CN103529205B - Quantitative determination method for titer of recombinant insect baculovirus - Google Patents
Quantitative determination method for titer of recombinant insect baculovirus Download PDFInfo
- Publication number
- CN103529205B CN103529205B CN201310521058.6A CN201310521058A CN103529205B CN 103529205 B CN103529205 B CN 103529205B CN 201310521058 A CN201310521058 A CN 201310521058A CN 103529205 B CN103529205 B CN 103529205B
- Authority
- CN
- China
- Prior art keywords
- cell
- culture media
- nutrient culture
- titre
- insect
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
- G01N2333/43552—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects
Abstract
The invention provides a quantitative determination method for the titer of recombinant insect baculovirus. The method utilizes the fact that the recombinant insect baculovirus interferes in growth and duplication of host cells after the recombinant insect baculovirus infects insect cells, and then the method detects the number of the cells by using an acidic phosphatase method so as to realize quantitative determination of the titer of the recombinant insect baculovirus. The method has the advantages of high sensitivity, good accuracy, usage of cheap and easily available reagents, simple and convenient operation, easy mastery of technology, small consumption of time and higher efficiency.
Description
Technical field
The present invention relates to virus titer to measure, particularly, relate to a kind of method of quantitative measurement recombinant baculovirus titre.
Background technology
After finding the strong promoter characteristic of polyhedron gene (polh) of Rhabdoviridae nuclear polyhedrosis virus the beginning of the eighties; Smith and Summer [Smith GE, MD Summers and MJ Fraser.Production of human beta interferon in insect cells infected with a baculovirus expression vector.Mol Cell Biol.1983; 3 (12): 2156-2165] baculovirus expression system (Baculovirus Expression Vector System, BEVS) is established first.Baculovirus expression system has become one of large expression system in current genetic engineering field four, compared with Escherichia coli, yeast mammalian cell expression system, BEVS has special researching value in following four: (1), as the eukaryotic gene expression system of ultra high efficiency, produces useful destination protein; (2) as genetically engineered virus pesticide, control of insect efficiency is improved; (3) 26S Proteasome Structure and Function of Baculovirus Gene group is studied; (4) expression and regulation mechanism of eukaryotic gene is studied.Rod string design has become the very effective and widely used instrument [Li Weiguo of the various protokaryon albumen of research and eukaryotic protein, Wang Houwei, Mu Zhimei, Shi Lianhui. Progress And Perspecfives of Research On The Obtaining Technique of Insect Recombinant Baculovirus. Journal of Shandong agri.Univ's (natural science edition) .2003,34 (1): 134-138].
The method detecting virus titer has Endpoint Dilution Method and Plaque Technique Detected.Endpoint Dilution Method is the conventional method detecting virus titer, has easy, feature fast.It virus is carried out gradient series dilution postoperative infection cell, by detecting 50% tissue cytopathogenic dose (TCID
50) judge virus titer.Plaque technic is set up [Dulbecco R.-Production of plaques in monolayer tissues by using single particles of animal virus.Proc Nat Acad Sci1952 by Dulbecco the earliest; 38 (8): 747-752], Hink and Vial was applied to this technology the research work of baculoviral afterwards.Plaque Technique Detected is by after appropriate virus infected cell, and virus is in the time multiplexed cell system infected, propagation discharge free virus particles, and these free virus particles, owing to being subject to the restriction of agarose film solid media, can only infect contiguous cell.After several infectious cycle, these infected cells are all dead and not by neutral red staining, living cells around is then dyed to redness, so formed a water white transparency region at first by the cell peripheral of virus infections, i.e. and plaque.Virus titer can be judged by the quantity counting plaque.
For the insect baculovirus of wild type, macroscopic inclusion body can be formed due to it in infection cell and in a short time peripheral cell disintegration be formed obvious plaque, all can determine its titre with Endpoint Dilution Method and Plaque Technique Detected.But for recombinant insect virus, because it has lacked polyhedron gene, it can not form macroscopic inclusion body as the baculoviral of wild type in infection cell, therefore can not can only measure the titre of recombinant baculovirus with Plaque Technique Detected with Endpoint Dilution Method.
