CN106853247A - A kind of method for preparing rabies live vector vaccine and products thereof and purposes - Google Patents
A kind of method for preparing rabies live vector vaccine and products thereof and purposes Download PDFInfo
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Abstract
The invention discloses a kind of method for preparing rabies live vector vaccine and products thereof and purposes.Methods described comprises the following steps：First, primer is designed, Rabies Virus Antigen Protein G gene and bacterial flagellin FIjB or FIiC gene is amplified；2nd, antigen protein G genes and FIjB or FIiC genes are inserted between replication-defective adenoviral shuttle vector specificity promoter and terminator, are built into the adenovirus shuttle vector containing gene G-FIjB or G-FIiC；3rd, the recombinant replication-defective adenoviral shuttle vector and adenoviral backbone carrier of acquisition are obtained into recombinant adenovirus plasmid by homologous recombination；4th, the homogeneous recombined adhenovirus of genome structure are produced with recombinant adenovirus plasmid.Additionally, the invention allows for the rabies live vector vaccine for thering is the above method to prepare.Compared to prior art, the live vector vaccine that the present invention is prepared is safer and effective, and fabrication cycle is shorter, will be had a good application prospect in rabic prevention or therapy field.
Description
Technical field
Method of viral vectors production rabies live vector vaccine and products thereof and purposes are the present invention relates to the use of, especially
It is that one kind expresses rabies virus G protein using adenovirus vector and bacterial flagellin prepares rabies live vector epidemic disease
Method of seedling and products thereof and purposes.
Background technology
Rabies (rabies) are connect by hydrophobin (rabies virus, the RV) Zoonosis that cause of infection are acute
Venereal disease infectious disease is touched, once morbidity, then 100% is dead.It is global that there are about 55000 people dies from the disease, China every year
Nearly 5 years about 3000 people of annual death or so, are only second to India, are mainly caused by dog bite.Therefore, prevent mad
The major measure of dog disease is the immunity inoculation that rabies vacciness is carried out to dog, cat and wild animal.Current China is for animals
Rabies vacciness mainly have inactivated vaccine and Attenuate vaccine.Although traditional vaccine still accounts for leading in rabies prophylaxis control
Status, but because production cost is expensive, and inactivation does not cause thoroughly to dissipate the presence of the factors such as the potential danger of poison,
Limit widely using for its.Therefore, the safe and efficient new rabies molecular vaccine of development still seems and very must
Will.
Live vector vaccine is one of focus of Recent study, it is considered to be a kind of vaccine for having very much a DEVELOPMENT PROSPECT.System
Toward insertion external source protectiveness genes of interest in live vector during seedling, because foreign gene is had become as the virus of carrier
Or a part for the self structure of bacterium, therefore produced immune response level is usually less than intact virus or bacterium
Immune intensity caused by corresponding composition.Recombinant poxvirus carrier and recombinant adenoviral vector have host range wide,
There is certain resistance to external world, it is considered to be the excellent carrier of recombinant vaccine.Although being carried to recombinant poxvirus
The research starting of body is earliest, but because vaccinia virus can be replicated in mammal body, bio-safety of the people to it
Property there is certain query, worry that the vaccinia virus similar with the variola virus application on animal can be evolved and had to the mankind
Pathogenic new virus.And replication-defective adenoviral is often lacked for early genes necessary to virus replication
E1 areas, make its not reproducible in animal body, therefore develop recombinant replication-defective adenoviral rabies vacciness more
It is safe and effective.
Rabies viruses has 5 kinds of structural proteins, the infection of wherein glycoprotein G and virus and immune has substantial connection.Grind
Study carefully and show that G is the albumen for uniquely inducing neutralizing antibody, can also stimulate cellullar immunologic response, can resist outside rabies viruses
Week and the attack of intracerebral approach.The rabies genetic engineering recombinant vaccine developed in recent years, mostly with glycoprotein cDNA
As genes of interest.Although these recombinant vaccines can stimulate the generation of specific antibody, and produce certain level
Protection, but, its antibody level that totally induction is produced of single glycoprotein G is not high, awaits improving it
Level.
The flagellin of bacterium is the main component in the thread portion of flagellum, in the virulence and the fortune of bacterium of bacterial pathogen
Played an important role in dynamic.In most of salmonella strains, there are two genes (fljB and fliC) to encode
Flagellar antigen.Phase flagellin FliC, the fljB coding two-phase flagellin FljB of fliC codings one.Flagellin
It is a kind of pathogen-associated molecular pattern (pathogen-associated molecular pattern, PAMP), Ke Yiyu
TLR5 on mammalian cell combines the conduction of mediation signal.Innate immune system can be known by TLRs
The PAMPs guarded on other bacterium, such as lipopolysaccharides and flagellin.These structures can induce strong inflammatory reaction,
And finally activate the acquired immune response.
