CN109652450A - One boar vaccine carrier - Google Patents

Abstract

The invention discloses a kind of swine fever vaccine carriers, belong to biomedicine field.Preparation method of the invention uses clone pig IgG Fc gene and E 2 gene of Classical Swine Fever, connect with baculoviral surface display vector, obtains recombinant transfer vector；Recombinant transfer vector obtains recombinant baculovirus Bacmid carrier by Tn7 swivel base；Bacmid liposome method transfection insect cell is recombinated, culture supernatant is collected, obtains recombinant baculovirus, recombinate shape virus infection insect cell collects cell and does Western blot analysis 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

One boar vaccine carrier

Technical field

The invention belongs to biomedicine fields, and in particular to a boar vaccine carrier.

Background technique

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 taking in mammalian cells 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 to protein than insect cell, 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 is able to enter certain cells in the mammalian body, but not can be carried out duplication 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 is able to suppress body and exempts to recombinant virus Epidemic disease response makes danger of the baculoviral recombinant in transmission process by being destroyed when serum.

Baculovirus Surface Display System is a kind of novel eucaryote surface display system that developed recently gets up, because of it The advantages that big with exogenous sequences length range of choice, value-added speed is fast, can keep 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, building 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, also cries peplomer albumen (peplomer protein), be entrenched in virus after mature 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 building are to be inserted into foreign gene, foreign gene between the signal peptide and maturation protein of its gp64 With gp64 protein fusion expression, the excision after processing because of signal peptide forms N-terminal fusion protein, is just entrenched in after this protein maturation 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 significantly increase some pairs of baculovirals The transduction efficiency of the mammalian cell strain of sense such as CHO, NIH3T3 etc., however cell can be increased after VSVG modifies baculoviral Toxicity simultaneously promotes cell fusion.Then, researcher is by VSV-GED (vesicular stomatitis virus ectodomain, 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 function, and is that IgG keeps longer by half in vivo Decline the phase the main reason for.Since Ig Fc has 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 has antigen presenting cell receptor and is easily absorbed by antigen presenting cell, so as to small for improving The immune response ability of molecular antigen.Baculoviral surface display Ig Fc can increase the combination of itself and antigen presenting cell.

Summary of the invention

It is an object of the invention to aiming at the shortcomings in the prior art, provide a kind of pig IgG 1Fc recombinant baculovirus conduct The preparation method of pig vaccine carrier.

The present invention is realized especially by following technical scheme: a kind of pig IgG 1Fc recombinant baculovirus is carried as pig with vaccine The preparation method of body, is completed by following steps:

1) clone pig IgG Fc gene 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 carrier by Tn7 swivel base；

3) Bacmid liposome method transfection insect cell is recombinated, culture supernatant is collected, obtains recombinant baculovirus, It observes cytopathy and extracts viral DNA and do PCR identification；

4) recombinate shape virus infection insect cell collects cell and does Western blot analysis 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 gene is the SEQ ID NO:1 in sequence table in the step 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:2 in sequence table in the step 1)；Its corresponding pig The amino acid sequence of pestivirus E2 is SEQ ID NO:4.

Control of the expression of E 2 gene of Classical Swine Fever by CMV promoter in the step 1)；

Baculoviral surface display vector is VSVG pseudotyping baculoviral in the step 1).

The literary californica nuclear polyhedrosis virus genome of Bacmid carrier 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 is analyzed in the step 4) are as follows: the insect cell for collecting virus infection uses cell It is mixed after lysate processing with albumen sample-loading buffer, 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, and most postposition transfers in buffer, mixes 10min；Protein After electrophoresis, protein is transferred on pvdf membrane using electroporation；By the pvdf membrane transferred with skimmed milk containing 50g/L Room temperature closes 1h, with washing buffer cleaning 3 times；It is incubated overnight with shaking table at diluted 4 DEG C of His label mouse monoclonal antibody, The next day with appropriate cleaning solution cleaning 3 times；The sheep anti-mouse igg antibody of diluted HRP label is added, at room temperature after shaking table effect 1h, With appropriate cleaning solution cleaning 3 times；Wash with distilled water, pvdf membrane is taken out, is slightly dried, dark place carries out chemiluminescence reaction (ECL), Equivalent ECL reagent I:II=1:1 is covered on after reacting 1min on pvdf membrane, carries out X-ray development analysis.

