CN110042087A - A kind of recombinant rabies virus rHEP- △ G-EGFP and its application - Google Patents
A kind of recombinant rabies virus rHEP- △ G-EGFP and its application Download PDFInfo
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Abstract
The present invention relates to antibody test technical field more particularly to a kind of recombinant rabies virus rHEP- △ G-EGFP and its applications.The recombinant rabies virus rHEP- △ G-EGFP is glycoprotein gene deletion form recombinant strain, and the glycoprotein gene deletion form recombinant strain has the property that a) the standard velogen strain glycoprotein containing cell origin；It b) being capable of infection cell and the expressing green fluorescent protein in cell；It c) can not self-replacation and diffusion in cell and animal body.Have following advantages using the hydrophobin neutralizing antibody level detection method that recombinant rabies virus rHEP- △ G-EGFP is established: one, the virus cannot pass on breeding in cell and living body, therefore safer；Two, the detection method is more economical convenient with direct fluorescence microscopy microscopic observation；Three, the measurement result of the detection method is relative to relatively reliable using the method for low virulent strain.
Description
Technical field
The present invention relates to antibody test technical field more particularly to a kind of recombinant rabies virus rHEP- △ G-EGFP and
It is applied.
Background technique
Rabies (rabies) are one caused by hydrophobin (rabies virus, RABV) by height neurotropism
The strong Arbo infectious disease of kind, case fatality rate are up to 100%.There are about 60,000 people to die of rabies in the annual whole world, so far rabies
Still threaten the world, especially developing country public health security (Fooks AR, Banyard AC, Horton DL,
et al.Current status of rabies and prospects for elimination[J].Lancet.2014,
384(9951):1389-1399).Chinese human world rabies morbidity number is only second to India, occupies the 2nd, the world.The current nothing of the disease
Specific short, and can prevent to infect completely by pre-exposure prophylaxis or timely post-exposure prophylaxis.The human rabies in China
In the infection sources, dog accounts for 95% or more.Therefore, prevent and control rabic key to be to establish perfect animal rabies in China
Prevention system is controlled mad by various canines of the vaccine inoculation including pet dog, wandering dog and wild animal from source
Dog disease.Planning, implementation and effect assessment of the foundation of prevention system dependent on vaccine immunity, and the monitoring to animal's antibody level
It is the important evidence for evaluating immune effect of vaccine.
Currently, the hydrophobin neutralizing antibody that the World Health Organization (WHO) and World Organization for Animal Health (OIE) are approved
Detection method be mainly rapid fluorescence stove Inhibition test (rapid fluorescent focus inhibition test,
) and fluorescence antibody virus neutralization tests (fluorescent antibody virus neutralization, FAVN) RFFIT
(World Health Organization,WHO expert consultation on rabies,third report[R],
WHO technical report series,2018).Both methods in terms of the consistency of testing result and stability all
It is relatively good.But both methods still remains some defects, they will be with hydrophobin standard fixed virus CVS-11
The neutralization test that strain carries out serum antibody is tested, which is pathogenic strain, and there are security risks.The operation of two methods simultaneously
Complexity to the more demanding of laboratory condition and operator, and needs expensive fluorescein-labeled antibody as real
Test material.Therefore, they are not suitable for common laboratory or base is used for the monitoring of hydrophobin neutralizing antibody level, now
It still needs research and development and establishes a kind of safe, economic and quick hydrophobin neutralizing antibody detection method.Have some grind at present
Study carefully using the recombinant rabies virus of expressing green fluorescent protein establish virucidin detection method (Hua Tao, Tao Lihong,
Ge Jinying's etc., expressing green fluorescent protein recombinant rabies virus Flury-LEP constructs and its for neutralizing antibody detection
It studies [J], Chinese Preventive Veterinary Medicine report, 2010,32 (8): 581-585；Xue Xianghong, Zheng Xuexing cover pico- etc., novel rabies
The foundation [J] of virucidin's detection method, Products in China magazine, 2013,26 (8): 1165-1174；Tang
Haibo,Lu Zhuanling,Wei Xiankai et al.A recombinant rabies virus expressing a
phosphoprotein–eGFP fusion is rescued and applied to the rapid virus
neutralization antibody assay[J],Journal of Virological Methods,2015,219:75-
83；Jillybeth Burgado,Lauren Greenberg,Mike Niezgoda et al.A high throughput
neutralization test based on GFP expression by recombinant rabies virus[J],
PLOS Neglected Tropical Diseases, 2018,12 (12): e0007011), but these methods all have one and ask
Topic is that recombinant strain used all derives from weak poison strain, these strains have certain on the glycoprotein sequence of virus surface
Difference, glycoprotein is the important antigenic protein of hydrophobin, therefore is using neutralizing antibody level measured by low virulent strain
It is no to can truly reflect the antibody level for neutralizing street strain or velogen strain? it is still necessary to further inquire into for this problem.On the other hand, though
These right methods use low virulent strain, but still remain potential viral spread risk.