The most frequently used method detecting recombinant insect virus is Plaque Technique Detected, but due to Plaque Technique Detected complex operation, longer (because it has lacked polyhedron gene, it makes the time needed for infected cell generation disintegration also greatly extend) consuming time, and the more difficult grasp of technology, error are larger.External have two kinds of methods detecting recombinant insect virus at present: a kind of is determine recombinant baculovirus titre [Dee by the expression of the cell surface marker albumen gp64 of immunological method detection recombinate shape virus infection; K.U; Shuler, M.L.Optimization of an assay for baculovirus titer and design of regimens for synchronous infection of insect cells.Biotechnol.Prog.1997; 13 (1): 14-24].The method is easy fast (completing in 48h), but expensive; One is directly by jellyfish green fluorescent albumen (Green Flurescent Protein; GFP) recombinant baculovirus is built as reporter gene; recombinant baculovirus screening and titer determination is made all to become very simple [Takehiko Saito a; Takashi Dojima b; Masaru Toriyama a, Enoch Y.Park The effect of cell cycle on GFPuv gene expression in the baculovirus expression system.Journal of Biotechnology.2002; 93 (2): 121-129].But the method relates to be done from the construction work of upstream, and workload is huge.Still there is no method that is easy, quantitative measurement recombinant insect virus titre rapidly at present.
Therefore, need in this area a kind of low cost, easy, fast method carry out quantitative measurement recombinant insect virus titre, be beneficial to the extensive Study and appliance of the shaft-like expression system of insect.
Summary of the invention
The present invention relates to a kind of quantitative, easy and method fast, for detecting the titre of recombinant baculovirus, specifically, the method carrys out the method for quantitative measurement recombinant baculovirus titre by the quantity that Acid Phosphatase Method detects cell.
Therefore, the object of this invention is to provide the method for quantitative measurement recombinant baculovirus titre, can disturb the growth of host cell after the method utilizes recombinant baculovirus infected insect cell and copy, the quantity being detected cell by Acid Phosphatase Method is quantitatively realized.Acid Phosphatase Method measuring principle is that the acid phosphatase in living cells accurately can reflect cell number., make membranolysis discharge acid phosphatase with scaling agent under certain condition, then after acting on a period of time with substrate pNPP (p-nitrophenyl phosphate), its catalysis can be generated yellow paranitrophenol.This is that colour reaction can detect by microplate reader, and the absorbance surveyed can represent activity of acid phosphatase, also indirectly reflects viable count.
The invention provides a kind of method by Acid Phosphatase Method quantitative measurement recombinant baculovirus titre.Comprise the following steps:
A (), by resuspended for the insect cell of exponential phase, after carrying out suitably dilution with nutrient culture media, inoculation insect cell is in 96 porocyte culture plates;
B virus titer standard items and virus liquid to be checked are carried out serial dilution by (), be inoculated in insect cell and infect;
C () adds substrate solution, after colour developing certain hour, microplate reader detects absorbance value;
D () is according to the absorbance value drawing standard curve of each dilution series virus titer standard items;
E () adopts calibration curve method, calculate the titre of virus liquid to be checked.
In embodiment of the present invention, wherein said recombinant baculovirus refers to the insect baculovirus having lacked polyhedron gene.
In embodiment of the present invention, described insect cell is bomyx mori cell, includes but not limited to Sf9 cell, Sf21 cell, Hi5 cell, most preferably Sf9 cell.The cell should adjust cell growth state and growth cycle before using, growth selection is in good condition, being in exponential phase is for experiment.
In embodiment of the present invention, Sf9 cell inoculation quantity is 2 × 10
3~ 3 × 10
3individual cells/well, preferably 3 × 10
3individual cells/well.
In embodiment of the present invention, described infection is, infects 1 ~ 2h, preferably under 25 DEG C of conditions, infect 2h under 15 ~ 25 DEG C of conditions.
In embodiment of the present invention, described nutrient culture media is TC-100 or Sf-900 II nutrient culture media, preferred TC-100 nutrient culture media.Can 1 ~ 20%(V/V be contained in TC-100 nutrient culture media) hyclone, preferably containing 10%(V/V) hyclone.
In embodiment of the present invention, after described virus titer standard items dilution, concentration range is 1.9 × 10
4~ 3.0 × 10
5pfu/mL.
In embodiment of the present invention, described color condition for act on 1h under 37 DEG C of conditions.
In embodiment of the present invention, be measure light absorption angle value under 405nm.
