CN105039406A - Method for preparing vaccine vectors for swine from swine IgG1 Fc recombinant baculoviruses - Google Patents
Method for preparing vaccine vectors for swine from swine IgG1 Fc recombinant baculoviruses Download PDFInfo
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Abstract
The invention discloses a method for preparing vaccine vectors for swine from swine IgG1 Fc recombinant baculoviruses, and belongs to the field of biological medicine. The method includes connecting cloned swine IgG Fc genes and swine fever virus E2 genes to baculovirus surface display vectors to obtain recombinant transfer vectors; enabling the recombinant transfer vectors to acquire recombinant baculovirus Bacmid vectors by means of Tn7 transposition; enabling the recombinant baculovirus Bacmid vectors to carry out transfection on insect cells by the aid of a liposome process, collecting culture supernatants to acquire recombinant baculoviruses, infecting the insect cells by the recombinant baculoviruses, and collecting cells to carry out Western blot analysis and confocal microscopic analysis on the cells; enabling recombinant baculovirus swine fever vectors to carry out vaccine efficacy tests on the swine. The method has the advantages that surface display swine IgG Fc recombinant baculoviruses prepared by the aid of the method is high in survival ability in swine serum and can be prevented from being removed by serum complements, and accordingly the contents of effective antigens of vaccine immunity can be increased.
Description
Technical field
The invention belongs to biomedicine field, be specifically related to the preparation method of a kind of pig IgG 1Fc recombinant baculovirus as pig vaccine carrier.
Background technology
The virus of baculovirus (Baculovirus) to be a kind of with insect be unique host, can be used as biotic pesticide or as expression vector mass expressing external albumen in insect cell, prepares vaccine.Research finds, the recombinant baculovirus carrying mammalian promoter in mammalian cell can start the expression of downstream foreign gene but virus can not be rised in value in mammalian cell, little to cytotoxicity, successful cell of transduceing can stablize the also effective expression foreign gene that goes down to posterity, mammalian cell has better posttranslational modification than insect cell to protein, and the protein structure given expression to is closer to native protein.Therefore, baculovirus can be used as a kind of novel mammalian cell gene transfer vector, for expression alien gene and as a kind of gene therapy vector, has great potential, day by day receives the concern of people.
Baculovirus can enter some cell in mammalian body, but can not copy in these cells.Researchist's utilize this feature to be transform as instrument that one is carried gene, and carry out certain modification processing, be transformed into the carrier that orientation is carried, carried gene, medicine is arrived the position of specifying.But, baculovirus is a foreign peoples after all concerning body, body has immunization to it, this is unfavorable for cell entry appointed part, in order to address this problem, just introduce Complement inhibition molecule in Baculovirus Gene inside, the introducing of Complement inhibition molecule can suppress body to the immunne response of recombinant virus, make baculovirus recombinant chou in transmitting procedure through serum by the danger eliminated.
Baculovirus Surface Display System is the novel eukaryote surface display system of one that developed recently gets up, because of it, to have exogenous sequences length range of choice large, value-added speed is fast, the advantages such as the original activity of foreign protein can be kept well, be widely used in new generation vaccine development, gene transfer and gene therapy at present, show complicated eukaryotic protein, built the fields such as the preparation of polypeptide libraries and antibody library and monoclonal antibody.Gp64 is the basis of current Baculovirus Surface Display System, the distinctive structural protein of budding pattern virus, also be peplomer albumen (peplomerprotein), be entrenched in after maturation on virus envelope, have dimer, tripolymer, the tetramer 3 kinds of existing waies.Present most popular baculovirus display systems is AcMNPV and BmNPV, the mode built inserts foreign gene between the signal peptide and maturation protein of its gp64, foreign gene and gp64 protein fusion expression, fusion rotein is held because the excision of signal peptide forms N after processing, just be entrenched in after this protein maturation on cytolemma or virus envelope, such foreign gene is just shown cell or virus surface, can obtain the baculovirus particle of expressing differential protein through simple screening process.