Summary of the invention
In order to overcome the shortcomings of Gao Chengben in two kinds of standard methods of RFFIT and FAVN, low-security and existing based on table
The disadvantages of up to green fluorescence recombinant virus detection method accuracy and inadequate safety, the present invention is using technique for gene engineering steady
Surely on the baby hamster kidney cell (baby hamster kidney, BHK-21) for expressing hydrophobin CVS-11 strain glycoprotein
It constructs and saves out a kind of hydrophobin recombinant strain rHEP- △ that glycoprotein gene is substituted for green fluorescence protein gene
G-EGFP。
The specific technical solution of the present invention is as follows:
One aspect of the present invention discloses a kind of recombinant rabies virus rHEP- △ G-EGFP, the recombinant rabies virus
RHEP- △ G-EGFP is glycoprotein gene deletion form recombinant strain, and the glycoprotein gene deletion form recombinant strain has as follows
Characteristic:
A) the standard velogen strain glycoprotein containing cell origin；
It b) being capable of infection cell and the expressing green fluorescent protein in cell；
It c) can not self-replacation and diffusion in cell and animal body.
The second aspect of the present invention discloses the method for preparation and reorganization hydrophobin rHEP- △ G-EGFP a kind of, utilizes
Technique for gene engineering constructs on the BHK-21 cell for stablizing expression hydrophobin CVS-11 strain glycoprotein and saves out one
Glycoprotein gene is substituted for the hydrophobin recombinant strain of green fluorescence protein gene by kind.
Genetic engineering, also known as gene splicing technology and DNA recombinant technique are using molecular genetics as theoretical basis, to divide
Sub- biology and microbiological modernism are means, by the gene of separate sources by the blueprint being pre-designed, structure in vitro
Hybrid DNA molecule is built, living cells is then introduced into, to change the original hereditary capacity of biology, obtain new varieties, production new product.
Preferably, the above method the following steps are included:
S1: the BHK-21 cells of expression CVS-11 glycoprotein (CVS-G) are stablized in screening；
S2: construction recombination plasmid pHEP- △ G-EGFP；
S3: rescue and screening recombinant rabies virus rHEP- △ G-EGFP.
It should be understood that the present invention is not limited to above-mentioned steps, the step of can also including other, such as before step S1,
It include also other additional steps between step S1 and S2, between step S2 and S3, after step S3, and without departing from the present invention
Protection scope.
Preferably, the S2 includes:
S21:PCR expands EGFP and carrier pHEP- △ G (missing glycoprotein gene) segment；
S22: plasmid is seamlessly connected, and is screened to obtain positive gram after connection product is converted to competent cell
Grand bacterium；
S23: it is identified after extracting the plasmid of positive colony bacterium.
It should be understood that the present invention is not limited to above-mentioned steps, the step of can also including other, such as before step S21,
Between step S21 and S22, between step S22 and S23, also include other additional steps after step S23, and without departing from this
The protection scope of invention.
It is furthermore preferred that the S21 includes:
S211: using plasmid pEGFP-N1 as template, carrying out PCR amplification using primer EGFP-F and primer EGFP-R, described
The nucleotide sequence of primer EGFP-F and primer EGFP-R are respectively as shown in SEQ NO:1 and SEQ NO:2；
S212: using plasmid pHEP-3.0 as template, PCR expansion is carried out using primer pHEP- △ G-F and primer pHEP- △ G-R
Increase；The nucleotide sequence of the primer pHEP- △ G-F and primer pHEP- △ G-R are respectively such as SEQ NO:3 and SEQ NO:4 institute
Show.