Instant invention overcomes shortcoming of the prior art, with titre logarithm value and absorbance for coordinate axis mapping drawing standard curve, determining recombinant baculovirus titre by calculating.Concrete grammar is: with the titre logarithm value of standard items for horizontal ordinate, with the absorbance of the 405nm of correspondence for ordinate, experimentally Plotting data typical curve, asks linear regression equation.The absorbance recording measuring samples is substituted into linear regression equation, result of calculation is multiplied by extension rate and is titre.All absorbances being positioned at the measuring samples of standard curve range are calculated titre respectively, takes the mean after addition, be the titre of this measuring samples.
The process eliminate Plaque Technique Detected and got ready the shortcoming counting plaque and easily cause subjective error by naked eyes, the result of acquisition is more considerable, accurate.The method realizes quantitative measurement recombinant baculovirus titre by the quantity detecting cell.Highly sensitive, accuracy is good, and required reagent is cheap and easy to get; Easy and simple to handle, technology is easily grasped; Consuming time shorter, efficiency is higher.
Accompanying drawing explanation
The typical curve that Fig. 1 draws for the testing result according to embodiment 3.
Embodiment
Be described below in detail embodiments of the invention, it should be noted that embodiment described below is exemplary, only for explaining the present invention, and can not limitation of the present invention be interpreted as.In addition, if do not clearly not stated, adopted in the following embodiments all reagent are, and market can be buied, or can according to text or the synthesis of known method, for the reaction conditions do not listed, be also that those skilled in the art easily obtain.
Embodiment 1: cell is inoculated
(1) material Sf9 cell, purchased from Chinese Typical Representative thing DSMZ; TC-100 nutrient culture media and hyclone, purchased from GIBCO company; 96 orifice plates, purchased from NUNC company.
(2) method of operating
The Sf9 adherent growth cell of taking the logarithm growth period, softly blows and beats, makes cell resuspended.1,000g(swing bucket rotor) centrifugal 8min.Abandon supernatant, by cell with containing 10%(V/V) the TC-100 nutrient culture media of hyclone is resuspended, makes cell suspension.Sampling, Trypan Blue counts.
With containing 10%(V/V) the TC-100 nutrient culture media of hyclone is by cell dilution to 3 × 10
4individual/mL, joins in 96 porocyte culture plates, every hole 100 microlitre.27 DEG C of standing 2h.
Embodiment 2: the dilution of recombinant baculovirus titre standard items and measuring samples
(1) material TC-100 nutrient culture media and hyclone, purchased from GIBCO company; Micropipettor, purchased from EPPENDORF company; EP pipe, rifle head, purchased from AXYGEN company.
(2) method of operating
With TC-100 nutrient culture media dilution recombinant baculovirus titre standard items, maximum concentration is 320 times of dilutions, below uses 2 times of gradient dilutions such as 640 times of dilutions, 1280 times of dilutions, totally 8 dilutabilitys; With TC-100 nutrient culture media dilution measuring samples, maximum concentration is 20 times of dilutions, below uses 4 times of gradient dilutions such as 80 times of dilutions, 320 times of dilutions, totally 5 dilutabilitys.
Embodiment 3: quantitative measurement recombinant baculovirus titre
(1) material hyclone, purchased from GIBCO company; PNPP, purchased from Amersco company; Triton X-100, sodium acetate, NaOH are purchased from traditional Chinese medicines; EDTA, tries purchased from Shanghai.
(2) method of operating
Sucked by nutrient solution in 96 porocyte culture plates, every hole adds the virus liquid 50 μ L of serial dilution successively, incubated at room 2h, allows viruses adsorption.Add containing 20%(V/V in each hole) the TC-100 nutrient culture media 50 μ L of hyclone, put 27 DEG C and cultivate 72h.Exhaust nutrient solution in each hole, add pNPP solution (1mg/mL pNPP, 0.06mol/L sodium acetate, 0.001mol/LEDTA, 0.2%Triton X-100, pH6.0) 100 μ L successively, place 1h for 37 DEG C.Every hole adds 1mol/L NaOH10 μ L, mixes, and room temperature places 5 ~ 20min.Absorbance is read, as shown in table 1, table 2 at BIO-TEK microplate reader 405nm place.Data according to obtaining make typical curve as shown in Figure 1.With the titre logarithm value of typical curve its absorbance corresponding, ask linear regression equation.The absorbance recording measuring samples is substituted into linear regression equation, result of calculation is multiplied by the titre that namely extension rate obtains measuring samples.All absorbances being positioned at the measuring samples of standard curve range are calculated titre respectively, takes the mean after addition, the titre recording this measuring samples is 3.1 × 10
7pfu/mL.The titre detecting this measuring samples with Plaque Technique Detected compares, and result fits like a glove.