Oral herpes viral G protein (VesicularstomatatisvirusGprotein, VSVG) for number of different types and tissue-derived mammalian cell, there is good versatility, significantly can strengthen some to the insensitive mammalian cell strain of baculovirus as the transduction efficiency of CHO, NIH3T3 etc., but can cytotoxicity be increased and impel cytogamy after VSVG modification baculovirus.Subsequently, VSV-GED (vesicular stomatitis virus ectodomain, by 21 Amino acid profiles, comprises trans-membrane region and cytoplasmic tail) amalgamation and expression in baculovirus envelope protein gp64, then can be ignored on the impact that cell causes by researchist.
Immunoglobulin Fc is not only the significant points determining Ig immunologic function, and be IgG keep in vivo compared with long half-lift major cause.Because IgFc has the effect of stabilize proteins, it is used to merge with multiple protein, ites is desirable to strengthen protein stability with this, extends its Half-life in vivo.IgFc can form many polymerizable moleculars, makes recombinant protein have stronger antigen bonding force, simultaneously IgFc have antigen presenting cell acceptor and easily absorb by antigen presenting cell, thus can be used for the immunne response ability improving small molecule antigens.Baculovirus surface display IgFc can increase the combination of itself and antigen presenting cell.
Summary of the invention
The object of the invention is to for deficiency of the prior art, provide a kind of pig IgG 1Fc recombinant baculovirus as the preparation method of pig vaccine carrier.
The present invention realizes especially by following technical scheme: a kind of pig IgG 1Fc recombinant baculovirus, as the preparation method of pig vaccine carrier, is completed by following steps:
1) clone pig IgGFc gene and E 2 gene of Classical Swine Fever, is connected with baculovirus surface display vector, obtains recombinant transfer vector;
2) recombinant transfer vector obtains recombinant baculovirus Bacmid carrier by Tn7 swivel base;
3) to recombinate Bacmid liposome method transfection insect cell, collect culture supernatant, obtain recombinant baculovirus, observation of cell pathology is also extracted viral DNA and is done PCR qualification;
4) recombinate shape virus infection insect cell, collecting cell is Westernblot and analyzes 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 at pig body.
Described step 1) in the sequence of pig IgG Fc gene be SEQIDNO:1 in sequence table; The aminoacid sequence of the pig IgG Fc of its correspondence is SEQIDNO:3;
Described step 1) in the sequence of E 2 gene of Classical Swine Fever be SEQIDNO:2 in sequence table; The aminoacid sequence of the Pestivirus suis E2 of its correspondence is SEQIDNO:4.
Described step 1) in the expression of E 2 gene of Classical Swine Fever by the control of CMV promoter;
Described step 1) in shaft-like virus surface display carrier be VSVG pseudotyping baculovirus.
Described step 2) in Bacmid carrier containing clover silver civilian californica nuclear polyhedrosis virus genome.
Described step 3) in insect cell be fall army worm gonad cell sf9.
Described step 4) in the step analyzed of Westernblot be: collect the insect cell of virus infection, mix with albumen sample-loading buffer with after cell pyrolysis liquid process, draw supernatant after boiling 10min and do SDS-PAGE electrophoresis; Pvdf membrane is immersed in 100% methyl alcohol, after 10s, moves to distilled water wash 5min, in the most rearmounted transfer printing damping fluid, mixing 10min; After protein electrophorese terminates, electroporation is utilized to be imprinted on pvdf membrane by protein transduction; By pvdf membrane good for transfer printing to close 1h containing 50g/L skimmed milk room temperature, clean 3 times with lavation buffer solution; With dilution His label mouse monoclonal antibody 4 DEG C at shaking table overnight incubation, the next day clean 3 times with appropriate washings; Add the sheep anti-mouse igg antibody of the HRP mark of dilution, under room temperature after shaking table effect 1h, clean 3 times with appropriate washings; With distilled water cleaning, take out pvdf membrane, slightly dry, dark place carries out chemiluminescence reaction (ECL), and equivalent ECL reagent I:II=1:1, covers after pvdf membrane reacts 1min, carries out X-ray development and analyzes.