Live vector vaccine has the advantages that safe efficient, producing cost is cheap, but not yet has rabies mobile load at present
Body vaccine is listed.The present Research of comprehensive rabies molecular vaccine, it is believed that with adenovirus as expression vector
Genetic engineering recombinant rabies vaccine (or live vector vaccine) will have a good application prospect.
The content of the invention
It is an object of the invention to provide one kind rabies virus G protein and bacterial flagellum egg are expressed using adenovirus vector
The white method for preparing rabies live vector vaccine, so as to obtain safe and efficient rabies vacciness.
The purpose of the present invention is achieved through the following technical solutions：
One kind of the invention expresses rabies virus G protein using adenovirus vector and bacterial flagellin prepares mad dog
The method of sick live vector vaccine, comprises the following steps：
(1) genes of interest is prepared：
Design primer, the antigen protein G of hydrophobin (Rabies Virus, RV) is amplified by the method for PCR
Gene and bacterial flagellin FIjB or FIiC gene；
(2) recombinant replication-defective adenoviral shuttle vector is built：
The antigen protein G genes for arriving that step (1) is expanded are with bacterial flagellin FIjB or FIiC gene together
It is inserted between replication-defective adenoviral shuttle vector specificity promoter and terminator, detects antigen protein G bases
Whether cause and FIjB or FIiC genes have been incorporated into adenovirus shuttle vector, and structure obtains containing G-FIjB
Or the recombinant replication-defective adenoviral shuttle vector of G-FIiC, be named as PAdTrack-CMV/G-FIjB or
PAdTrack-CMV/G-FIiC；
(3) replication-defective adenoviral plasmid is obtained：
Will obtain recombinant replication-defective adenoviral shuttle vector PAdTrack-CMV/G-FIjB or
Between PAdTrack-CMV/G-FIiC and adenoviral backbone carrier PAdEasy-1 is by Escherichia coli carrying out plasmid
Homologous recombination is so as to obtain recombinant adenovirus plasmid；
(4) the homogeneous recombined adhenovirus of genome structure, including following operation are produced with recombinant adenovirus plasmid：
1) recombinant adenovirus plasmid obtained with Pac I digestions step (3),
2) rotaring redyeing 293 cell,
3) 293 cells are inoculated with,
4) the homogeneous recombined adhenovirus of genome structure are obtained, the sick cell of virus infection is collected, is frozen together with supernatant
Deposit, obtain final product.
In the present invention, it is preferred to, expansion of antigen GFP G and bacterial flagellin are used in step (1)
The amplimer of FIjB and FIiC genes is：
GF：5 ' GCAGATCTGCCACCATGGTTCCTCAAGCTCTTTTG3 ' (contain the enzymes of Bgl II
Enzyme site)
GR：5 ' GCGTCGACTCACAGTCTGATCTCACC 3 ' (containing the restriction enzyme sites of Sal I)
FIjBF：5 ' ATTGTCGACGCCACCATGGCACAAGTAATCAACACT 3 ' (contain
The sites of Sal I)
FIjBR：5 ' GCTCTAGATTAACGTAACAGAGACAGC 3 ' (contain the digestions of Xba I position
Point)
FIiCF：5 ' ATTGTCGACGCCACCATGGCACAAGTCATTAATACA 3 ' (contain
The sites of Sal I)
FIiCR：5 ' GCAAGCTTTTAACGCAGTAAAGAGAG 3 ' (containing the restriction enzyme sites of Hind III)
Wherein, ATG is initiation codon；Hydrophobin G genes are used by template of rabies viruses aG strains
Primer pair GF/GR is expanded；Bacterial flagellin FIjB or FIiC gene are respectively with salmonella typhimurium
Flagellin gene is expanded for template using primer pair FIjBF/FIjBR or FIiCF/FIiCR.
