid, the viral genome is packaged in, virus particles with infectious capability are formed, and finally, the host cells are cracked and released out, thus completing the life cycle of adenovirus. Retrovirus, spherical, enveloped, 80-120 nm, single-stranded positive-strand RNA virus containing reverse transcriptase. Lentiviruses are subfamilies in the family retrovirus, which invade host cells and then integrate into the chromosome of the host cells by reverse transcriptase with their own RNA as a template after DNA cyclization, and replicate in the host cells as proviruses.
When using viral packaging nucleic acids, not all viruses are properly packaged, often there are damaged, non-infectious viruses, which "null" viruses can affect subsequent applications, and therefore the determination of viral packaging efficiency is important. In the existing detection system, the traditional methods for detecting the packaging efficiency include a plaque detection method, a dilution fluorescence counting method, a quantitative PCR method, an ELISA method (for detecting the content of P24 protein) and the like, wherein the plaque detection method is a gold standard, but the methods are time-consuming and labor-consuming or have high cost, cannot reflect the situation in the packaging process of viruses in real time, and seriously influence the efficiency of the production and manufacturing process of the viruses. Thus, there is a need for a simple and accurate method for detecting viral packaging efficiency.
The ZETA potential (ZETA potential) is also known as the ZETA potential or electrokinetic potential (ZETA potential or ZETA potential). One of the main uses of Zeta potential is to detect the amount of charge on the surface of particles. For example, the surface of a viral particle is charged, which in a certain solution system will form a certain ZETA potential, which changes when the charge of the viral surface changes. The nucleic acid packaged by the virus is negatively charged, so that when the virus completely packages the nucleic acid, the surface charge is changed under the influence of the nucleic acid, and the ZETA potential is changed, so that the integrity of the virus package can be effectively distinguished by measuring the change of the ZETA potential on the surface of the virus particle.
The measurement of ZETA potential can be based on TRPS technology (Tunable resistive pulse sensing, adjustable resistance pulse sensing technology), and particles suspended in electrolyte replace the electrolyte with the same volume when the electrolyte passes through a small hole pipe, so that the resistance between the inner electrode and the outer electrode of the small hole pipe is instantaneously changed in a constant current designed circuit to generate potential pulses. The size and number of pulses is proportional to the size and number of particles. When the virus particles pass through the electrolyte system, as the virus surface is charged, when the intensity of the electric field is adjusted by changing the voltage, the velocity of the virus particles passing through the electric field changes, and when the change is compared with particles of known ZETA potential and size, the ZETA potential and size of the virus particles can be determined. The results of TRPS detection can be shown using a scatter plot with particle size (nm scale) on the abscissa and the ZETA potential on the ordinate. The size of the completely packaged viral particles is substantially the same as that of the viral particles of the unpackaged nucleic acid, corresponding to approximately the same region on the abscissa; while the difference in charge results in a difference in the ZETA potential of the two viral particles, corresponding to different regions on the ordinate. The sample volume required by the detection based on the TRPS technology is about 30ul, the detection time is 2min, and compared with the traditional virus packaging efficiency detection technology, the efficiency is greatly improved. Thus, TRPS technology is a useful method for performing viral nucleic acid packaging efficiency assays.
Disclosure of Invention
The invention provides a method for detecting virus packaging efficiency, which is based on that surface charge changes after the virus successfully packages nucleic acid, and the number of virus particles for successfully packaging the nucleic acid is judged by detecting the relative condition of the surface charge of the virus, so that the virus particle packaging efficiency is calculated.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
cell culture was performed by conventional methods, when cells grew to 70-80% saturation, 3 plasmids constituting lentiviral vectors were transfected, the complete culture solution was changed after 6-8 hours of transfection, after about 48-60 hours of further culture, virus culture supernatant was collected, and after centrifugation, virus pellet was resuspended in PBS filtered in advance. Detecting the virus heavy suspension by using a surface charge measuring instrument, analyzing the charges carried by the detected viruses, judging the number of virus particles for successfully packaging the nucleic acid according to the charge difference, and further calculating to obtain the virus particle packaging efficiency.