Described step 4) in the step of Laser Scanning Confocal Microscope analysis be: in 6 well culture plates, put into sterilized slide glass, cell is first inoculated on aseptic slide glass, and cell access amount is that every hole adds 1 × 10
6; After cell adsorption 1h, inhale and abandon substratum, add recombinant virus and infect under MOI=10; Inhale after 48h and abandon substratum, then add the methyl alcohol of 1mL: the mixed solution of acetone=1:1 fixes 5min under-20 DEG C of refrigerators, adds 1mLPBS damping fluid shaking speed 50r/min afterwards again and cleans 2 times, each 5min; At 37 DEG C, 30min is reacted with the 20mL/L bovine serum albumin of 1mL; Add 1mLPBS damping fluid shaking speed 50r/min and clean 3 times, each 5min; Next add His antibody (1:100) and react 1h at 37 DEG C, to demarcate the IgGFc of surface display; Add after 1mLPBS damping fluid shaking speed 50r/min cleans 3 times, then react 1h at the rabbit anti-mouse igg (1:100) 37 DEG C adding FITC mark; Add after 1mLPBS damping fluid shaking speed 50r/min cleans 3 times, observe under Laser Scanning Confocal Microscope, the cytolemma of display protein can be dyeed by FITC and present green fluorescence.
Described step 5) in the method that detects of complement antagonistic activity be: adopt health pig precaval vein blood separation of serum, serum is divided into two parts: a through 56 DEG C of deactivation 30min, another part not deactivation; Two parts of serum are hatched 60min with recombinant baculovirus 37 DEG C respectively, then utilizes Endpoint Dilution Method to measure its virus titer; Viral survival rate is non-inactivation treatment group/inactivation treatment group.
Described step 6) in vaccine potency inspection step be: double-negative for antigen-antibody health pig is divided into 4 groups: the recombinant baculovirus swine Fever Vaccine group of not showing IgGFc: 10
8pfu/mL recombinant baculovirus 2mL; Show the recombinant baculovirus swine Fever Vaccine group of IgGFc: 10
8pfu/mL recombinant baculovirus 2mL; Hog cholera lapinised virus vaccine group: 1 part/2mL; PBS group: PBS2mL; Immune programme for children is that swinery is observed after one week and carries out immunity, and humoral immunization and cell immunization experiments analysis are carried out in 7d, 14d blood sampling before immunity and after immunity; After observing immunity swinery whether there will be apocleisis, spirit depressed, generate heat, tremble, hemorrhage, diarrhoea, the untoward reaction such as constipation; Humoral immunization is analyzed: utilize ELISA kit to detect antibody horizontal; Neutralizing antibody level is detected with reference to OIE handbook; Cellular immunization is analyzed: utilize ELISA kit to detect IFN-γ level.
The present invention has following advantage and effect relative to prior art:
An advantage of the present invention is, the baculovirus surface display vector of use utilizes TM and CTD of VSVG (VSVG-ED) to substituted for TM and CTD of gp64, improves the transduction efficiency to mammalian cell and the antagonistic ability to serum complement;
Another advantage of the present invention is, utilizes the antagonistic ability of pFc raising to serum complement shown, and is conducive to improving immune efficacy with the combination of antigen presenting cell;
Be even more important, have no adverse reaction after recombinant baculovirus immune animal, and effectively can stimulate generation humoral immunization and cellular immunization.
Accompanying drawing explanation
Below in conjunction with in the detailed description of accompanying drawing, above-mentioned feature and advantage of the present invention will be more obvious, wherein:
Fig. 1. recombinant baculovirus transfer vector structural representation.
Fig. 2 .WesternBlot analyzes pFc protein expression result figure.
Fig. 3. Laser Scanning Confocal Microscope analyzes the position result figure of pFc in cell, and wherein, A represents natural light picture, and B represents FITC fluorescence picture, C representative superposition picture.
Fig. 4. recombinant virus is to the antagonistic action result figure of serum complement deactivation.