In the present invention, it is preferred to, in step (2), described adenovirus vector promoter is CMV；Eventually
Only son is PolyA；The structure of described recombinant replication-defective adenoviral shuttle vector includes following operation：
1) the genes of interest G for obtaining will be expanded through Bgl II, the double digestions of Sal I, the purpose with cohesive end is formed
Genetic fragment；
2) while, by adenovirus shuttle vector PAdTrack-CMV through Bgl II, the double digestions of Sal I, obtain linear
The carrier of change；
3) cohesive end connection is carried out to above-mentioned product using T4DNA ligases；
4) connection product is converted into ETEC according to a conventional method, is screened on the agar plate containing that resistance of card,
After selected clone, plasmid is extracted；
5) identified using digestion, PCR amplification method, obtain positive recombinant plasmid, be named as PAdTrack-CMV/G；
6) the genes of interest FIjB that obtains will be reclaimed through Sal I, the double digestions of Xba I, genes of interest FIiC through Sal I,
The double digestions of Hind III, form the genes of interest fragment with cohesive end；
7) while, by positive recombinant plasmid PAdTrack-CMV/G through Sal I, the double digestions of Xba I or Sal I,
The double digestions of Hind III, the carrier for being linearized；
8) cohesive end connection is carried out to the product of step (6) and (7) using T4DNA ligases；It is routinely square
Connection product is converted ETEC JM109 by method, and screening, selects gram on the agar plate containing that resistance of card
After grand, plasmid is extracted；Identified using digestion, PCR amplification method, obtain positive recombinant plasmid, be named as
PAdTrack-CMV/G-FIjB or PAdTrack-CMV/G-FIiC.
In the present invention, it is preferred to, in step (3), obtain the process bag of replication-defective adenoviral plasmid
Include following operation：
1) by recombinant replication-defective adenoviral shuttle vector PAdTrack-CMV/G-FIjB or
PAdTrack-CMV/G-FIiC Pme I single endonuclease digestions are linearizing；
2) PAdEasy-1 and step 1 are taken) recombinant replication-defective adenoviral shuttle vector after the linearisation that obtains
Mixing, high-tension electricity conversion E.coli BJ5183；
3) electricity recovers cell after turning in nutrient solution, takes cell and is painted into the culture plate containing kanamycins, cultivates；
4) kalamycin resistance clone is selected, DNA is extracted in a small amount, electricity is carried out with 0.8% Ago-Gel
Swimming mobility analysis, and carry out analysis of Restriction Endonuclease Profile；
5) the recombinant adenovirus plasmid high-tension electricity of gained is transformed into Host Strains, a large amount of height can be obtained in this Host Strains
The plasmid of quality, picking purpose clone.Wherein, it is preferred that described Host Strains are DH10B.
In the present invention, it is preferred to, in step (4), produce genome structure equal with recombinant adenovirus plasmid
The specific practice of each operation of one recombined adhenovirus is：
1) take the recombinant adenovirus plasmid that step (3) is obtained, with Pac I digestions, digest completely with cut including
Ori and Kan resistant genes expose its inverted terminal repetitive sequence (ITR) in interior plasmid constructs；
2) liposome-mediated transfection, 293 cells is inoculated with tissue culture dishes and is transfected；
3) viral supernatants are taken and is inoculated with 293 cells, with the complete DMEM nutrient solutions containing 2% calf serum in culture
Cultivated in case, day by day observation of cell lesion, to the complete lesion of 293 cells；
4) sick cell of virus infection is collected, whether detection antigen protein G-FIjB and G-FIiC gene obtains table
Reach, finally frozen together with supernatant.
In the present invention, it is preferred to, step 2) in, described liposome-mediated transfection includes following operation step
Suddenly：
1. in recombinant adenovirus plasmid to the DMEM nutrient solutions of dilution Pac I linearization for enzyme restriction；
2. in and then Liposomal suspensions being added into DMEM nutrient solutions, fully the two is mixed, is incubated at room temperature；
3. inhale during this period and abandon cell original fluid, cell is washed once with DMEM nutrient solutions；
4. add DNA/ liposome complexes toward per plate, inhaled after being incubated in 5%CO2 incubators and abandoned containing DNA/
The nutrient solution of liposome complex,
5. the DMEM complete culture solutions containing 10% calf serum are added, continues to cultivate, collected after liposome transfection
Cell, multigelation.
In the present invention, it is preferred to, step 4) in by observing the method for egfp expression, including profit
The expression of cytopathy and fluorescence microscopy reporter gene green fluorescent protein is observed with inverted microscope come really
Determine whether antigen protein G-FIjB and G-FIiC gene are expressed.
In the present invention, it is preferred to, identified by using PCR and restriction enzyme in step (2), carry out
The method of agarose gel electrophoresis analysis detection determines whether are antigen protein G genes and FIjB or FIiC genes
It has been incorporated into adenovirus vector.