It should be understood that the present invention is not limited to above-mentioned steps, the step of can also including other, such as step S211 it
Before, include also other additional steps, and without departing from protection of the invention between step S211 and S212, after step S212
Range.
Third aspect of the present invention discloses the recombinant rabies virus rHEP- △ G-EGFP that above-mentioned method obtains.
The 4th aspect of the present invention discloses above-mentioned recombinant rabies virus rHEP- △ G-EGFP in detection antibody level
In application.
The 5th aspect of the present invention discloses in above-mentioned recombinant rabies virus rHEP- △ G-EGFP detection serum
With the method for antibody level, comprising the following steps:
S1: test serum is subjected to inactivation treatment；
S2: recombinant rabies virus rHEP- △ G-EGFP is added and carries out incubation neutralization reaction；
S3: being added BHK-21 cell suspension, directly counts under the microscope in fluorescence microscopy after culture 24-48 hours.
In a specific embodiment of the invention, by the test serum of acquisition 56 DEG C water-bath inactivation treatment 30 minutes.?
3 times of gradient dilutions are carried out to test serum in 96 porocyte culture plates, each sample is detected using 4 × 6 bore regions.Dilution
100 μ L culture mediums are added in region apertures, serum is added in 4 hole of the 1st column after inactivation, every 50 μ L of hole, and it draws 50 μ L and the 2nd column is added,
The 6th column are diluted to method, finally abandon 50 μ L.Standard positive serum and negative serum are carried out according to the above method in control wells dilute
It releases.Virus CVS-11 or rHEP- △ G-EGFP to 100TCID50/50 μ L is diluted with culture medium, 50 μ L are added in every hole, and 37 DEG C incubate
It educates and neutralizes 60min.50 μ L (2 × 10 of cell suspension is added in every hole later4A cell), 37 DEG C are set, 5%CO2Culture 48 in incubator
Hour.It can directly be counted under the microscope in fluorescence microscopy using the test group of rHEP- △ G-EGFP measurement, CVS-11 test group is abandoned
Cells and supernatant adds -20 DEG C of acetone soln fixed 30min of 80% pre-cooling, after abandoning fixer, natural air drying 10min.It is added
Rabies virus N protein fluorescence antibody is incubated for 1 hour and is marked, and calculates the hole of fluorescent staining after washing under fluorescence microscope
Number.By in the half of test serum and extension rate is divided by the half of standard positive serum and extension rate, multiplied by standard blood
Clear potency (0.5IU/mL) is test serum rabies neutralization titer.
The 6th aspect of the present invention discloses application of the above-mentioned method in evaluation immune effect of vaccine.On specifically,
The method of stating can be used for the monitoring to animal's antibody level.
The 7th aspect of the present invention discloses a kind of kit, and the kit includes above-mentioned recombinant rabies virus
rHEP-△G-EGFP.It preferably, further include specification in the kit.The kit can be used for detecting hydrophobin
Neutralizing antibody it is horizontal.
On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination, and without departing from structure of the invention
Think of and protection scope.
The present invention has following remarkable advantage and effect compared with the existing technology:
First is that recombinant strain rHEP- △ G-EGFP energy expressing green fluorescent protein, can directly exist after finishing neutralization test
Fluorescence microscopy under the microscope, is detected without fixed cell and using fluorescein-labeled antibody, both economy or when can save
Between；Second is that recombinant strain rHEP- △ G-EGFP can be in the BHK-21 cell of expression hydrophobin CVS-11 strain glycoprotein
Middle breeding, and the CVS-11 strain glycoprotein that acquisition cell is expressed when sprouting release, CVS-11 are RFFIT and two kinds of FAVN marks
Fixed virus used in quasi- method.Therefore, it is examined using the hydrophobin neutralizing antibody that recombinant strain rHEP- △ G-EGFP is established
Survey method is in accuracy better than the detection method established using low virulent strain；Third is that the base of recombinant strain rHEP- △ G-EGFP
Because lacking glycoprotein gene in group, therefore it can not replicate and spread in ordinary cells and animal body, be a kind of replication defect type
Strain has very high safety.