Embodiment
By the following examples and accompanying drawing illustrate the preparation of surface display pig IgG Fc recombinant baculovirus of the present invention as pig vaccine carrier further.Following examples just illustrate that helping those skilled in the art understands further, 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. the structure of baculovirus surface display vector and checking
With AmMultiBac plasmid for template, increase GP64SP and GP64TM-CTD respectively, and between GP64SP and GP64TM-CTD, add 6 × His label and NheI, SalI, SacI, EcoRI tetra-restriction enzyme sites so that detect and the insertion of foreign gene; After GP64SP and GP64TM-CTD being inserted successively PFBDM transfer vector p10 promotor; With pDsred-Monomer-C1 plasmid for template, after amplification CMV-dsred fragment inserts PFBDM transfer vector ph promotor; In order to verify whether pSur-gp64 carrier successfully constructs, enhanced green fluorescence protein EGFP being inserted in multiple clone site and checks.
2. gene clone
VSVG-ED gene, obtains for primer PCR increases with VSVGTM-F and VSVGTM-R for template with pMD2.G carrier; PUC57-pFc plasmid is synthesized by Jin Weizhi company; Raq gene, obtains for primer PCR increases with E2-F and E2-R for template with CSFV Strain Shimen virus cDNA.
3. the structure of the baculovirus recombinant plasmid of surface display pFc
By TM and the CTD district of gp64TM and the CTD district VSVG of pSur-gp64 replace and complement inhibitor pFc gene clone is entered carrier P10 promotor downstream multiple clone site in obtain pSur-VSVG-ED-pFc recombinant baculovirus plasmid.Swine fever raq gene is cloned in the multiple clone site under CMV promoter and obtains final recombinant baculovirus plasmid pSur-VSVG-ED-pFc-CMV-E2.
4.Tn7 swivel base obtains recombinant baculovirus plasmid
CaCl
2legal system is for SW106 (AmMultiBac) competent cell.When being cultured to OD600=0.25, add the L-arabinose induction that final concentration is 0.1%, continue to be cultured to OD600=0.5, ordinary method prepares competent cell.Add pSur-VSVG-ED-pFc-CMV-E2 to transform, 32 DEG C of shaking culture 8h, the LB be coated with containing Kan/Tet/Gm/IPTG/X-Gal is dull and stereotyped, and 32 DEG C of constant temperature culture 18 ~ 24h occur to blue hickie, picking hickie also to be rule confirmation again, and hickie is verified with each gene primer PCR again.
In the present invention, the primer sequence information is as shown in table 1:
The primer sequence information used in table 1 the present invention
5. recombinant baculovirus plasmid BacmidDNA transfection sf9 cell
(1) in 6 orifice plates, sf9 insect cell 9 × 10 is added in each hole
5cells, substratum 2mLGrace ' s insect perfect medium is 0.5 × final concentration (50 μ g/mL penicillin, 50 μ g/mL Streptomycin sulphates) containing dual anti-concentration.Cell is the logarithmic phase cell of cultivation 3 ~ 4d, and vigor is greater than 97%.Put 1h in 28 DEG C of incubators.
(2) get 2 1.5mLEppendorf pipes and be designated as A, B, add the Grace's insect perfect medium nutrient solution of 100 μ L antibiotic-frees respectively, in A pipe, add the restructuring Bacmid of 5 μ L, mixing; 6 μ L lipofectamine (Cellfectin) are added, mixing (notice that liposome is the suspended substance of a grease, may precipitate, put upside down pipe before using 6 ~ 8 times, be mixed even) in B pipe.A pipe liquid is poured into B pipe, fully mix, put room temperature 45min, make it form Cellfectin-DNA mixture.
(3) get the sf9 monolayer cell that adherent growth is good, abandon old nutrient solution, wash 1 time with Grace ' the s insect perfect medium nutrient solution of antibiotic-free.