The step of Laser Scanning Confocal Microscope is analyzed in the step 4) are as follows: 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 every hole is added 1 × 10⁶；After cell adsorbs 1h, inhales and abandons culture medium, 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 speed 50r/min cleaning 2 times later, every time 5min；30min is reacted at 37 DEG C with the 20mL/L bovine serum albumin(BSA) of 1mL；1mL PBS buffer solution shaking speed 50r/ is added Min is cleaned 3 times, each 5min；Next His antibody (1:100) is added and reacts 1h at 37 DEG C, to demarcate surface display IgG Fc；Be added 1mL PBS buffer solution shaking speed 50r/min clean 3 times after, add FITC label rabbit anti-mouse igg (1: 100) 1h is reacted at 37 DEG C；After being added 1mL PBS buffer solution shaking speed 50r/min cleaning 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 that complement antagonistic activity detects in the step 5) are as follows: adopt health pig vena cava anterior blood system from serum, blood Sorting is two parts: portion passes through 56 DEG C of inactivation 30min, another is not inactivated；By two parts of serum respectively with 37 DEG C of recombinant baculovirus It is incubated for 60min, then measures its virus titer using Endpoint Dilution Method；Viral survival rate is non-inactivation treatment group/inactivation treatment group.

The step of vaccine potency is examined in the step 6) are as follows: the double-negative health pig of antigen-antibody is divided into 4 groups: not being opened 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 group: PBS 2mL；Immune programme is observed for swinery and is immunized after a week, before being immunized and rear 7d is immunized, 14d blood sampling carries out humoral immunity It is analyzed with cell immunization experiments；After observation is immune swinery whether will appear anorexia, spirit is depressed, generate heat, tremble, bleeding, diarrhea, The adverse reactions such as constipation；Humoral immunity analysis: antibody level is detected using ELISA kit；It is neutralized referring to the detection of OIE handbook anti- Body is horizontal；Cellular immunity analysis: 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 TM and the CTD (VSVG- of VSVG ED the TM and CTD of gp64) is substituted, improves the transduction efficiency to mammalian cell and the antagonistic ability to serum complement；

It is another advantage of the present invention that improving the antagonistic ability to serum complement using the pFc shown, and 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.

Detailed description of the invention

From detailed description with reference to the accompanying drawing, features described above of the invention and advantage be will be apparent from, in which:

Fig. 1 recombinant baculovirus transfer vector structural schematic diagram.

Fig. 2 .Western Blot analyzes pFc protein expression result figure.

Fig. 3 Laser Scanning Confocal Microscope analyzes position result figure of the pFc in cell, wherein A represents natural light picture, B generation Table FITC fluorescence picture, C represent superposition picture.

The antagonism result figure that Fig. 4 recombinant virus inactivates serum complement.

Specific embodiment

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. the building and verifying of baculoviral surface display vector

Using AmMultiBac plasmid as template, GP64SP and GP64TM-CTD is expanded respectively, and in GP64SP and GP64TM- Tetra- restriction enzyme sites of 6 × His label and NheI, SalI, SacI, EcoRI are added between CTD in order to detect and foreign gene Insertion；GP64SP and GP64TM-CTD are sequentially inserted into after PFBDM transfer vector p10 promoter；With pDsred-Monomer- C1 plasmid is template, and amplification CMV-dsred segment is inserted into after PFBDM transfer vector ph promoter；In order to verify pSur-gp64 Whether carrier constructs success, and enhanced green fluorescence protein EGFP is inserted into multiple cloning sites and is examined.

2. gene cloning

VSVG-ED gene is expanded as primer PCR using VSVGTM-F and VSVGTM-R and is obtained using pMD2.G carrier as template； PUC57-pFc plasmid is synthesized by Jin Weizhi company；Raq gene is with E2-F and E2-R using CSFV crossdrift strain virus cDNA as template Primer PCR amplification obtains.

3. the building of the baculoviral recombinant plasmid of surface display pFc

By the area TM and CTD of the VSVG replacement of the area gp64TM and CTD of pSur-gp64 and by complement inhibitor pFc gene It is cloned into the multiple cloning sites in carrier P10 promoter downstream and obtains pSur-VSVG-ED-pFc recombinant baculovirus plasmid. 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 base obtains 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 conversion, the LB plate of 32 DEG C of shaken cultivation 8h, Tu Han 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 confirmation, hickie is again with each gene primer PCR verifying.

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 transfects sf9 cell

(1) sf9 insect cell 9 × 10 is added in each hole in 6 orifice plates⁵Cells, culture medium is with 2mL Grace ' s elder brother Worm complete medium is 0.5 × final concentration (50 μ g/mL penicillin, 50 μ g/mL streptomysins) containing dual anti-concentration.Cell is training The logarithmic growth phase cell of 3~4d is supported, vigor is greater 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 insect for being separately added into 100 μ L antibiotic-frees is complete The recombination Bacmid of 5 μ L is added in A pipe, mixes for full culture medium culture solution；6 μ L lipofectamines are added in B pipe (Cellfectin), it mixes and (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 pipe, mixes well, sets room temperature 45min, it is mixed to form it into Cellfectin-DNA Close object.