Detailed description of the invention
Fig. 1 is the building schematic diagram of recombinant strain rHEP- △ G-EGFP；
Fig. 2 is that cell strain BHK-CVS-G stablizes expression hydrophobin CVS-11 glycoprotein figure (in figure: the RT-PCR that A is
Figure, B are Immunofluorescence test figure)；
Fig. 3 is that (in figure: the PCR amplification figure that A is EGFP, B are the PCR amplification of carrier pHEP- △ G segment to PCR amplification figure
Figure)；
Fig. 4 is rescue figure of the recombinant virus rHEP- △ G-EGFP in BHK-CVS-G cell line；
Fig. 5 is growth in vitro curve graph of the recombinant virus rHEP- △ G-EGFP on BHK-CVS-G cell；
Fig. 6 is for recombinant virus rHEP- △ G-EGFP through intracranial injection mode to the impact effect figure at mouse weight；
Fig. 7 is the virus infection fluorogram after the effect of different neutralization titer serum；
Fig. 8 is the measurement chart of dog serum sample neutralizing antibody level；
Fig. 9 is the measurement chart of human serum sample's neutralizing antibody level.
Specific embodiment
Technical solution of the present invention is described in detail with reference to the accompanying drawings and examples, but therefore will be not of the invention
It is limited among the embodiment described range
In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or says according to commodity
Bright book selection.The reagents and materials used in the present invention are commercially available.
The technical scheme is that passing through seamless clone and reverse genetics manipulation technology for hydrophobin low virulent strain base
Because the glycoprotein gene green fluorescence protein gene in group replaces (construction strategy is shown in Fig. 1), and fixed stablizing expression standard
The recombinant strain is saved out in the cell strain of strain CVS-11 glycoprotein.It is established using the recombinant strain safe, economical, convenient
Hydrophobin neutralizing antibody detection method.
Embodiment 1
Present embodiment discloses a kind of methods for constructing recombinant virus rHEP- △ G-EGFP, specifically includes the following steps:
1. stablizing the BHK-21 cells screening of expression CVS-11 glycoprotein (CVS-G)
1.1 cell transfecting
(1) cultivate BHK-21 cell: 6 porocyte culture plates are inoculated with BHK-21 cell (by College of Veterinary Medicine, South China Agricultural University
Veterinary microbiology teaching and research room provides), cell density is 3-5 × 105A/hole contains 10% fetal calf serum in culture medium DMEM；37
DEG C culture 12~16h, until cell 70%-90% converges.
(2) according to the transfection reagent Lipofectamine of Thermo Fisher Scientific company
The specification of 3000Reagent Protocol is prepared rotaring redyeing system and is transfected, the method is as follows:
(3) Opti-MEM culture medium (125 hole μ L/) and Lipofectamine 3000Reagent (7.5 hole μ L/) are prepared
Dilution mixes well.
(4) DNA dilution is prepared by system, by (5 holes μ g/, by Agricultural University Of South China's veterinary science plasmid pcDNA3-CVS-G
Veterinary microbiology teaching and research room, institute constructs and saves) it is diluted in Opti-MEM culture medium (125 hole μ L/), add P3000 examination
Agent (10 hole μ L/), mixes well.
(5) the dilution 1:1 in step (3) and (4) is mixed, is stored at room temperature 10-15 minutes.It is directly added into every hole later
In cell, 4 holes of parllel screening, setting untransfected compares 2 holes.
The screening of 1.2 resisting cells
(1) after culture 24 hours, the DMEM of antibiotic containing G418 (concentration is 800 μ g/mL) is added in above-mentioned Transfected cells
Fresh culture, replacement in every 3-5 days only replace the half of original culture medium once containing the screening and culturing medium of G418 every time.
(2) after screening about 15 days, the around-France monoclonal for being combined with limit dilution method picking resisting cell of set to 24 orifice plates is utilized
In, it is added and continues to cultivate containing the maintenance screening and culturing medium that concentration is 400 μ g/mL G418.