Further, the invention allows for the rabies live vector that the method as described in any of the above is prepared
The purposes of vaccine and described rabies live vector vaccine in preparing prevention or treating rabic medicine.
Rabies virus G protein gene is passed through IRES by the present invention with bacterial flagellin FIjB or FIiC gene respectively
Sequence is connected, and structure obtains containing rabies virus G gene and flagellin FIjB and G gene and flagellin
The recombinant adenovirus plasmid of FIiC, and then obtain the homogeneous recombined adhenovirus of genome structure.It is prepared by the inventive method
The rabies live vector vaccine for obtaining, its based on rabies virus glycoprotein G, while in the participation of flagellin
Under, can induce body and produce more comprehensively immune response, obtain more preferable immune effect.
Build recombined adhenovirus work in rate-limiting step be that genes of interest is integrated into adenoviral gene group, at present this
Mostly realize there is certain difficulty in homologous recombination mode in mammalian cell in one critical process.Meanwhile,
Because recombination efficiency is not high, the recombinant virus of gained usually needs Plaque-purified, therefore experimental period is more long, nor
Can guarantee that the homogeneity of recombined adhenovirus genome structure.The present invention is fast using growth cycle, large intestine easy to operate
Bacillus bacterium realize it is homologous between plasmid so that the homogeneous recombinant adenoviral vector of acquisition quickly.
In sum, the recombinant replication-defective adenoviral rabies vacciness that the present invention is obtained is than existing rabies epidemic disease
Seedling is safer and effective, and fabrication cycle is shorter.
Brief description of the drawings
Fig. 1 be recombinant shuttle vector PAdTrack-CMV/G-FIjB and PAdTrack-CMV/G-FIiC digestion and
PCR qualification figures；
Fig. 2 is recombinant adenovirus plasmid qualification figure；
Fig. 3 is to observe cytopathy figure and using fluorescence microscopy green fluorescent protein using inverted microscope
Expression figure；
Fig. 4 is the structure figure for expressing shuttle vector of adenovirus；
Fig. 5 is neutralizing antibody testing result.
Specific embodiment
The present invention is further described with reference to specific embodiment, advantages of the present invention and feature will be with description
And it is apparent.But embodiment is only exemplary, does not constitute any limitation to the scope of the present invention.This area
Technical staff should be understood that without departing from the spirit and scope of the invention can be to technical solution of the present invention
Details and form are modified or are replaced, but these modifications and replacement are each fallen within protection scope of the present invention.
The preparation of the rabies live vector vaccine of embodiment 1
The replication-defective adenoviral rabies for directly inoculation animal can be prepared with the present embodiment method
Live vector vaccine.Including procedure below, reference picture 4：
First, genes of interest is prepared
Design primer, hydrophobin (Rabies Virus, RV) antigen protein G is amplified by the method for PCR
Gene and bacterial flagellin FIjB genes or FIiC genes；Designed antigenic protein gene G and bacterial flagellum
The amplimer of albumen FIjB genes or FIiC genes is：
GF：5 ' GCAGATCTGCCACCATGGTTCCTCAAGCTCTTTTG3 ' (contain the enzymes of Bgl II
Enzyme site)
GR：5 ' GCGTCGACTCACAGTCTGATCTCACC 3 ' (containing the restriction enzyme sites of Sal I)
FIjBF：5 ' ATTGTCGACGCCACCATGGCACAAGTAATCAACACT 3 ' (contain
The sites of Sal I)
FIjBR：5 ' GCTCTAGATTAACGTAACAGAGACAGC 3 ' (contain the digestions of Xba I position
Point)
FIiCF：5 ' ATTGTCGACGCCACCATGGCACAAGTCATTAATACA 3 ' (contain
The sites of Sal I)
FIiCR：5 ' GCAAGCTTTTAACGCAGTAAAGAGAG 3 ' (containing the restriction enzyme sites of Hind III)
Wherein, ATG is initiation codon；Hydrophobin G genes are used by template of rabies viruses aG strains
Primer pair GF/GR is expanded；Bacterial flagellin FIjB or FIiC gene are respectively with salmonella typhimurium
Flagellin gene is expanded for template using primer pair FIjBF/FIjBR or FIiCF/FIiCR.
GF is located at rabies viruses aG plants of complete sequence 3318；GR is located at rabies viruses aG plants of complete sequence 4893
Place；
FIjBF is located at the 1 of flagellin FIjB genes；FIjBR is located at the 1521 of flagellin FIjB genes
Place；
FIiCF is located at the 1 of flagellin FIiC genes；FIjBR is located at the 1488 of flagellin FIiC genes
Place.