(4) get above-mentioned Cellfectin-DNA mixture, add 0.8mL not containing antibiotic Grace's insect perfect medium, mix.From cell, sucking-off discards the substratum of cleaning, and the liposome mixed and recombinant DNA mixture are covered cell, puts 28 DEG C and cultivates 6h.
(5) sucking-off discards mixture, adds and continues to cultivate containing antibiotic Grace ' s insect perfect medium nutrient solution, every day observation of cell form and fluorescence performance thereof.Observe cytopathy and do following judgement: 1. transfection early lesion, 25% ~ 50% cell dia increases or karyon is full of cell; 2. transfection pathology in mid-term, cell stops growing, occur vesica in cell, occur that viral inclusion body or cell come off from culture dish; 3. transfection advanced lesions, there is cracking in cell.Collecting cell, analyzes the situation of expressing protein.Collect culture supernatant as former seed culture of viruses, packing, puts-20 DEG C of storages, uses as lower subinfection sf9 cell simultaneously.
6. the PCR qualification of recombinant baculovirus
Get the cell culture 2mL of virus, multigelation 3 times, the centrifugal 15min of 12000r/min, gets supernatant 437.5 μ L, adds 12.5 μ L Proteinase Ks (20mg/mL) and 50 μ LSDS liquid (10%), 37 DEG C of water-bath 30min; Equal-volume phenol/chloroform once, gets supernatant; Equal-volume chloroform is carried again, gets supernatant; Add 1/10 volume NaAc (2mol/L) and 2 times of volume dehydrated alcohols, place 2h at-20 DEG C; At 4 DEG C with the centrifugal 15min of 15000r/min, get precipitation; Once, precipitation, with 30 μ LTE solubilize, is the DNA of extraction, puts-70 DEG C and save backup 70% washing with alcohol.With recombinant baculovirus DNA for template, use universal primer amplifying target genes.
7. recombinant baculovirus propagation and Endpoint Dilution Method measure virus titer
Recombinant baculovirus is inserted suspension culture in the sf9 insect cell of the Grace substratum containing 100mL/L foetal calf serum.Automatically can multiply daughter of virus after virus infection insect cell and be released among substratum, after infecting 3 ~ 4d, collecting the process that supernatant liquor just completes virus quantity amplification.Suspension culture insect cell treats that cell concn reaches 1 × 10
6cells/mL, with the recombinant baculovirus amount infected insect cell of MOI=1 (Multiplicityofinfection, MOI), utilizes virus can copy the object reaching virus quantity amplification in host insect cell.Centrifugal removing cell and cell debris after infection 3 ~ 4d, collect supernatant liquor.Virus concentration utilizes Endpoint Dilution Method (End-pointdilutionmethod).
8. the Westernblot of recombinant protein analyzes
SDS-PAGE electrophoresis: 12% separation gel, pvdf membrane is immersed in 100% methyl alcohol by 5% concentrated glue, after 10s, moves to distilled water wash 5min, in the most rearmounted transfer printing damping fluid, mixing 10min.After protein electrophorese terminates, on the pvdf membrane utilizing Westernblot to be imprinted on by protein transduction to infiltrate with methyl alcohol, at 350mA transfer printing 2.5h.By pvdf membrane good for transfer printing to close containing 50g/L skimmed milk 1 × PBS fluid-tight, to block the reaction of non-specific antigen antibody.1h is acted on, with lavation buffer solution (1.0g/LTween20inPBSBuffer) 10min/ cleaning 3 times under room temperature.Add antibody incubation below.At each reaction adds the primary antibodie 4 DEG C of suitably diluting through 1 × PBSBuffer (containing 1.0g/L skimmed milk) respectively, shaking table sense is spent the night, and the next day cleans 3 times for 10min/ time with appropriate washings.Wash away non-specific combination.Then add and resist through two of the suitable dilution of 1 × PBS liquid (containing 1.0g/L skimmed milk), as HRP marks the anti-pig antibody of rabbit, under room temperature after shaking table effect 1h, with appropriate washings 10min/ cleaning 3 times.Finally with distilled water cleaning, take out pvdf membrane, slightly dry, dark place carries out chemiluminescence reaction (ECL), and equivalent ECL reagent I:II=1:1, covers after pvdf membrane reacts 1min, carries out X-ray development and analyzes.