(3) after cytotostatic passed for 5 generations, expression can be stablized using the methods of PCR, RT-PCR, cellular immunofluorescence identification
This cell strain is named as BHK-CVS-G by the cell strain of CVS-G albumen, and seed cell is stored in liquid nitrogen after DMSO is added.Benefit
With RT-PCR and rabies virus glucoprotein monoclonal antibody (by veterinary microbiology teaching and research room, College of Veterinary Medicine, South China Agricultural University
Preparation) P-glycoprotein expression situation in BHK-CVS-G cell is detected as shown in Fig. 2, rabies virus glucoprotein is stablized in cell
Expression, a large amount of albumen are distributed on cell membrane.
2. the building of recombinant plasmid pHEP- △ G-EGFP
2.1PCR expands EGFP and carrier pHEP- △ G segment
(1) with plasmid pEGFP-N1 (being saved by veterinary microbiology teaching and research room, College of Veterinary Medicine, South China Agricultural University) for mould
Plate carries out PCR amplification using primer EGFP-F and EGFP-R, and the enzyme used is the Q5High-Fidelity DNA of NEB company
Polymerase, amplification program are as follows: 95 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 30s, operation
30 circulations；Last 72 DEG C of extensions 8min.After reaction, PCR product carries out electrophoresis in 1% Ago-Gel and utilizes
The plastic recovery kit of QIAGEN company is recycled.PCR products electrophoresis map is shown in Fig. 3 A.
(2) with plasmid pHEP-3.0 (being presented by College of Veterinary Medicine, South China Agricultural University professor Guo Xiaofeng) for template, using drawing
Object pHEP- △ G-F and pHEP- △ G-R carries out PCR amplification, and the enzyme used is the Q5High-Fidelity DNA of NEB company
Polymerase, amplification program are as follows: 95 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 7min, fortune
Row 30 circulations；Last 72 DEG C of extensions 8min.After reaction, PCR product carries out electrophoresis in 1% Ago-Gel and utilizes
The plastic recovery kit of QIAGEN company is recycled.PCR products electrophoresis map is shown in Fig. 3 B.
Wherein, the nucleotide sequence of primer is as follows:
EGFP-F:ctcaaaagacttaaggaaagatggtgagcaagggcgag (SEQ NO:1)
EGFP-R:gtcaaaaggatgaccggcctcttgtacagctcgtccatgc (SEQ NO:2)
PHEP- △ G-F:aggccggtcatccttttg (SEQ NO:3)
PHEP- △ G-R:ctttccttaagtcttttgaggg (SEQ NO:4)
The seamless connection and conversion of 2.2 plasmids
(1) using the Gibson Assembly Cloning kit of NEB company by above-mentioned EGFP segment (15ng) and
PHEP- △ G segment (100ng) is seamlessly connected, and Gibson Assembly Master Mix is added after two segment mixing
10 μ L of reagent adds ultrapure water to 20 μ L, and condition of contact is 50 DEG C, 30min.
(2) connection product is converted into DH5 α competent cell, is screened on the plate of amicillin resistance positive
Clone.
(3) after the plasmid for extracting positive colony bacterium, recombinant plasmid is identified by the method for digestion, PCR and sequencing.
3. the rescue and screening of recombinant rabies virus rHEP- △ G-EGFP
The rescue of 3.1 recombinant viruses
(1) BHK-CVS-G cell is cultivated: the cell strain BHK- for stablizing expression CVS-11 glycoprotein that the above method is obtained
CVS-G is inoculated in 12 orifice plates, and cell density is 1 × 105A/hole contains 10% fetal calf serum in DMEM culture solution；37 DEG C of cultures 12
~16h, until cell 60%-80% converges.
(2) according to the transfection reagent Lipofectamine of Thermo Fisher Scientific company
The specification of 3000Reagent Protocol is prepared rotaring redyeing system and is transfected.Each system is done 11 in 12 orifice plates and is put down
Row, a hole is as blank control, specific full-length cDNA plasmid pHEP- △ G-EGFP and helper plasmid pH-N, pH-P, pH-L
The dosage and proportion of (helper plasmid is presented by College of Veterinary Medicine, South China Agricultural University professor Guo Xiaofeng herein) are as shown in table 1:
Table 1
(3) preparation Opti-MEM culture medium and Lipofectamine 3000Reagent dilution, every 100 μ L of hole, sufficiently
It mixes.
(4) DNA dilution is prepared by system, by P3000 reagent dilutions into Opti-MEM culture medium, then by system addition
DNA is mixed well.