2nd, recombinant replication-defective adenoviral shuttle vector is built
The rabies virus G gene for obtaining will be expanded to be inserted respectively into again with bacterial flagellin FIjB or FIiC gene
PolyA between defective adenoviral shuttle vector specificity promoter CMV processed and terminator, is built into and contains base
Because of the recombinant replication-defective adenoviral shuttle vector of G-FIjB or G-FIiC, it is named as
PAdTrack-CMV/G-FIjB or PAdTrack-CMV/G-FIiC；
Specifically include following operating procedure：
1) the genes of interest G for obtaining will be reclaimed through Bgl II, the double digestions of Sal I, the purpose with cohesive end is formed
Gene；
2) while, by adenovirus shuttle vector PAdTrack-CMV through Bgl II, the double digestions of Sal I, obtain linear
The carrier of change；
3) cohesive end connection is carried out to above-mentioned product using T4DNA ligases；
4) connection product is converted into ETEC JM109 according to a conventional method, in the agar plate containing that resistance of card
Upper screening, after selected clone, extracts plasmid；
5) identified using digestion, PCR amplification method, obtain positive recombinant plasmid PAdTrack-CMV/G.
6) the genes of interest FIjB that obtains will be reclaimed through Sal I, the double digestions of Xba I, genes of interest FIiC through Sal I,
The double digestions of Hind III, form the genes of interest with cohesive end.
7) while, by positive recombinant plasmid PAdTrack-CMV/G through Sal I, the double digestions of Xba I or Sal I,
The double digestions of Hind III, the carrier for being linearized.
8) cohesive end connection is carried out to above-mentioned product using T4DNA ligases；Connection product is turned according to a conventional method
Change ETEC JM109, screened on the agar plate containing that resistance of card, after selected clone, extract plasmid；
Using digestion, PCR amplification method identify, obtain positive recombinant plasmid PAdTrack-CMV/G-FIjB or
PAdTrack-CMV/G-FIiC。
Described detection G-FIjB or G-FIiC gene has been incorporated into adenovirus vector, using PCR and limitation
Property restriction endonuclease identification, enter row agarose gel electrophoresis analysis.The restructuring containing genes of interest G-FIjB that will be obtained
Shuttle vector PAdTrack-CMV/G-FIjB can cut out the mesh of about 3.0KB through Bgl II, the identification of Xba I double digestions
Fragment, while PCR identifications also it is amplifiable go out the G genes that are consistent of size and FIjB genes purpose fragment (Fig. 1 is left)；
The recombinant shuttle vector PAdTrack-CMV/G-FIiC containing genes of interest G-FIiC that will be obtained through Bgl II,
The identification of the double digestions of Hind III can cut out the purpose fragment of about 3.0KB, at the same PCR identifications also it is amplifiable go out size phase
The G genes and FIiC genes purpose fragment (Fig. 1 is right) of symbol, can determine whether by being contrasted with standard molecular weight.
3rd, replication-defective adenoviral plasmid is obtained
Will obtain recombinant replication-defective adenoviral shuttle vector PAdTrack-CMV/G-FIjB or
PAdTrack-CMV/G-FIiC and adenoviral backbone carrier PAdEasy-1 is by homologous between plasmid in Escherichia coli
Restructuring specifically includes following operating procedure so as to obtain recombinant adenovirus plasmid：
1) by recombinant shuttle vector respectively with Pme I single endonuclease digestions linearizing；
2) take 0.1mg PAdEasy-1 to mix with 1.0mg linearisation recombinant shuttle vectors, high-tension electricity conversion large intestine bar
Bacterium BJ5183；
3) electricity turn after by cell in LB nutrient solutions in 37 DEG C of recovery 30min, take cell and be painted into card containing 50mg/Ml
In the culture plate of that mycin, 16~20h is cultivated in 37 DEG C；
4) kalamycin resistance clone is selected, DNA is extracted in a small amount, electrophoresis is carried out with 0.8% Ago-Gel
Mobility analysis, and analysis of Restriction Endonuclease Profile is carried out, as a result as shown in Fig. 2 same as seen from Figure 2
Testing goal gene has been integrated into adenovirus vector after the restructuring of source, and visible restructuring is analyzed by agarose gel electrophoresis
Plasmid lags significantly behind skeleton carrier, the band of visible about 4.5KB and 33KB after digestion with restriction enzyme, its
Result is consistent with expection；The positive recombinant plasmid for obtaining is named as PAdEasy-G+FIiC or PAdEasy-G+
FIjB；
5) recombinant adenovirus plasmid of gained is transformed into Host Strains DH10B (Rec A, End A) with high-tension electricity,
A large amount of high-quality plasmids, picking purpose clone can be obtained in this Host Strains.