(3) the good sf9 cell monolayer of adherent growth is taken, old culture solution is abandoned, with Grace ' the s insect of antibiotic-free Complete medium culture solution washs 1 time.

(4) above-mentioned Cellfectin-DNA mixture is taken, the not antibiotic Grace's insect of 0.8mL is added and trains 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 insect complete medium culture solution is added and continues to cultivate, Observation cellular morphology and its fluorescence performance daily.It observes that cytopathy is changed into following judgement: 1. transfecting early lesion, 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 is cracked.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 cell With.

6. the PCR of recombinant baculovirus is identified

The cell culture 2mL of virus is taken, multigelation 3 times, 12000r/min is centrifuged 15min, 437.5 μ L of supernatant is taken, 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 mentions again, takes supernatant；1/10 volume NaAc (2mol/L) and 2 times of volume dehydrated alcohols are added, In -20 DEG C of placement 2h；15min is centrifuged with 15000r/min at 4 DEG C, takes precipitating；70% ethanol washing is primary, 30 μ of precipitating The dissolution of LTE solution, the DNA as extracted set -70 DEG C and save backup.Using recombinant baculovirus DNA as template, universal primer is used Amplifying target genes.

7. recombinant baculovirus proliferation and Endpoint Dilution Method measure virus titer

The insect that recombinant baculovirus merging suspension is incubated at the Grace culture medium containing 100mL/L fetal calf serum is thin Born of the same parents sf9.Daughter of virus out can be multiplied after virus infection insect cell automatically and is discharged to culture medium, is 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 Duplication achievees the purpose that virus quantity amplifies in host insect cell.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 of recombinant protein is analyzed

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, mixes 10min.After protein electrophorese, Western blot is utilized Protein is transferred on the pvdf membrane infiltrated with methanol, transfers 2.5h in 350mA.By the pvdf membrane transferred to contain 50g/ L skimmed milk 1 × PBS fluid-tight is closed, to block the reaction of non-specific antigen antibody.1h is acted on, 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 antibody of 1 × PBS Buffer (skimmed milk containing 1.0g/L), the next day to wash in right amount Liquid 10min/ times is washed to clean 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 appropriate cleaning solution 10min/ times cleaning 3 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 reagent I: II=1:1 is covered on 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 membrane

(1) sterilized slide glass is put into 6 well culture plates, cell is first inoculated on sterile glass slide, cell access amount 1 × 10 is added for every hole⁶。

(2) it after cell absorption 1h, inhales and abandons culture medium, recombinant virus is added and is infected at MOI=10.

(3) it is inhaled after 2d and abandons culture medium, then add methanol/acetone (1:1) methanol acetone of 1mL in -20 DEG C of ice 5min is fixed under case, adds 1mL PBS (phosphate-buffered saline) shaking speed 50r/min cleaning 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 speed 50r/min cleaning 2 is added after It is secondary, each 5min.

(6) His antibody (1:100) is next added and reacts 1h at 37 DEG C, to demarcate the circovirus cyst membrane egg of expression It is white.

(7) it after being cleaned 3 times with the PBS of 1mL, adds anti-at 37 DEG C of the rabbit anti-mouse igg (1:100Sigma) of FITC label 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

Take healthy Swine serum 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, its virus titer then 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 group, the 2nd group is BV-VSVGED-pFc-CMV-E2 vaccine immunity group, and the 3rd group is hog cholera lapinised virus epidemic disease Seedling immune group, the 4th group is PBS control group.Wherein 1.0 × 10 are injected in 0DPI and 14DPI for the 1st group⁸Pfu/mL recombination is rod-shaped Viral BV-VSVGED-CMV-E2, the 2nd group in 0DPI and 14DPI injection 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 isometric PBS of the 4th group of injection is as control. Experimental animal is in immune 0,7,14d blood was collected separation serum.Its detection project has: (1) measurement body temperature and observation period face daily Bed symptom, such as anorexia, spirit it is depressed, generate heat, tremble, bleeding, diarrhea, constipation；(2) immunological indices detect: serum is special The detection of heterogenetic antibody level 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) building and observation of recombination bacillary viral vector

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 also can enter duplication in sf9 cell and generate recombinant virus, while expression alien gene albumen. The Bacmid pSur-gp64-pFc of 0.8 μ g is transfected into insect sf9 cell with dedicated liposome Cellfectin.Transfection is opened The CPE for beginning to observe transfection 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 is analyzed

In order to prove the expression of pFc, Western blot analysis is carried out.Primary antibody is His monoclonal antibody, secondary antibody A549 The rabbit-anti pig IgG of label.As a result visible complement inhibitor proteins are shown at insect cell surface (Fig. 2).