(5) the dilution 1:1 in step (3) and (4) is mixed, is stored at room temperature 10min.In the meantime, it is softly washed with PBS
It washs cell 3 times.
(6) in 12 porocyte plates, 1mL DMEM culture medium is added in every hole, and transfection procedure is added by the amount of every 100 μ L of hole
(5) mixed liquor.
(7) transfection plate is placed in 37 DEG C, 5%CO2Under the conditions of cultivate 6~8h.
After (8) 6~8h, culture medium is discarded, is softly washed with PBS cell 2~3 times, adds fresh DMEM medium, 37
DEG C 5%CO2Under the conditions of cultivate.
The screening of 3.2 recombinant viruses
(1) after cultivating about 7 days, above-mentioned cell plates are encased with masking foil, multigelation cell 3 times, cooling time 1h, room temperature
Dissolution.Taking 40 μ L supernatants and the fresh BHK-21 cell of 100 μ L, (cell density is 4 × 105A/mL) 96 orifice plates are mixed in,
Thereafter in 37 DEG C of cultures 48h, 37 DEG C of culture 96h.
(2) incline cell culture fluid, and the acetone of 80% pre-cooling is filled it up in every hole, and quickly incline acetone, fills it up with acetone, tin again
Foil paper encases culture plate, sets 30min at -20 DEG C.Acetone is discarded, slight empty dry, 30 μ L anti-rabies virus N proteins are added in every hole
Fluorescence antibody, 37 DEG C of incubation 1h, discards Incubating Solution, washs cell twice with 1 × PBS, each 5min, then quick with deionized water
It is primary to wash cell.
(3) it spontaneously dries, fluorescence microscopy can see a large amount of green fluorescence spots under the microscope, as save successfully.It saves
The viral nomenclature for rescuing acquisition is rHEP- △ G-EGFP.The recombinant virus fluorescence picture saved out is shown in Fig. 4.
The passage of 2 recombinant virus rHEP- △ G-EGFP of embodiment and growth curve measurement
Present embodiment discloses the passages and growth curve measuring method of a kind of recombinant virus rHEP- △ G-EGFP, specifically
The following steps are included:
One, recombinant virus rHEP- △ G-EGFP is passed on and is cultivated on BHK-CVS-G cell strain, utilize RT-
The method of PCR identification and fluorescence microscope evaluates the genetic stability of recombinant virus.
Two, the recombinant virus rHEP- △ G-EGFP of secondary culture is utilized into BHK-CVS-G cell, parent plant rHEP-Flur
It is bent that (being saved by veterinary microbiology teaching and research room, College of Veterinary Medicine, South China Agricultural University) then draws growth in vitro using BH-21 cell
Line.It is 2 × 10 with cell nutrient solution adjustment cell density5/ mL, by recombinant rabies poison rHEP- △ G-EGFP and parent plant
After rHEP-Flury virus infection concentration is adjusted to MOI (multiplicity of infection) as 3,37 DEG C of infection 1h, abandon
Supernatant is removed, fresh complete culture solution (10%FBS) is added in 5%CO2It is cultivated in 37 DEG C of incubators, co-cultures 120h；Often
Every collecting 100 μ L supernatant culture solutions for 24 hours, the virus titer of different time points is measured, parent plant carries out direct immunofluorescence antibody
Detection, recombinant strain then directly in fluorescence microscopy microscopic observation, are depicted as growth curve.Recombinant virus rHEP- △ G-EGFP and
The growth curve of parent plant rHEP-Flury is as shown in figure 5, as can be seen from Figure 5, the duplication titre of rHEP- △ G-EGFP is slightly below
rHEP-Flury。
3 recombinant virus rHEP- △ G-EGFP of embodiment is to the pathogenic of suckling mouse
The present embodiment studies recombinant virus rHEP- △ G-EGFP to the pathogenic of suckling mouse, the specific steps are as follows:
Recombinant virus rHEP- △ G-EGFP is to the SPF grade within pathogenic each 3 age in days of dilution intercerebral inoculation of suckling mouse
Kunming system suckling mouse (is bought from Nanfang Medical Univ's Experimental Animal Center), and 50 μ L after being placed in baking oven drying with high pressure sterilization are micro-
It measures sample injector and injects 30 μ L virus liquids, the morbidity and death condition of continuous 15 days observation suckling mouses calculate suckling mouse half with Karber method
Number lethal dose LD50.The suckling mouse encephalic median lethal dose of parent plant rHEP-Flury is 10 as the result is shown-4.58LD50/ 0.03mL, weight
It is not lethal after group strain rHEP- △ G-EGFP intercerebral inoculation suckling mouse, illustrate that it has higher safety.