4th, the homogeneous recombined adhenovirus of genome structure, including following operation are produced with recombinant adenovirus plasmid：
1) digestion, take from DH10B extract each recombinant adenovirus plasmid PAdEasy-G+FIiC or
The μ g of PAdEasy-G+FIjB 4, with Pac I digestions, digest to cut the plasmids such as Ori and Kan resistant genes completely
Component, and expose its inverted terminal repetitive sequence ITR；
2) transfect, liposome-mediated transfection is inoculated with 293 cells in 35mm tissue culture dishes and is transfected,
Described liposome-mediated transfection includes following point of operation：
1. in μ g to the 250ul DMEM nutrient solutions of recombinant adenovirus plasmid 4 of dilution Pac I linearization for enzyme restriction；
2. in and then the μ l of liposome LipofectAMINE2000 suspensions 10 being added into 250ul DMEM nutrient solutions,
Fully the two is mixed, 20min is incubated at room temperature；
3. inhale during this period and abandon cell original fluid, cell is washed once with DMEM nutrient solutions；
4. add 500ul DNA/ liposome complexes toward per plate, at 37 DEG C, 3~5h is incubated in 5%CO2 incubators
Inhale afterwards and abandon the nutrient solution containing DNA/ liposome complexes,
5. DMEM complete culture solutions of the 3ml containing 10% calf serum is added, continues to cultivate 7 days, liposome turns
After dye, cell, multigelation 3 times are collected.
3) be inoculated with, take viral supernatants be inoculated with 293 cells, with the complete DMEM nutrient solutions containing 2% calf serum in
37 DEG C, cultivate in 5%CO2 incubators, observation of cell lesion CPE day by day, to the complete lesion of 293 cells；
4) freeze, collect the sick cell of virus infection, frozen in -20 DEG C together with supernatant, structure obtain containing anti-
Former Protein G gene and the recombined adhenovirus of bacterial flagellin FIjB or FIiC gene, are named as Ad-RV-G-FljB
Or Ad-RV-G-FliC.
Construct the recombined adhenovirus only containing antigen protein G genes according to the method described above simultaneously, be named as
Ad-RV-G。
Whether detection G-FIjB or G-FIiC genes are expressed, using the method for observation egfp expression
Cytopathy is observed using inverted microscope, it is seen that compared with control cells normal growth, and transfectional cell occurs substantially
Cytopathy.The expression of reporter gene green fluorescent protein can be directly observed using fluorescence microscope.Its result
Shown in Fig. 3.
Recombined adhenovirus qualified after testing are made rabies live vector vaccine, animal is immunized direct injection.
The immune efficacy experimental animal test data of the rabies live vector vaccine of embodiment 2
The tri- kinds of recombined adhenovirus of Ad-RV-G, Ad-RV-G-FljB, Ad-RV-G-FliC built with embodiment 1 are lived
Carrier respectively in 0,14 days Balb/c small white mouses of immune 6-8 week old, 7 after immune, 14,21,28,35,
42 take a blood sample respectively, determine neutralizing antibody with RFFIT methods, as a result as shown in Figure 5.Result shows that 3 kinds recombinate glands
After virus is immune at 2 times, neutralizing antibody begins to ramp up, and wherein recombined adhenovirus Ad-RV-G-FljB stimulates generation
The horizontal highest of neutralizing antibody, and the neutralizing antibody level that recombined adhenovirus Ad-RV-G-FliC is produced is relatively low, but
Higher than Ad-RV-G.Result shows, by flagellin FljB or FliC and the weight of rabies virus G protein coexpression
The neutralizing antibody level that group adenovirus has obvious adjuvant enhancement effect, its immune generation can resist rabies viruses
The attack of lethal dose.
42 days rabies virus challenges with 30LD50 are immunized, observe 14 days, as a result all immune mouses acquisitions
100%.These results suggest that of the invention a kind of based on recombined adhenovirus expression rabies virus G protein and flagellum egg
White rabies live vector vaccine has a good application prospect.