Example 1 Virus preparation
293T cells were cultured according to the conventional method, when the cells were grown to 70-80% saturation, 3 plasmids (containing eGFP gene) constituting lentiviral vectors were transfected, the complete culture solution was changed after 6-8 hours of transfection, and after about 48-60 hours of culture, the virus culture supernatant was collected, centrifuged, and virus pellet was resuspended in PBS filtered in advance.
EXAMPLE 2Real-time PCR detection of viral particle quantity
And (3) performing DNase treatment on 2 mu L of virus suspension, adding proteinase K and other treatments into the treated mixed solution, extracting with phenol and chloroform, precipitating with absolute ethyl alcohol, and dissolving with DEPC water. The real-time quantitative PCR is used for measuring the copy number of the virus LTR, and the software analyzes fluorescence detection data.
TABLE 1 PCR primers and probes
| Primer and probe | Sequence(s) |
| LTR-F | 5′-ACAGCCGCCTAGCATTTCAT-3′ |
| LTR-R | 5′-GAGAGCTCCCAGGCTCAGATC-3′ |
| LTR-Probe | 5′-ACATGGCCCGAGAGCTGCATCC-3′ |
TABLE 2 PCR reaction conditions
| Reaction system | |
| Primer and probe | 1μL |
| PCR Mix | 5μL |
| Sample of | 2μL |
| H2O | 2μL |
| Total reaction System | 10μL |
| Reaction conditions | |
| 95℃ | Denaturation for 5min |
| 95℃30s，59℃30s， | 40 cycles |
| 59℃ | Detection of fluorescence at 520nm |
The real-time quantitative PCR measurement result shows that the number of virus particles is 5.78X10 11 /ml。
EXAMPLE 3 Virus-infected cells
The 293T cells were infected with the virus at different doses, and 0.1. Mu.L of the virus suspension (lentiviral particle number: 5.78X10) was taken by 10-fold dilution 7 、5.78×10 6 ) By using
After dilution of the culture medium, 293T cells were infected (1.5X10) 6 /well), cells were cultured for 2d, and then digested with pancreatin to collect cell pellet.
EXAMPLE 4Real-time PCR detection of infected cell efficiency
The collected 293T cells were treated with proteinase K, extracted with phenol and chloroform, precipitated with absolute ethanol, and dissolved in DEPC water. The viral LTR copy number was determined using real-time quantitative PCR, the reaction conditions were the same as in example 2, and the software analyzed for fluorescence detection data.
After infection of cells according to one virus and integration of the foreign gene into the genome, each DNA single strand contains two complete LTRs, and therefore, one lentiviral particle = LTR copy number/4 in the genome of the cell being integrated. 5.78*10 7 、5.78*10 6 After virus particle infection, the copy number of exogenous gene integration in the cell is 1.15X10 respectively 6 、1.96×10 5 。
According to the formula: infection efficiency = number of effective infectious viruses/total number of viral particles of infected cells,
viral infection efficiency = (1.45×10) 6 /5.78×10 7 +2.16×10 5 /5.78×10 6 )/2×100％＝3.12％.。
Since only whole virus particles can be packed to infect cells and function, the packing efficiency of the present virus=virus infection efficiency=3.12%.
Example 5 surface Charge detection of viral particles and viral surface charges
The virus suspension prepared in example 1 was diluted 1000 times and then subjected to ZETA potential measurement using a qNano surface Charge measuring device manufactured by IZON Inc. of New Zealand. CPC200 was used as a standard particle with known diameter, concentration, and surface charge. The number of virus particles and the surface charge distribution were obtained by one detection (Table 3). The data of 500 virus particles, specifically 500 virus particle size distribution and virus surface charge, are counted in the test, see fig. 1 and 2. Wherein the size of the virus particles is distributed between 75-159nm, and more than 90% of the particles are distributed between 100-139nm, which accords with the size range of the slow virus particles.
TABLE 3 determination of viral particle data using a surface Charge meter
FIG. 1 viral particle size distribution
FIG. 2 surface Charge Condition of viral particles
Example 6 calculation of viral particle packaging efficiency
500 virus particles tested in example 5 had surface charges with Zeta potential from-89.51 to-13.83 mV, the number of bits being-33.77 mV, the Zeta potential of most of the virus particles being near the median (black circle), the surface charges of the virus particles being changed after successful packaging of nucleic acid by the virus, setting Zeta potential of 2 times the median as threshold, the Zeta potential of the virus surface reaching the threshold range, i.e. less than-67.54 mV, being considered successful packaging of nucleic acid by the virus, and the total 18 virus particles were counted as having charges between-67.54 to-89.51 (red circle), so that 18 virus particles were successfully packaged and the packaging efficiency was 18/500×100% = 3.60%.