4 recombinant virus rHEP- △ G-EGFP of embodiment is influenced at mouse weight
The present embodiment studies recombinant virus rHEP- △ G-EGFP to be influenced at mouse weight, the specific steps are as follows:
SPF grades of Kunming female mices of 6 week old (buying from Nanfang Medical Univ's Experimental Animal Center) are grouped, are respectively divided into
3 groups, every group 8.The titre of hydrophobin rHEP-Flury and rHEP- △ G-EGFP is adjusted to 1.0 × 107FFU/mL,
30 μ L of intracranial injection Mice Inoculated.After infection, observes the incidence of each group mouse and dead situation occur, and daily to small
Mouse is weighed, and by the weight before respectively injecting on the weight ratio of all mouse, studies body weight ratio situation of change.As the result is shown
RHEP- △ G-EGFP strain is weaker compared with rHEP-Flury to the pathogenicity of mouse, as shown in Figure 6.
Embodiment 5 is horizontal using the neutralizing antibody in recombinant virus rHEP- △ G-EGFP detection animal and human serum
It is anti-using the neutralization in recombinant virus rHEP- △ G-EGFP detection animal and human serum that present embodiment discloses a kind of
The method of body level, comprising the following steps:
Measure the FAVN S.O.P. of neutralizing antibody horizontal reference Britain OIE rabies reference laboratory in serum.
By the test serum acquired on dog and people 56 DEG C water-bath inactivation treatment 30 minutes.To test serum in 96 porocyte culture plates
3 times of gradient dilutions are carried out, each sample is detected using 4 × 6 bore regions.It dilutes and 100 μ L culture mediums is added in region apertures, go out
Serum is added in 4 hole of the 1st column after work, every 50 μ L of hole, draws 50 μ L and the 2nd column are added, and is diluted to the 6th column with method, finally abandons 50 μ L.
Standard positive serum and negative serum are diluted according to the above method in control wells.With culture medium dilute virus CVS-11 or
RHEP- △ G-EGFP to 100TCID5050 μ L are added in/50 μ L, every hole, and 37 DEG C of incubations neutralize 60min.BHK- is added in every hole later
21 cell suspension, 50 μ L (2 × 104A cell), 37 DEG C are set, 5%CO2Culture 48 hours in incubator.Utilize rHEP- △ G-EGFP
The test group of measurement can be counted directly in fluorescence microscopy under the microscope, and CVS-11 test group abandons cells and supernatant, be added 80% pre-
- 20 DEG C of acetone soln cold fixed 30min, after abandoning fixer, natural air drying 10min.It is anti-that rabies virus N protein fluorescence is added
Body is incubated for 1 hour and is marked, and calculates the hole count of fluorescent staining after washing under fluorescence microscope.It will be in the half of test serum
With extension rate divided by the half of standard positive serum and extension rate, multiplied by standard serum potency (0.5IU/mL) be to
Survey serum rabies neutralization titer.The virus fluorescence picture such as Fig. 7 detected in the serum of different neutralizing antibody levels and after virus
It is shown.The serum of 20 immune dogs and 3 not immune dogs uses CVS-11 (FAVN method) and recombinant strain rHEP- △ G-EGFP respectively
The neutralizing antibody level of (FAVN- △ G-EGFP method) measurement is shown in Fig. 8.15 parts of serum are acquired from immune crowd and crowd is not immunized
3 parts of serum are acquired, the neutralizing antibody level measured respectively with above two method is shown in Fig. 9.Compare this with the method for paired t-test
The consistency of the measured value of two methods does not have between them significant difference (p > 0.05) as the result is shown, the two consistency compared with
It is good.