Table 1: standard items (virus of known titre)
| Extension rate | 320 | 640 | 1280 | 2560 | 5120 | 10240 | 20480 | 40960 |
| The logarithm of titre | 5.477 | 5.176 | 4.875 | 4.574 | 4.273 | 3.972 | 3.671 | 3.370 |
| A405 | 1.369 | 1.686 | 1.937 | 2.352 | 2.651 | OUT ※ | OUT | OUT |
※: testing result is more than 3.0, exceeds range of readings.
Table 2: virus to be checked and contrast
#: replace recombinant baculovirus to infect with TC-100.
Although illustrate and describe embodiments of the invention above, be understandable that, above-described embodiment is exemplary, can not be interpreted as limitation of the present invention, those of ordinary skill in the art can change above-described embodiment within the scope of the invention when not departing from principle of the present invention and aim, revising, replacing and modification.
Claims (13)
1. a method for quantitative measurement recombinant baculovirus titre, comprises the following steps:
A (), by resuspended for the insect cell of exponential phase, after carrying out suitably dilution with nutrient culture media, inoculation insect cell is in 96 porocyte culture plates;
B virus titer standard items and virus liquid to be checked are carried out serial dilution by (), be inoculated in insect cell and infect;
C () adds pNPP substrate solution, after colour developing certain hour, microplate reader detects absorbance value;
D () is according to the absorbance value drawing standard curve of each dilution series virus titer standard items;
E () adopts calibration curve method, calculate the titre of virus liquid to be checked,
Wherein,
Described recombinant baculovirus refers to the insect baculovirus having lacked polyhedron gene,
Light absorption angle value is measured under 405nm.
2. method according to claim 1, is characterized in that: described insect cell is bomyx mori cell, and described bomyx mori cell is Sf9 cell, Sf21 cell or Hi5 cell.
3. method according to claim 2, is characterized in that: described bomyx mori cell is Sf9 cell.
4. method according to claim 3, is characterized in that: described Sf9 cell inoculation quantity is 2 × 10
3~ 3 × 10
3individual cells/well.
5. method according to claim 4, is characterized in that: described Sf9 cell inoculation quantity is 3 × 10
3individual cells/well.
6. method according to claim 1, is characterized in that: described infection for infecting 1 ~ 2h under 15 ~ 25 DEG C of conditions.
7. method according to claim 6, is characterized in that: described infection for infect 2h under 25 DEG C of conditions.
8. method according to claim 1, is characterized in that: described nutrient culture media is TC-100 or Sf-900 II nutrient culture media.
9. method according to claim 8, is characterized in that: described nutrient culture media is TC-100 nutrient culture media.
10. method according to claim 9, is characterized in that: described TC-100 nutrient culture media contains 1 ~ 20% (V/V) hyclone.
11. methods according to claim 10, is characterized in that: described TC-100 nutrient culture media contains 10% (V/V) hyclone.
12. methods according to claim 1, is characterized in that: after the dilution of virus titer standard items, concentration range is 1.9 × 10
4~ 3.0 × 10
5pfu/mL.