Claims (10)
1. it is a kind of to express rabies virus G protein and bacterial flagellin preparation rabies work using adenovirus vector
The method of carrier bacterin, it is characterised in that comprise the following steps：
(1) genes of interest is prepared：
Design primer, the antigen protein G of hydrophobin (Rabies Virus, RV) is amplified by the method for PCR
Gene and bacterial flagellin FIjB or FIiC gene；
(2) recombinant replication-defective adenoviral shuttle vector is built：
The antigen protein G genes for arriving that step (1) is expanded are with bacterial flagellin FIjB or FIiC gene together
It is inserted between replication-defective adenoviral shuttle vector specificity promoter and terminator, detects antigen protein G bases
Whether cause and FIjB or FIiC genes have been incorporated into adenovirus shuttle vector, and structure obtains containing G-FIjB
Or the recombinant replication-defective adenoviral shuttle vector of G-FIiC, be named as PAdTrack-CMV/G-FIjB or
PAdTrack-CMV/G-FIiC；
(3) replication-defective adenoviral plasmid is obtained：
Will obtain recombinant replication-defective adenoviral shuttle vector PAdTrack-CMV/G-FIjB or
Between PAdTrack-CMV/G-FIiC and adenoviral backbone carrier PAdEasy-1 is by Escherichia coli carrying out plasmid
Homologous recombination is so as to obtain recombinant adenovirus plasmid；
(4) the homogeneous recombined adhenovirus of genome structure, including following operation are produced with recombinant adenovirus plasmid：
1) recombinant adenovirus plasmid obtained with Pac I digestions step (3),
2) rotaring redyeing 293 cell,
3) 293 cells are inoculated with,
4) the homogeneous recombined adhenovirus of genome structure are obtained, the sick cell of virus infection is collected, is frozen together with supernatant
Deposit, obtain final product.
2. method according to claim 1, it is characterized in that being used for expansion of antigen albumen base in step (1)
Because the amplimer of G and bacterial flagellin FIjB or FIiC gene is：
GF：5’GCAGATCTGCCACCATGGTTCCTCAAGCTCTTTTG 3’
GR：5’GCGTCGACTCACAGTCTGATCTCACC 3’
FIjBF：5’ATTGTCGACGCCACCATGGCACAAGTAATCAACACT 3’
FIjBR：5’GCTCTAGATTAACGTAACAGAGACAGC 3’
FIiCF：5’ATTGTCGACGCCACCATGGCACAAGTCATTAATACA…3’
FIiCR：5’GCAAGCTTTTAACGCAGTAAAGAGAG 3’
Wherein, ATG is initiation codon；Hydrophobin G genes are used by template of rabies viruses aG strains
Primer pair GF/GR is expanded；Bacterial flagellin FIjB or FIiC gene are respectively with salmonella typhimurium
Flagellin gene is expanded for template using primer pair FIjBF/FIjBR or FIiCF/FIiCR.
3. method according to claim 1, it is characterised in that in step (2), described adenovirus is carried
Body promoter is CMV；Terminator is PolyA；The structure of described recombinant replication-defective adenoviral shuttle vector
Including following operation：
1) the genes of interest G for obtaining will be expanded through Bgl II, the double digestions of Sal I, the purpose with cohesive end is formed
Genetic fragment；
2) while, by adenovirus shuttle vector PAdTrack-CMV through Bgl II, the double digestions of Sal I, obtain linear
The carrier of change；
3) cohesive end connection is carried out to above-mentioned product using T4DNA ligases；
4) connection product is converted into ETEC according to a conventional method, is screened on the agar plate containing that resistance of card,
After selected clone, plasmid is extracted；
5) identified using digestion, PCR amplification method, obtain positive recombinant plasmid, be named as PAdTrack-CMV/G；
6) the genes of interest FIjB that obtains will be reclaimed through Sal I, the double digestions of Xba I, genes of interest FIiC through Sal I,
The double digestions of Hind III, form the genes of interest fragment with cohesive end；
7) while, by positive recombinant plasmid PAdTrack-CMV/G respectively through Sal I, the double digestions of Xba I or Sal I,
The double digestions of Hind III, the carrier for being linearized；
8) cohesive end connection is carried out to the product of step (6) and (7) using T4DNA ligases；It is routinely square
Connection product is converted ETEC JM109 by method, and screening, selects gram on the agar plate containing that resistance of card
After grand, plasmid is extracted；Identified using digestion, PCR amplification method, obtain positive recombinant plasmid, be named as
PAdTrack-CMV/G-FIjB or PAdTrack-CMV/G-FIiC.
