CN105039406B - A kind of preparation method of 1 Fc recombinant baculovirus of pig IgG as pig vaccine carrier - Google Patents
A kind of preparation method of 1 Fc recombinant baculovirus of pig IgG as pig vaccine carrier Download PDFInfo
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Abstract
A kind of preparation method the invention discloses pig IgG 1Fc recombinant baculovirus as pig vaccine carrier, belongs to biomedicine field.The preparation method of the present invention uses clone pig IgG Fc genes and E 2 gene of Classical Swine Fever, is connect with baculoviral surface display vector, obtains recombinant transfer vector；Recombinant transfer vector obtains recombinant baculovirus Bacmid carriers by Tn7 swivel bases；Bacmid liposome method transfection insect cells are recombinated, culture supernatant is collected, obtain recombinant baculovirus, recombinate shape virus infection insect cell collects cell and does Western blot analyses and Laser Scanning Confocal Microscope analysis；Recombinant baculovirus swine fever carrier does vaccine potency inspection in pig body.Surface display pig IgG Fc recombinant baculovirus prepared by the present invention has stronger survival ability in Swine serum, the removing that can be escaped to serum complement to virus, to improve the Effective Antigens content of vaccine immunity.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to a kind of 1 Fc recombinant baculovirus of pig IgG is as pig vaccine
The preparation method of carrier.
Background technology
Baculoviral (Baculovirus) be it is a kind of with insect be unique host virus, can be used as biological insecticides or
As expression vector in insect cell mass expressing external albumen, prepare vaccine.The study found that being taken in mammalian cell
Recombinant baculovirus with mammalian promoter can start the expression of downstream foreign gene but virus cannot be thin in mammal
Rise in value in born of the same parents, small to cytotoxicity, successful cell of transduceing can stablize passage and effective expression foreign gene, and mammal is thin
Born of the same parents have better posttranslational modification than insect cell to protein, and the protein structure given expression to is closer to native protein.Therefore,
Baculoviral can be used as a kind of novel mammalian cell gene transfer vector, for expression alien gene and as a kind of base
Because of therapy vector, there is great potential, be increasingly subject to the concern of people.
Baculoviral can enter certain cells in the mammalian body, but cannot be replicated into the cell at these.
Researcher is transform as the tool of a carrying gene using this feature, and carries out certain modification processing, is changed
The carrier for causing orientation to carry carries gene, drug reaches specified position.But baculoviral is after all for body
One foreign peoples, body have immunization to it, this is unfavorable for cell entry appointed part, in order to solve this problem, just in bar
Complement inhibition molecule is introduced inside shape viral gene, the introducing of Complement inhibition molecule can inhibit body to exempt from recombinant virus
Epidemic disease response, the danger being destroyed when baculoviral recombinant being made to pass through serum in transmission process.
Baculovirus Surface Display System is a kind of novel eucaryote surface display system that developed recently gets up, because of it
Big with exogenous sequences length range of choice, value-added speed is fast, the advantages that can keeping the original activity of foreign protein well,
It has been widely used in new generation vaccine development, gene transfer and gene therapy at present, has shown that complicated eukaryotic protein, structure are more
The fields such as the preparation of peptide library and antibody library and monoclonal antibody.Gp64 is the base of current Baculovirus Surface Display System
Plinth is the distinctive structural proteins of budding pattern virus, is also peplomer albumen (peplomer protein), virus is entrenched in after ripe
On cyst membrane, there are 3 kinds of dimer, tripolymer, tetramer existing ways.Most popular baculoviral display systems are now
AcMNPV and BmNPV, the mode of structure are to be inserted into foreign gene, foreign gene between the signal peptide and maturation protein of its gp64
It is just entrenched in after this protein maturation because the excision of signal peptide forms N-terminal fusion protein after processing with gp64 protein fusion expressions
On cell membrane or virus envelope, such foreign gene is just shown cell or virus surface, can be obtained through simple screening process
There is the baculovirus particle of differential protein to expression.