9. Laser Scanning Confocal Microscope analyzes the performance of recombinant protein on sf9 cytolemma
(1) in 6 well culture plates, put into sterilized slide glass, cell is first inoculated on aseptic slide glass, and cell access amount is that every hole adds 1 × 10
6.
(2), after cell adsorption 1h, inhale and abandon substratum, add recombinant virus and infect under MOI=10.
(3) after 2d, substratum is abandoned in suction, methanol/acetone (1:1) methanol acetone adding 1mL again fixes 5min under-20 DEG C of refrigerators, add 1mLPBS (phosphate-bufferedsaline) shaking speed 50r/min afterwards again and clean 2 times, each 5min.
(4) then at 37 DEG C, 30min is reacted with the 20mL/LBSA of 1mL (bovineserumalbumin).
(5) add 1mLPBS (phosphate-bufferedsaline) shaking speed 50r/min again after and clean 2 times, each 5min.
(6) next add His antibody (1:100) and react 1h at 37 DEG C, to demarcate the PCV-II envelope protein of expression.
(7) after cleaning 3 times with the PBS of 1mL, then add FITC mark rabbit anti-mouse igg (1:100Sigma) 37 DEG C at react 1h.
(8) finally 3 times are cleaned with the PBS of 1mL again.Observe under Laser Scanning Confocal Microscope, the cytolemma of display protein can be dyeed by FITC and present green fluorescence.
10. suppress complement activity to detect
Health pig serum is taked to be divided into two parts: a through 56 DEG C of deactivation 30min, another part not deactivation.Two parts of serum are hatched 60min with recombinant baculovirus 37 DEG C respectively, then utilizes Endpoint Dilution Method to measure its virus titer.Viral survival rate is non-inactivation treatment group/inactivation treatment group.
11. animal immune experiments
Get the negative pig of 8 surrounding CSFV in age antigen-antibodies and be divided into 4 groups at random, often organize 2; 1st group is BV-VSVGED-CMV-E2 vaccine immunity group, and the 2nd group is BV-VSVGED-pFc-CMV-E2 vaccine immunity group, and the 3rd group is hog cholera lapinised virus vaccine immune group, and the 4th group is PBS control group.Wherein inject 1.0 × 10 at 0DPI and 14DPI for the 1st group
8pfu/mL recombinant baculovirus BV-VSVGED-CMV-E2, the 2nd group in 0DPI and 14DPI injection 1.0 × 10
8pfu/mL recombinant baculovirus BV-VSVGED-pFc-CMV-E2, injects 1 part hog cholera lapinised virus vaccine for the 3rd group, and the 4th group of injection equal-volume PBS in contrast.Laboratory animal carries out blood sampling separation of serum at immunity 0,7,14d.Its test item has: (1) take temperature every day and observation period clinical symptom, as depressed in apocleisis, spirit, generate heat, tremble, hemorrhage, diarrhoea, constipation etc.; (2) immunological indices detects: serum specific antibody level detection and serum neutralizing antibody level detection; (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 vectors, reproducible in specific escherichia coli SW106Bac, also can enter in sf9 cell and copy generation recombinant virus, simultaneously expression alien gene albumen.With the BacmidpSur-gp64-pFc transfection insect sf9 cell of special liposome Cellfectin by 0.8 μ g.Namely transfection starts to observe the CPE change of transfectional cell: cell dia obviously increases, and vesica and viral inclusion body appear in cell internal cause and particle increases, part lysis, death.Illustrate in transfection process, by liposome-mediated, there is homologous recombination with baculovirus DNA, create the recombinant baculovirus with infection activity in restructuring BacmidpSur-gp64-pFc in insect cell.
2) Westernblot analyzes
In order to prove the expression of pFc, carry out Westernblot analysis.Primary antibodie is His monoclonal antibody, and two resist the anti-pig IgG of rabbit for A549 mark.The visible complement inhibitor proteins of result is illustrated in insect cell surface (Fig. 2).