The hydrophobin neutralizing antibody level detection side established using recombinant virus rHEP- △ G-EGFP in the present embodiment
Method has following advantages: 1. safeties are good, and the virus used is Gene Deletion virus, although energy infection cell cannot be thin
Breeding is passed in born of the same parents and living body, experimental evidence also indicates that the recombinant virus is pathogenic to all not having at mouse and suckling mouse.2. economical
Convenient, recombinant virus energy expressing green fluorescent protein does not need to carry out virus signature using the more expensive fluorescence antibody of price, simultaneously
Also save the time.3. accurate and reliable, the glycoprotein on recombinant virus surface is the glycoprotein of standard fixed virus CVS-11, is
Major antigen in conjunction with neutralizing antibody, therefore the result measured is relative to relatively reliable using the method for low virulent strain.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Sequence table
<110>Agricultural University Of South China
<120>a kind of recombinant rabies virus rHEP- △ G-EGFP and its application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 38
<212> DNA
<213> Artificial Sequence
<400> 1
ctcaaaagac ttaaggaaag atggtgagca agggcgag 38
<210> 2
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 2
gtcaaaagga tgaccggcct cttgtacagc tcgtccatgc 40
<210> 3
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 3
aggccggtca tccttttg 18
<210> 4
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 4
ctttccttaa gtcttttgag gg 22
Claims (10)
1. a kind of recombinant rabies virus rHEP- △ G-EGFP, which is characterized in that the recombinant rabies virus rHEP- △ G-
EGFP is glycoprotein gene deletion form recombinant strain, and the glycoprotein gene deletion form recombinant strain has the property that
A) the standard velogen strain glycoprotein containing cell origin；
It b) being capable of infection cell and the expressing green fluorescent protein in cell；
It c) can not self-replacation and diffusion in cell and animal body.
2. a kind of method of preparation and reorganization hydrophobin rHEP- △ G-EGFP, which is characterized in that existed using technique for gene engineering
Stablize and constructs and save out a kind of by glycoprotein gene on the BHK-21 cell of expression hydrophobin CVS-11 strain glycoprotein
It is substituted for the hydrophobin recombinant strain of green fluorescence protein gene.
3. according to the method described in claim 2, characterized by comprising the following steps:
S1: the BHK-21 cells of expression CVS-11 glycoprotein are stablized in screening；
S2: construction recombination plasmid pHEP- △ G-EGFP；
S3: rescue and screening recombinant rabies virus rHEP- △ G-EGFP.
4. according to the method described in claim 3, it is characterized in that, the S2 includes:
S21:PCR expands EGFP and carrier pHEP- △ G (missing glycoprotein gene) segment；
S22: plasmid is seamlessly connected, and is screened to obtain positive colony after connection product is converted to competent cell
Bacterium；
S23: it is identified after extracting the plasmid of positive colony bacterium.
5. according to the method described in claim 4, it is characterized in that, the S21 includes:
S211: using plasmid pEGFP-N1 as template, PCR amplification, the primer are carried out using primer EGFP-F and primer EGFP-R
The nucleotide sequence of EGFP-F and primer EGFP-R are respectively as shown in SEQ NO:1 and SEQ NO:2；
S212: using plasmid pHEP-3.0 as template, PCR amplification is carried out using primer pHEP- △ G-F and primer pHEP- △ G-R；
The nucleotide sequence of the primer pHEP- △ G-F and primer pHEP- △ G-R are respectively as shown in SEQ NO:3 and SEQ NO:4.
6. the recombinant rabies virus rHEP- △ G-EGFP obtained according to method described in claim 2-5.
7. recombinant rabies virus rHEP- △ G-EGFP according to claim 1 or 6 answering in detection antibody level
With.
8. a kind of neutralization using in the detection serum of recombinant rabies virus rHEP- △ G-EGFP described in claim 1 or 6
The method of antibody level, which comprises the following steps:
S1: test serum is subjected to inactivation treatment；
S2: recombinant rabies virus rHEP- △ G-EGFP is added and carries out incubation neutralization reaction；
S3: being added BHK-21 cell suspension, directly counts under the microscope in fluorescence microscopy after culture 24-48 hours.
9. application of the method according to claim 11 in evaluation immune effect of vaccine.
10. a kind of kit, which is characterized in that the kit includes recombinant rabies virus described in claim 1 or 6
rHEP-△G-EGFP。
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Application publication date: 20190723 |