Oral herpes viral G protein (Vesicular stomatatis virus G protein, VSVG) is for a variety of
Different type and tissue-derived mammalian cell have preferable versatility, and it is unwise to baculoviral to significantly increase some
The transduction efficiency of the mammalian cell strain of sense such as CHO, NIH3T3 etc., however can increase cell after VSVG modifies baculoviral
Toxicity simultaneously promotes cell fusion.Then, researcher is by VSV-GED (vesicular stomatitis virus ectodomains, by 21 amino acid
Constituting, including trans-membrane region and cytoplasmic tail) amalgamation and expression in baculoviral envelope protein gp64, influences then caused by cell
It is negligible.
Immunoglobulin Fc is not only to determine the significant points of Ig immune functions, and is that IgG keeps longer by half in vivo
Decline the phase the main reason for.Since Ig Fc have the function of stablizing albumen, it is used to merge with multiple protein, it is desirable to be increased with this
Strong protein stability extends its Half-life in vivo.Ig Fc can form more polymerizable moleculars, and recombinant protein is made to have stronger antigen
Binding force, while Ig Fc have antigen presenting cell receptor and are easily absorbed by antigen presenting cell, it is small so as to be used to improve
The immune response ability of molecular antigen.Baculoviral surface display Ig Fc can increase the combination of itself and antigen presenting cell.
Invention content
It is an object of the invention to be directed to deficiency in the prior art, a kind of 1 Fc recombinant baculovirus of pig IgG work is provided
For the preparation method of pig vaccine carrier.
The present invention is realized especially by following technical scheme：A kind of 1 Fc recombinant baculovirus of pig IgG is as pig vaccine
The preparation method of carrier, is completed by following steps：
1) clone pig IgG Fc genes and E 2 gene of Classical Swine Fever connect with baculoviral surface display vector, are weighed
Group transfer vector；
2) recombinant transfer vector obtains recombinant baculovirus Bacmid carriers by Tn7 swivel bases；
3) Bacmid liposome method transfection insect cells are recombinated, culture supernatant is collected, obtain recombinant baculovirus,
It observes cytopathy and extracts viral DNA and do PCR identifications；
4) recombinate shape virus infection insect cell collects cell and does Western blot analyses and Laser Scanning Confocal Microscope point
Analysis；
5) recombinant baculovirus does the detection of complement antagonistic activity；
6) recombinant baculovirus swine fever carrier does vaccine potency inspection in pig body.
The sequence of pig IgG Fc genes is the SEQ ID NO in sequence table in the step 1):1；Its corresponding pig IgG
The amino acid sequence of Fc is SEQ ID NO:3；
The sequence of E 2 gene of Classical Swine Fever is the SEQ ID NO in sequence table in the step 1):2；Its corresponding pig
The amino acid sequence of pestivirus E2 is SEQ ID NO:4.
The expression of E 2 gene of Classical Swine Fever is controlled by CMV promoter in the step 1)；
Baculoviral surface display vector is VSVG pseudotyping baculovirals in the step 1).
The literary californica nuclear polyhedrosis virus genome of Bacmid carriers silver containing clover in the step 2).
Insect cell is fall army worm gonad cell sf9 in the step 3).
The step of Western blot analyses, is in the step 4)：The insect cell for collecting virus infection, uses cell
It is mixed with albumen sample-loading buffer after lysate processing, absorption supernatant does SDS-PAGE electrophoresis after boiling 10min；Pvdf membrane is soaked
Bubble is in 100% methanol, after 10s, moves to distillation water washing 5min, most postposition transfers in buffer solution, mixing 10min；Protein
After electrophoresis, protein is transferred on pvdf membrane using electroporation；By the pvdf membrane transferred to contain 50g/L skimmed milks
Room temperature closes 1h, is cleaned 3 times with washing buffer；It is incubated overnight with shaking table at diluted 4 DEG C of His labels mouse monoclonal antibody,
The next day with appropriate cleaning solution clean 3 times；The sheep anti-mouse igg antibody of diluted HRP labels is added, at room temperature after shaking table effect 1h,
It is cleaned 3 times with appropriate cleaning solution；Wash with distilled water, pvdf membrane is taken out, is slightly dried, dark place carries out chemiluminescence reaction (ECL),
Equivalent ECL reagents I:II=1:1, it is covered in after reacting 1min on pvdf membrane, carries out X-ray development analysis.