4. method according to claim 1, it is characterised in that in step (3), obtains replication defect type
The process of adenoviral plasmid includes following operation：
1) by recombinant replication-defective adenoviral shuttle vector PAdTrack-CMV/G-FIjB or
PAdTrack-CMV/G-FIiC Pme I single endonuclease digestions are linearizing；
2) PAdEasy-1 and step 1 are taken) recombinant replication-defective adenoviral shuttle vector after the linearisation that obtains
Mixing, high-tension electricity conversion E.coli BJ5183；
3) electricity recovers cell after turning in nutrient solution, takes cell and is painted into the culture plate containing kanamycins, cultivates；
4) kalamycin resistance clone is selected, DNA is extracted in a small amount, electricity is carried out with 0.8% Ago-Gel
Swimming mobility analysis, and carry out analysis of Restriction Endonuclease Profile；
5) the recombinant adenovirus plasmid high-tension electricity of gained is transformed into Host Strains, a large amount of height can be obtained in this Host Strains
The plasmid of quality, picking purpose clone, it is preferred that described Host Strains are DH10B.
5. method according to claim 1, it is characterised in that in step (4), use recombinant adenovirus toxin
Grain produces the specific practice of each operation of the homogeneous recombined adhenovirus of genome structure to be：
1) take the recombinant adenovirus plasmid that step (3) is obtained, with Pac I digestions, digest completely with cut including
Ori and Kan resistant genes expose its inverted terminal repetitive sequence (ITR) in interior plasmid constructs；
2) liposome-mediated transfection, 293 cells is inoculated with tissue culture dishes and is transfected；
3) viral supernatants are taken and is inoculated with 293 cells, with the complete DMEM nutrient solutions containing 2% calf serum in culture
Cultivated in case, day by day observation of cell lesion, to the complete lesion of 293 cells；
4) sick cell of virus infection is collected, whether detection antigen protein G-FIjB and G-FIiC gene obtains table
Reach, finally frozen together with supernatant.
6. method according to claim 5, it is characterised in that step 2) in, described is liposome-mediated
Transfection includes following operating procedure：
1. in recombinant adenovirus plasmid to the DMEM nutrient solutions of dilution Pac I linearization for enzyme restriction；
2. in and then Liposomal suspensions being added into DMEM nutrient solutions, fully the two is mixed, is incubated at room temperature；
3. inhale during this period and abandon cell original fluid, cell is washed once with DMEM nutrient solutions；
4. add DNA/ liposome complexes toward per plate, inhaled after being incubated in 5%CO2 incubators and abandoned containing DNA/
The nutrient solution of liposome complex,
5. the DMEM complete culture solutions containing 10% calf serum are added, continues to cultivate, collected after liposome transfection
Cell, multigelation.
7. method according to claim 5, it is characterised in that step 4) in by observing green fluorescent protein
The method of expression, observes cytopathy and fluorescence microscopy reporter gene green is glimmering using inverted microscope
The expression of photoprotein determines whether antigen protein G-FIjB and G-FIiC gene are expressed.
8. method according to claim 1, it is characterised in that by using PCR and limitation in step (2)
Property restriction endonuclease identification, enter row agarose gel electrophoresis analysis detection method come determine antigen protein G genes and
Whether FIjB or FIiC genes have been incorporated into adenovirus vector.
9. the method according to claim any one of 1-8 prepare containing antigen protein G genes with it is thin
The rabies live vector vaccine of bacterium flagellin FIjB or FIiC gene.
10. the rabies live vector vaccine described in claim 9 is in preparing prevention or treating rabic medicine
Purposes.
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| CN115029347A (en) * | 2022-05-11 | 2022-09-09 | 珠海中科先进技术研究院有限公司 | Molecular monitoring sequence for recognizing and regulating liver and kidney cell fibrosis, recombinant plasmid and virus inhibition |
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| WO2006127956A2 (en) * | 2005-05-23 | 2006-11-30 | Vaxin, Inc. | Rapid production of adenovirus-free recombinant adenovirus vectors |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108531511A (en) * | 2018-03-28 | 2018-09-14 | 扬州大学 | A kind of expression African swine fever virus E183L gene recombinant adenovirus vectors, construction method and recombined adhenovirus preparation method |
| CN115029347A (en) * | 2022-05-11 | 2022-09-09 | 珠海中科先进技术研究院有限公司 | Molecular monitoring sequence for recognizing and regulating liver and kidney cell fibrosis, recombinant plasmid and virus inhibition |
| CN115029347B (en) * | 2022-05-11 | 2024-02-20 | 珠海中科先进技术研究院有限公司 | Molecular monitoring sequence for recognizing and regulating hepatic and renal cell fibrosis, recombinant plasmid and virus inhibition |
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