Example 7 comparison of the merits of the two methods
The detection of virus packaging efficiency was carried out by using the method of real-time fluorescence quantitative PCR and the method of particle surface charge analyzer in the previous examples, respectively, and the values obtained by the detection of the two methods were 3.12% and 3.60%, respectively, which are substantially identical. The two methods are analyzed, the method of real-time fluorescence quantitative PCR not only needs to detect the collected virus particles, but also needs to collect the infected cells for detection, the whole detection period is longer than 1 week, and related reagents such as related probes, primers, cell culture and the like need to be purchased, and used instruments need a cell incubator and the like besides the real-time fluorescence quantitative PCR instrument, so that the whole process is long in time consumption and high in cost; the particle surface charge tester is used for detection, virus particles are only required to be diluted and put on the machine for detection, the time required for one-time detection is about 1 hour, the size of the virus particles, the virus concentration and the number of virus carried charges can be obtained at the same time, and the virus packaging efficiency can be directly calculated through the obtained values. In particular in the field of gene therapy, lentiviruses are used as gene carrying vectors, and detection and monitoring in the production process of the lentiviruses need a rapid and easy method, and obviously, the method of the patent is more suitable.
Claims (4)
1. A method for detecting virus packaging efficiency is characterized in that after the nucleic acid is successfully packaged by the virus, the nucleic acid is charged, so that the charge on the surface of the virus is changed, the number of virus particles for successfully packaging the nucleic acid is judged by detecting the relative condition of the charge on the surface of the virus, and then the virus particle packaging efficiency is calculated;
detecting surface charges of a unit number of viral particles by a charge detection instrument, wherein the surface charges are represented by a symbol Z, and the viral particles with Zi > =2zaverage are judged to be packaged with nucleic acid;
viral packaging efficiency= (Zi > = total number of viral particles of 2 Zaverage)/(total number of all viral particles detected this time) ×100%;
the virus is a lentivirus.
2. The method of claim 1, wherein the change in surface charge after successful packaging of the nucleic acid by the virus comprises both an increase in surface charge and a decrease in surface charge due to the difference in charge of the nucleic acid.
3. The method of claim 1, wherein the method is used to evaluate packaging nucleic acids and deoxyribonucleic acids using viruses.
4. The method according to claim 1, which is useful in the field of transgenesis, gene therapy.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002002738A2 (en) * | 2000-07-03 | 2002-01-10 | Gala Design, Inc. | Host cells containing multiple integrating vectors |
| CN1839201A (en) * | 2003-06-18 | 2006-09-27 | 吉恩勒克斯公司 | Modified recombinant vaccinia viruses and other microorganisms, uses thereof |
| CN104797717A (en) * | 2012-07-26 | 2015-07-22 | 韦克塔里斯公司 | Method for detecting or measuring the impact of a viral vector composition on eukaryotic cells and biomarkers used thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002002738A2 (en) * | 2000-07-03 | 2002-01-10 | Gala Design, Inc. | Host cells containing multiple integrating vectors |
| CN1839201A (en) * | 2003-06-18 | 2006-09-27 | 吉恩勒克斯公司 | Modified recombinant vaccinia viruses and other microorganisms, uses thereof |
| CN104797717A (en) * | 2012-07-26 | 2015-07-22 | 韦克塔里斯公司 | Method for detecting or measuring the impact of a viral vector composition on eukaryotic cells and biomarkers used thereof |
Non-Patent Citations (1)
| Title |
|---|
| Lu Yang et al..Quantification of Virus Particles Using Nanopore-Based Resistive-Pulse Sensing Techniques.Frontiers in Microbiology.2016,第7卷第1-7页. * |
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Denomination of invention: A method for detecting the efficiency of virus packaging Granted publication date: 20230804 Pledgee: The Bank of Shanghai branch Caohejing Limited by Share Ltd. Pledgor: SHANGHAI WAYEN BIOMEDICAL TECHNOLOGY CO.,LTD. Registration number: Y2024980018233 |