The step of Laser Scanning Confocal Microscope analysis, is in the step 4)：Sterilized slide glass is put into 6 well culture plates, carefully
Born of the same parents are first inoculated on sterile glass slide, and cell access amount is that 1 × 10 is added per hole6；After cell adsorbs 1h, culture medium is abandoned in suction,
Recombinant virus is added to infect at MOI=10；It is inhaled after 48h and abandons culture medium, then add the methanol of 1mL：Acetone=1:1 mixed liquor
5min is fixed under -20 DEG C of refrigerators, adds 1mL PBS buffer solution shaking speeds 50r/min cleanings 2 times later, every time
5min；With the 20mL/L bovine serum albumin(BSA)s of 1mL 30min is reacted at 37 DEG C；1mL PBS buffer solution shaking speeds 50r/ is added
Min is cleaned 3 times, each 5min；Next His antibody (1 is added:100) 1h is reacted at 37 DEG C, to demarcate surface display
IgG Fc；After addition 1mL PBS buffer solution shaking speeds 50r/min is cleaned 3 times, the rabbit anti-mouse igg (1 of FITC labels is added:
100) 1h is reacted at 37 DEG C；After addition 1mL PBS buffer solution shaking speeds 50r/min is cleaned 3 times, seen under Laser Scanning Confocal Microscope
It examines, the cell membrane of display protein can be dyed by FITC and green fluorescence is presented.
The method of complement antagonistic activity detection is in the step 5)：Health pig vena cava anterior blood system is adopted from serum, blood
Sorting is two parts：By 56 DEG C of inactivation 30min, another is not inactivated portion；By two parts of serum respectively with recombinant baculovirus 37
DEG C be incubated 60min, then measure its virus titer using Endpoint Dilution Method；Viral survival rate is at non-inactivation treatment group/inactivation
Reason group.
The step of vaccine potency inspection, is in the step 6)：The double-negative health pig of antigen-antibody is divided into 4 groups：It does not open up
Show the recombinant baculovirus hog cholera vaccine group of IgG Fc：108Pfu/mL recombinant baculovirus 2mL；Show the recombination bar of IgG Fc
Shape virus hog cholera vaccine group：108Pfu/mL recombinant baculovirus 2mL；Hog cholera lapinised virus vaccine group：1 part/2mL；PBS groups：
PBS 2mL；Immune programme is observed for swinery and is immunized after a week, before being immunized and rear 7d is immunized, 14d blood samplings carry out humoral immunity
It is analyzed with cell immunization experiments；After observation is immune swinery whether will appear apocleisis, spirit is depressed, generate heat, tremble, bleeding, diarrhea,
The adverse reactions such as constipation；Humoral immunity is analyzed：Antibody level is detected using ELISA kit；It is neutralized with reference to the detection of OIE handbooks anti-
Body is horizontal；Cellular immunity is analyzed：It is horizontal using ELISA kit detection IFN-γ.
The present invention has the following advantages and effects with respect to the prior art：
An advantage of the present invention is that the baculoviral surface display vector used utilizes the TM and CTD of VSVG
(VSVG-ED) TM and CTD of gp64 is substituted, improves the transduction efficiency to mammalian cell and the antagonism to serum complement
Ability；
It is another advantage of the present invention that the antagonistic ability to serum complement is improved using the pFc of displaying, and it is advantageous
In and antigen presenting cell combination improve immune efficacy；
It is even more important, recombinant baculovirus has no adverse reaction after animal is immunized, and can effectively stimulate generation body
Liquid is immunized and cellular immunity.
Description of the drawings
From detailed description below in conjunction with the accompanying drawings, features described above of the invention and advantage will be apparent from, wherein：
Fig. 1 recombinant baculovirus transfer vector structural schematic diagrams.
Fig. 2 .Western Blot analyze pFc protein expression result figures.
Fig. 3 Laser Scanning Confocal Microscopes analyze position result figures of the pFc in cell, wherein A represents natural light picture, B generations
Table FITC fluorescence pictures, C represent superposition picture.
The antagonism result figure that Fig. 4 recombinant viruses inactivate serum complement.
Specific implementation mode
By the following examples and attached drawing carrys out the surface display pig IgG Fc recombinant baculovirus that the present invention is furture elucidated
Preparation as pig vaccine carrier.Following embodiment is merely illustrative that those skilled in the art is helped further to manage
Solution, but do not limit the present invention.Those skilled in the art can modify and improve without departing from the inventive concept of the premise.