3) Laser Scanning Confocal Microscope analyzes the performance of recombinant protein on sf9 cytolemma
In order to prove that pFc gene is illustrated on the cytolemma of insect cell, carry out Laser Scanning Confocal Microscope analysis.Primary antibodie is His monoclonal antibody, and two resist the anti-pig IgG of rabbit for A549 mark.The results are shown in Figure 4, can see that complement inhibitor proteins is illustrated in insect cell surface (Fig. 3).
4) virus titer of recombinant virus after animal serum inactivation of complement
In order to detect the antagonistic action of different recombinant virus to serum complement, health pig serum is taked respectively to be divided into two parts: a through 56 DEG C of deactivation 30min, another part not deactivation.Two parts of serum are hatched 60min with recombinant baculovirus 37 DEG C respectively, then utilizes Endpoint Dilution Method to measure its virus titer.Viral survival rate is non-inactivation treatment group/inactivation treatment group.Result shows, shows the recombinant virus of pFc survival ability the strongest (Fig. 4) in porcine blood serum.
5) immunological indices detects: serum specific antibody level detection and serum neutralizing antibody level detection are as shown in Table 1 and Table 2; Immunological Markers for Cell-Mediated Immunity detects: serum cytokines IFN-γ assay is as shown in table 3.
Table 1 antibody level of serum detects
Table 2 serum neutralizing antibody level detection
IFN-γ level detection in table 3 serum
More than show and describe ultimate principle of the present invention and principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (8)
1. pig IgG 1Fc recombinant baculovirus is as a preparation method for pig vaccine carrier, it is characterized in that: comprise the following steps:
1) clone pig IgGFc gene and E 2 gene of Classical Swine Fever, is connected with baculovirus surface display vector, obtains recombinant transfer vector;
2) recombinant transfer vector obtains recombinant baculovirus Bacmid carrier by Tn7 swivel base;
3) restructuring Bacmid liposome method transfection insect cell, collects culture supernatant, obtains recombinant baculovirus;
4) recombinate shape virus infection insect cell, collecting cell is Westernblot and analyzes 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 at pig body.
2. pig IgG 1Fc recombinant baculovirus according to claim 1 is as the preparation method of pig vaccine carrier, it is characterized in that:
Described step 1) in pig IgG Fc gene order be SEQIDNO:1 in sequence table;
Described step 1) in E 2 gene of Classical Swine Fever sequence be SEQIDNO:2 in sequence table;
Described step 1) in the expression of E 2 gene of Classical Swine Fever by the control of CMV promoter;
Described step 1) in shaft-like virus surface display carrier be VSVG pseudotyping baculovirus.
3. pig IgG 1Fc recombinant baculovirus according to claim 1 is as the preparation method of pig vaccine carrier, it is characterized in that: described step 2) in Bacmid carrier containing clover silver civilian californica nuclear polyhedrosis virus genome.
4. pig IgG 1Fc recombinant baculovirus according to claim 1 is as the preparation method of pig vaccine carrier, it is characterized in that: described step 3) in insect cell be fall army worm gonad cell sf9.
5. pig IgG 1Fc recombinant baculovirus according to claim 1 is as the preparation method of pig vaccine carrier, it is characterized in that: the step that in described step (4), Westernblot analyzes is: the insect cell collecting virus infection, mix with albumen sample-loading buffer with after cell pyrolysis liquid process, draw supernatant after boiling 10min and do SDS-PAGE electrophoresis; Pvdf membrane is immersed in 100% methyl alcohol, after 10s, moves to distilled water wash 5min, in the most rearmounted transfer printing damping fluid, mixing 10min; After protein electrophorese terminates, electroporation is utilized to be imprinted on pvdf membrane by protein transduction; By pvdf membrane good for transfer printing to close 1h containing 50g/L skimmed milk room temperature, clean 3 times with lavation buffer solution; With dilution His label mouse monoclonal antibody 4 DEG C at shaking table overnight incubation, the next day clean 3 times with appropriate washings; Add the sheep anti-mouse igg antibody of the HRP mark of dilution, under room temperature after shaking table effect 1h, clean 3 times with appropriate washings; With distilled water cleaning, take out pvdf membrane, slightly dry, dark place carries out chemiluminescence reaction, and equivalent ECL reagent I:II=1:1, covers after pvdf membrane reacts 1min, carries out X-ray development and analyzes.