13. methods according to claim 1, is characterized in that: described color condition for act on 1h under 37 DEG C of conditions.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310521058.6A CN103529205B (en) | 2013-10-29 | 2013-10-29 | Quantitative determination method for titer of recombinant insect baculovirus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310521058.6A CN103529205B (en) | 2013-10-29 | 2013-10-29 | Quantitative determination method for titer of recombinant insect baculovirus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103529205A CN103529205A (en) | 2014-01-22 |
| CN103529205B true CN103529205B (en) | 2015-06-17 |
Family
ID=49931383
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310521058.6A Active CN103529205B (en) | 2013-10-29 | 2013-10-29 | Quantitative determination method for titer of recombinant insect baculovirus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103529205B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106048090B (en) * | 2016-07-05 | 2019-06-07 | 华中农业大学 | The quickly method of measurement ascovirus titre |
| CN106802349A (en) * | 2016-12-30 | 2017-06-06 | 广东华南联合疫苗开发院有限公司 | Sf insect cell host cell proteins Double-antibody sandwich enzymelinked immunosorbent detection kits and method |
| CN110412028B (en) * | 2018-04-27 | 2020-10-16 | 中国科学院动物研究所 | Counting method of insect particle viruses |
-
2013
- 2013-10-29 CN CN201310521058.6A patent/CN103529205B/en active Active
Non-Patent Citations (5)
| Title |
|---|
| A rapid method for titrating baculovirus stocks using the Sf-9 easy titer cell line.;Ralph F.Hopkins等;《BioTechniques》;20091231;第47卷(第3期);第785-788页 * |
| Automated baculovirus titration assay based on viable cell growth monitoring using a colorimetric indicator.;Yann Pouliquen等;《BioTechniques》;20061231;第40卷;第282页右侧栏-281页，图1，图2 * |
| Error assessment in recombinant baculovirus titration: Evaluation of different methods.;António Roldão等;《Journal of Virological Methods》;20091231;第159卷;第71页第2.3-2.4节 * |
| Titration of non-Occluded baculovirus using a cell viability assay.;Jimmy A.Mena等;《BioTechniques》;20031231;第34卷(第2期);第260页右侧栏-262页，图1，图2 * |
| 黎路林.杆状病毒在昆虫培养细胞中的增殖、滴定和纯化.《杆状病毒表达载体系统》.1996, * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103529205A (en) | 2014-01-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103529205B (en) | Quantitative determination method for titer of recombinant insect baculovirus | |
| CN102925136B (en) | Zn<2+> ratiometric fluorescent probe compound and preparation method and use thereof | |
| CN106872695B (en) | A kind of H7 subtype avian influenza virus hemagglutinin antibody indirect ELISA detection kit | |
| CN104280551A (en) | Duck tembusu virus E-ELISA (E-enzyme-linked immuno sorbent assay) detection kit and preparation method thereof | |
| CN103773898A (en) | Triple detection kit for human herpes viruses HSV-1, HSV-2 and HCMV | |
| CN110308145A (en) | A kind of chlamydia trachomatis glycogen detection reagent, chlamydia trachomatis glycogen test strip and preparation method thereof | |
| CN202005754U (en) | Fluorescent optical fiber sensor capable of monitoring dissolved oxygen partial pressure and carbon dioxide partial pressure in blood | |
| CN103364560A (en) | Method for determining baculovirus titer | |
| Karlsson et al. | Measuring influenza virus infection using bioluminescent reporter viruses for in vivo imaging and in vitro replication assays | |
| CN105002298A (en) | Fluorescent quantitative PCR detection method of spring viraemia of carp virus | |
| CN104458361B (en) | The automatic collection method of tail gas of a kind of constant pressure type closed loop control and device | |
| Sheng et al. | Development and application of a rapid semiquantitative immunochromatographic test strip to detect white spot syndrome virus | |
| CN104531895B (en) | A kind of method for determining virus titer | |
| CN107686865A (en) | Detect primer pair, probe, kit and the method for I type bovine herpes virus | |
| CN106048090B (en) | The quickly method of measurement ascovirus titre | |
| Stewart et al. | The enumeration of aerosolised Saccharomyces cerevisiae using bioluminescent assay of total adenylates | |
| CN102192862A (en) | Method for measuring solid content in heavy black liquid | |
| CN105177176A (en) | Adenovirus titer detection method | |
| EP2646828A1 (en) | Method for determining the titre of viruses by using infectious standards | |
| CN101408510A (en) | Method for measuring dioxins in fly ash by ultraviolet spectrophotometer | |
| CN106047932B (en) | Methyl-B-cyclodextrin is improving the application in baculoviral exogenous protein expression amount | |
| CN105132586A (en) | Application of recombinant adenovirus containing porcine circovirus type 2 ORF2 genes as standard sample in nucleic acid amplification testing | |
| CN108845043A (en) | Microcapsule phycotoxin MC-LR Rapid Quantification in a kind of Growth of Microcystis aeruginosa period | |
| CN104330288A (en) | High-sensitivity detecting method of free biotin | |
| CN103529010A (en) | Bi-color fluorescence detection method for cell viability |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder |
Address after: Building b3-4, Guanggu biological city, 666 Gaoxin Avenue, Donghu New Technology Development Zone, Wuhan City, Hubei Province, 430075 Patentee after: Sino US huashitong biomedical technology (Wuhan) Co.,Ltd. Address before: Building b3-4, Guanggu biological city, 666 Gaoxin Avenue, Donghu New Technology Development Zone, Wuhan City, Hubei Province, 430075 Patentee before: WATERSTONE PHARMACEUTICALS(WUHAN) Co.,Ltd. |
|
| CP01 | Change in the name or title of a patent holder |