Embodiment 1
1. structure and the verification of baculoviral surface display vector
Using AmMultiBac plasmids as template, GP64SP and GP64TM-CTD is expanded respectively, and in GP64SP and GP64TM-
Tetra- restriction enzyme sites of 6 × His labels and NheI, SalI, SacI, EcoRI are added between CTD in order to detect and foreign gene
It is inserted into；GP64SP and GP64TM-CTD are sequentially inserted into after PFBDM transfer vector p10 promoters；With pDsred-Monomer-
C1 plasmids are template, and amplification CMV-dsred segments are inserted into after PFBDM transfer vector ph promoters；In order to verify pSur-gp64
Whether carrier builds success, and enhanced green fluorescence protein EGFP is inserted into multiple cloning sites and is examined.
2. gene cloning
VSVG-ED genes are expanded as primer PCR using VSVGTM-F and VSVGTM-R and are obtained using pMD2.G carriers as template；
PUC57-pFc plasmids are synthesized by Jin Weizhi companies；Raq gene is with E2-F and E2-R using CSFV crossdrift strain virus cDNA as template
Primer PCR amplification obtains.
3. the structure of the baculoviral recombinant plasmid of surface display pFc
The areas gp64TM and CTD of pSur-gp64 are replaced with the areas TM and CTD of VSVG and by complement inhibitor pFc genes
It is cloned into the multiple cloning sites in carrier P10 promoters downstream and obtains pSur-VSVG-ED-pFc recombinant baculovirus plasmids.
Final recombinant baculovirus plasmid pSur- will be obtained in multiple cloning sites that swine fever raq gene is cloned under CMV promoter
VSVG-ED-pFc-CMV-E2。
4.Tn7 swivel bases obtain recombinant baculovirus plasmid
CaCl2Method prepares SW106 (AmMultiBac) competent cell.When culture to OD600=0.25, final concentration is added
It is induced for 0.1% L-arabinose, continues culture to OD600=0.5, conventional method prepares competent cell.PSur- is added
VSVG-ED-pFc-CMV-E2 is converted, 32 DEG C of shaken cultivation 8h, LB tablet of the painting containing Kan/Tet/Gm/IPTG/X-Gal, 32 DEG C
Constant temperature incubation 18~occur for 24 hours to blue hickie, picking hickie and again scribing line confirm that hickie is verified with each gene primer PCR again.
The primer sequence information is as shown in table 1 in the present invention：
Primer sequence information used in 1 present invention of table
5. recombinant baculovirus plasmid Bacmid DNA transfect sf9 cells
(1) sf9 insect cells 9 × 10 are added in each hole in 6 orifice plates5Cells, culture medium is with 2mL Grace ' s elder brothers
Worm complete medium contains dual anti-a concentration of 0.5 × final concentration (50 μ g/mL penicillin, 50 μ g/mL streptomysins).Cell is training
The exponential phase cell of 3~4d is supported, vigor is more than 97%.Set 1h in 28 DEG C of incubators.
(2) 2 1.5mL Eppendorf pipes are taken to be designated as A, B, the Grace's insects for being separately added into 100 μ L antibiotic-frees are complete
The recombination Bacmid of 5 μ L, mixing are added in A pipes for full medium culture liquid；6 μ L lipofectamines are added in B pipes
(Cellfectin), mixing (notices that liposome is the suspended matter of a grease, may precipitate, use preceding reverse pipe 6~8
It is secondary, be mixed even).A pipe liquid is poured into B pipes, mixes well, sets room temperature 45min, it is mixed to form it into Cellfectin-DNA
Close object.
(3) the good sf9 cell monolayers of adherent growth are taken, old culture solution is abandoned, with Grace ' the s insects of antibiotic-free
Complete medium culture solution washs 1 time.
(4) above-mentioned Cellfectin-DNA mixtures are taken, the not antibiotic Grace's insects of 0.8mL are added and train completely
Base is supported, is uniformly mixed.The culture medium for discarding cleaning is sucked out from cell, uniformly mixed liposome and recombinant DNA are mixed
Object covers cell, sets 28 DEG C of culture 6h.