6. pig IgG 1Fc recombinant baculovirus according to claim 1 is as the preparation method of pig vaccine carrier, it is characterized in that: described step 4) in the step of Laser Scanning Confocal Microscope analysis be: in 6 well culture plates, put into sterilized slide glass, cell is first inoculated on aseptic slide glass, and cell access amount is that every hole adds 1 × 10
6; After cell adsorption 1h, inhale and abandon substratum, add recombinant virus and infect under MOI=10; Inhale after 48h and abandon substratum, then add the methyl alcohol of 1mL: the mixed solution of acetone=1:1 fixes 5min under-20 DEG C of refrigerators, adds 1mLPBS damping fluid shaking speed 50r/min afterwards again and cleans 2 times, each 5min; At 37 DEG C, 30min is reacted with the 20mL/L bovine serum albumin of 1mL; Add 1mLPBS damping fluid shaking speed 50r/min and clean 3 times, each 5min; Next add His antibody and react 1h at 37 DEG C, to demarcate the IgGFc of surface display; Add after 1mLPBS damping fluid shaking speed 50r/min cleans 3 times, then react 1h at the rabbit anti-mouse igg 37 DEG C adding FITC mark; Add after 1mLPBS damping fluid shaking speed 50r/min cleans 3 times, observe under Laser Scanning Confocal Microscope, the cytolemma of display protein can be dyeed by FITC and present green fluorescence.
7. pig IgG 1Fc recombinant baculovirus according to claim 1 is as the preparation method of pig vaccine carrier, it is characterized in that: described step 5) in the method that detects of complement antagonistic activity be: gather health pig precaval vein blood separation of serum, serum is divided into two parts: a through 56 DEG C of deactivation 30min, another part not deactivation; Two parts of serum are hatched 60min with recombinant baculovirus 37 DEG C respectively, then utilizes Endpoint Dilution Method to measure its virus titer; Viral survival rate is non-inactivation treatment group/inactivation treatment group.
8. pig IgG 1Fc recombinant baculovirus according to claim 1 is as the preparation method of pig vaccine carrier, it is characterized in that: described step 6) in vaccine potency checking procedure be: be divided into 4 groups to carry out immunity double-negative for antigen-antibody sodium selenite: the recombinant baculovirus swine Fever Vaccine group of not showing IgGFc: 10
8pfu/mL recombinant baculovirus 2mL; Show the recombinant baculovirus swine Fever Vaccine group of IgGFc: 10
8pfu/mL recombinant baculovirus 2mL; Hog cholera lapinised virus vaccine group: 1 part/2mL; PBS group: PBS2mL; Immune programme for children is that swinery is observed after one week and carries out immunity, and humoral immunization and cell immunization experiments analysis are carried out in 7d, 14d blood sampling before immunity and after immunity; After observing immunity swinery whether there will be apocleisis, spirit depressed, generate heat, tremble, hemorrhage, diarrhoea, constipation untoward reaction; Humoral immunization is analyzed: utilize ELISA kit to detect antibody horizontal; Neutralizing antibody level is detected with reference to OIE handbook; Cellular immunization is analyzed: utilize ELISA kit to detect IFN-γ level.
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| 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 |
| CN112135630A (en) * | 2018-09-19 | 2020-12-25 | 巴伊沃爱普有限公司 | Swine fever vaccine composition and preparation method thereof |
| CN112135630B (en) * | 2018-09-19 | 2024-02-27 | 巴伊沃爱普有限公司 | Swine fever vaccine composition and preparation method thereof |
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