(5) it is sucked out and discards mixture, antibiotic Grace ' s insects complete medium culture solution is added and continues to cultivate,
Observation cellular morphology and its fluorescence performance daily.Observe that cytopathy is changed into following judgement：1. early lesion is transfected, 25%~
50% cell dia increases or karyon is full of cell；2. transfecting mid-term lesion, cell stops growing, occurs vesica in cell, goes out
Existing viral inclusion body or cell fall off from culture dish；3. transfecting advanced lesions, cell cracks.Collect cell, analysis expression
The case where albumen.Culture supernatant is collected simultaneously as former seed culture of viruses, packing sets -20 DEG C of storages, as lower subinfection sf9 cells
With.
6. the PCR of recombinant baculovirus is identified
The cell culture 2mL of virus, multigelation 3 times, 12000r/min is taken to centrifuge 15min, take 437.5 μ L of supernatant,
12.5 μ L Proteinase Ks (20mg/mL) and 50 μ L SDS liquid (10%), 37 DEG C of water-bath 30min are added；Isometric phenol/chloroform
Once, supernatant is taken；Isometric chloroform carries again, takes supernatant；1/10 volume NaAc (2mol/L) and 2 times of volume absolute ethyl alcohols are added,
2h is placed at -20 DEG C；15min is centrifuged with 15000r/min at 4 DEG C, takes precipitation；70% ethyl alcohol washed once, 30 μ of precipitation
LTE solution dissolves, and the DNA as extracted sets -70 DEG C and saves backup.Using recombinant baculovirus DNA as template, universal primer is used
Amplifying target genes.
7. recombinant baculovirus is proliferated and Endpoint Dilution Method measures virus titer
The insect that recombinant baculovirus merging suspension is incubated to the Grace culture mediums containing 100mL/L fetal calf serums is thin
Born of the same parents sf9.Daughter of virus can be multiplied after viral infected insect cell automatically and discharged to culture medium, received after infecting 3~4d
Collection supernatant just completes the process of virus quantity amplification.The culture insect cell that suspends waits for cell concentration up to 1 × 106Cells/mL, with
The recombinant baculovirus amount infected insect cell of MOI=1 (Multiplicity of infection, MOI) utilizes viral meeting
It is replicated in host insect cell and achievees the purpose that virus quantity amplifies.It is centrifuged off cell and cell fragment after 3~4d of infection, is received
Collect supernatant.Virus concentration is to utilize Endpoint Dilution Method (End-point dilution method).
8. the Western blot analyses of recombinant protein
SDS-PAGE electrophoresis：Pvdf membrane is immersed in 100% methanol, after 10s, moves to by 12% separation gel, 5% concentration glue
Water washing 5min is distilled, most postposition transfers in buffer solution, mixing 10min.After protein electrophorese, Western blot are utilized
Protein is transferred on the pvdf membrane infiltrated with methanol, 2.5h is transferred in 350mA.By the pvdf membrane transferred to contain 50g/
L skimmed milk 1 × PBS fluid-tights are closed, to block the reaction of non-specific antigen antibody.1h is acted at room temperature, with washing buffer
(1.0g/L Tween 20in PBS Buffer) 10min/ cleaning 3 times.Below plus antibody incubation.Each reaction is separately added into
Stayed overnight through shaking table sense at appropriate 4 DEG C of the diluted primary antibodies of 1 × PBS Buffer (skimmed milk containing 1.0g/L), the next day to wash in right amount
Wash 10min/ cleaning of liquid 3 times.Wash away non-specific combination.It is appropriate through 1 × PBS liquid (skimmed milk containing 1.0g/L) to be subsequently added into
Diluted secondary antibody, if HRP marks rabbit-anti pig antibody, at room temperature after shaking table effect 1h, with the 10min/ cleaning 3 of appropriate cleaning solution
It is secondary.Finally wash with distilled water, pvdf membrane is taken out, is slightly dried, dark place carries out chemiluminescence reaction (ECL), equivalent ECL reagents I:
II=1:1, it is covered in after reacting 1min on pvdf membrane, carries out X-ray development analysis.
9. Laser Scanning Confocal Microscope analyzes performance of the recombinant protein on sf9 cell membranes
(1) sterilized slide glass is put into 6 well culture plates, cell is first inoculated on sterile glass slide, cell access amount
It is added 1 × 10 for every hole6。
(2) after cell absorption 1h, culture medium is abandoned in suction, and recombinant virus is added and is infected at MOI=10.
(3) it is inhaled after 2d and abandons culture medium, then add the methanol/acetone (1 of 1mL:1) methanol acetone is in -20 DEG C of ice
5min is fixed under case, adds 1mL PBS (phosphate-buffered saline) shaking speeds 50r/min cleanings 2 later
It is secondary, each 5min.
(4) 30min is then reacted at 37 DEG C with the 20mL/L BSA of 1mL (bovine serum albumin).
(5) 1mL PBS (phosphate-buffered saline) shaking speeds 50r/min cleanings 2 are added after
It is secondary, each 5min.
(6) His antibody (1 is next added:100) 1h is reacted at 37 DEG C, with the circovirus cyst membrane egg of calibration expression
In vain.
(7) after being cleaned 3 times with the PBS of 1mL, the rabbit anti-mouse igg (1 of FITC labels is added:It is 100Sigma) anti-at 37 DEG C
Answer 1h.
(8) last to be cleaned 3 times with the PBS of 1mL again.It is observed under Laser Scanning Confocal Microscope, the cell membrane of display protein can quilt
FITC is dyed and green fluorescence is presented.
10. inhibiting complement activity detection
Healthy Swine serum is taken to be divided into two parts：By 56 DEG C of inactivation 30min, another is not inactivated portion.By two parts of serum
Respectively with 37 DEG C of incubation 60min of recombinant baculovirus, then its virus titer is measured using Endpoint Dilution Method.Viral survival rate is
Non- inactivation treatment group/inactivation treatment group.
11. animal immune experiment
8 four week old CSFV antigen-antibody feminine gender pigs are taken to be randomly divided into 4 groups, every group 2；1st group is BV-VSVGED-
CMV-E2 vaccine immunity groups, the 2nd group is BV-VSVGED-pFc-CMV-E2 vaccine immunity groups, and the 3rd group is hog cholera lapinised virus epidemic disease
Seedling immune group, the 4th group is PBS control group.Wherein the 1st group in 0DPI and 14DPI injections 1.0 × 108Pfu/mL recombinations are rod-shaped
Viral BV-VSVGED-CMV-E2, the 2nd group in 0DPI and 14DPI injections 1.0 × 108Pfu/mL recombinant baculovirus BV-
VSVGED-pFc-CMV-E2,1 part hog cholera lapinised virus vaccine of the 3rd group of injection, the 4th group of isometric PBS of injection is as a contrast.
Experimental animal is in immune 0,7,14d blood was collected separation serum.Its detection project has：(1) it measures body temperature daily and the observation period faces
Bed symptom, such as apocleisis, spirit it is depressed, generate heat, tremble, bleeding, diarrhea, constipation；(2) immunological indices detect：Serum is special
Heterogenetic antibody level detects and the detection of serum neutralizing antibody level；(3) Immunological Markers for Cell-Mediated Immunity detects：Serum cytokines IFN-γ
Assay.
The result of above-mentioned detection is as follows：
1) structure of recombination bacillary viral vector and observation
Recombination bacillary viral vector pSur-VSVG-ED-pFc-CMV-E2 (Fig. 1) is shuttle vector, in specific large intestine angstrom
Reproducible in uncommon bacterium SW106Bac can also enter in sf9 cells and replicate generation recombinant virus, while expression alien gene albumen.
The Bacmid pSur-gp64-pFc of 0.8 μ g are transfected into insect sf9 cells with dedicated liposome Cellfectin.Transfection is opened
The CPE for beginning to observe transfectional cell changes：Cell dia significantly increases, and particle increases due to there is vesica and viral inclusion body into the cell
It is more, part cell cracking, death.Illustrating in transfection process, BacmidpSur-gp64-pFc is by liposome-mediated for recombination,
Homologous recombination occurs in insect cell with baculovirus DNA, produces the recombinant baculovirus with infection activity.
2) Western blot are analyzed
In order to prove the expression of pFc, Western blot analyses are carried out.Primary antibody is His monoclonal antibodies, secondary antibody A549
The rabbit-anti pig IgG of label.As a result visible complement inhibitor proteins are illustrated in insect cell surface (Fig. 2).
3) performance of the Laser Scanning Confocal Microscope analysis recombinant protein on sf9 cell membranes
In order to prove that pFc genes are illustrated on the cell membrane of insect cell, Laser Scanning Confocal Microscope analysis is carried out.Primary antibody is
His monoclonal antibodies, secondary antibody are the rabbit-anti pig IgG of A549 labels.As a result see Fig. 4, it can be seen that complement inhibitor proteins are shown
At insect cell surface (Fig. 3).
4) virus titer of the recombinant virus after animal blood serum inactivation of complement
In order to detect antagonism of the different recombinant viruses to serum complement, healthy Swine serum is taken respectively to be divided into two parts：One
By 56 DEG C of inactivation 30min, another is not inactivated part.By two parts of serum respectively with 37 DEG C of recombinant baculovirus incubation 60min, so
Afterwards its virus titer is measured using Endpoint Dilution Method.Viral survival rate is non-inactivation treatment group/inactivation treatment group.The result shows that
The recombinant virus of displaying pFc survival ability in Swine serum is most strong (Fig. 4).
5) immunological indices detect：Serum specific antibody level detects and serum neutralizing antibody level detection such as table 1
Shown in table 2；Immunological Markers for Cell-Mediated Immunity detects：Serum cytokines IFN-γ assay is as shown in table 3.
1 antibody level of serum of table detects
2 serum neutralizing antibody level of table detects
IFN-γ level detects in 3 serum of table
The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications,
Equivalent substitute mode is should be, is included within the scope of the present invention.
Claims (3)
1. a kind of preparation method of pig IgG 1Fc recombinant baculovirus as swine fever vaccine carrier, it is characterised in that：Including with
Lower step：
1) clone pig IgG Fc genes and E 2 gene of Classical Swine Fever are connect with baculoviral surface display vector, are obtained recombination and are turned
Transfer body；
2) recombinant transfer vector obtains recombinant baculovirus Bacmid carriers by Tn7 swivel bases；
3) Bacmid liposome method transfection insect cells are recombinated, culture supernatant is collected, obtain recombinant baculovirus；
4) recombinate shape virus infection insect cell collects cell and does Western blot analyses and Laser Scanning Confocal Microscope analysis；
5) recombinant baculovirus does the detection of complement antagonistic activity；
6) recombinant baculovirus swine fever carrier does vaccine potency inspection in pig body；
Pig IgG Fc gene orders are the SEQ ID NO in sequence table in the step 1):1；
E 2 gene of Classical Swine Fever sequence is the SEQ ID NO in sequence table in the step 1):2；
The expression of E 2 gene of Classical Swine Fever is controlled by CMV promoter in the step 1)；
Baculoviral surface display vector is VSVG pseudotyping baculovirals in the step 1)；
The TM of gp64 is substituted using the TM and CTD of VSVG for the baculoviral surface display vector that the recombinant baculovirus uses
And CTD, improve the transduction efficiency to mammalian cell and the antagonistic ability to serum complement.
2. preparation method of the pig IgG 1Fc recombinant baculovirus according to claim 1 as swine fever vaccine carrier,
It is characterized in that：The literary californica nuclear polyhedrosis virus genome of Bacmid carriers silver containing clover in the step 2).
3. preparation method of the pig IgG 1Fc recombinant baculovirus according to claim 1 as swine fever vaccine carrier,
It is characterized in that：Insect cell is fall army worm gonad cell sf9 in the step 3).
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| CN201810938346.4A CN109652450A (en) | 2015-07-10 | 2015-07-10 | One boar vaccine carrier |
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| CN106519041B (en) * | 2016-11-24 | 2019-07-05 | 唐山怡安生物工程有限公司 | The construction method and its preparation method and application of pig immune globulin Fc segment and swine fever E2 fusion protein in Chinese hamster ovary celI strain |
| CN108059685B (en) * | 2018-01-25 | 2022-03-25 | 中国农业科学院兰州兽医研究所 | Pig foot-and-mouth disease virus A-type Fc polypeptide vaccine and preparation method and application thereof |
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