3) performance of the Laser Scanning Confocal Microscope analysis recombinant protein on sf9 cell membrane

In order to prove that pFc gene is shown on the cell membrane of insect cell, Laser Scanning Confocal Microscope analysis is carried out.Primary antibody is His monoclonal antibody, secondary antibody are the rabbit-anti pig IgG of A549 label.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 different recombinant viruses to the antagonism of serum complement, healthy Swine serum is taken respectively to be divided into two parts: one Part passes through 56 DEG C of inactivation 30min, another is not inactivated.By two parts of serum respectively with 37 DEG C of incubation 60min of recombinant baculovirus, so Its virus titer is measured using Endpoint Dilution Method afterwards.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: the detection of serum specific antibody level and serum neutralizing antibody level detection such as table 1 With shown in table 2；Immunological Markers for Cell-Mediated Immunity detection: serum cytokines IFN-γ assay is as shown in table 3.

The detection of 1 antibody level of serum of table

The detection of 2 serum neutralizing antibody level of table

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, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention, It should be equivalent substitute mode, be included within the scope of the present invention.

Sequence table

<110>Shaanxi Nowe Li Hua Biotechnology Co., Ltd

Wuhan Zhong Tuokangming Biotechnology Co., Ltd

Trace to the source agricultural development Co., Ltd in Shaanxi

Yellow light east

<120>one boar vaccine carriers

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<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 4

Met Arg Leu Ala Cys Lys Glu Asp Tyr Arg Tyr Ala Leu Ser Ser Thr

1 5 10 15

Asn Glu Ile Gly Leu Leu Gly Ala Gly Gly Leu Thr Thr Thr Trp Glu

20 25 30

Glu Tyr Ser His Asp Leu Gln Leu Asn Asp Gly Thr Val Lys Ala Ile

35 40 45

Cys Val Ala Gly Ser Phe Lys Val Thr Ala Leu Asn Val Val Ser Arg

50 55 60

Arg Tyr Leu Ala Ser Leu His Lys Gly Ala Leu Leu Thr Ser Val Thr

65 70 75 80

Phe Glu Leu Leu Phe Asp Gly Thr Asn Pro Ser Thr Glu Glu Met Gly

85 90 95

Asp Asp Phe Gly Phe Gly Leu Cys Pro Phe Asp Thr Ser Pro Val Val

100 105 110

Lys Gly Lys Tyr Asn Thr Thr Leu Leu Asn Gly Ser Ala Phe Tyr Leu

115 120 125

Val Cys Pro Ile Gly Trp Thr Gly Val Ile Glu Cys Thr Ala Val Ser

130 135 140

Pro Thr Thr Leu Arg Thr Glu Val Val Lys Thr Phe Arg Arg Glu Lys

145 150 155 160

Pro Phe Pro His Arg Met Asp Cys Val Thr Thr Thr Val Glu Asn Glu

165 170 175

Asp Leu Phe Tyr Cys Lys Leu Gly Gly Asn Trp Thr Cys Val Lys Gly

180 185 190

Glu Pro Val Val Tyr Thr Gly Gly Gln Val Lys Gln Cys Lys Trp Cys

195 200 205

Gly Phe Asp Phe Asn Glu Pro Asp Gly Leu Pro His Tyr Pro Ile Gly

210 215 220

Lys Cys Ile Leu Ala Asn Glu Thr Gly Tyr Arg Ile Val Asp Ser Thr

225 230 235 240

Asp Cys Asn Arg Asp Gly Val Val Ile Ser Ala Glu Gly Ser His Glu

245 250 255

Cys Leu Ile Gly Asn Thr Thr Val Lys Val His Ala Ser Asp Glu Arg

260 265 270

Leu Gly Pro Met Pro Cys Arg Pro Lys Glu Ile Val Ser Ser Ala Gly

275 280 285

Pro Val Arg Lys Thr Ser Cys Thr Phe Asn Tyr Ala Lys Thr Leu Lys

290 295 300

Asn Lys Tyr Tyr Glu Pro Arg Asp Ser Tyr Phe Gln Gln Tyr Met Leu

305 310 315 320

Lys Gly Glu Tyr Gln Tyr Trp Phe Asp Leu Asp Val Tyr Pro Tyr Asp

325 330 335

Val Pro Asp Tyr Ala

340

<210> 5

<211> 40

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

ccccaaaacc cgggatgcta cttgtaaatc agtcacacca 40

<210> 6

<211> 41

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

ctagctagca tgatgatgat gatgatgcgc aaaggcagaa t 41

<210> 7

<211> 41

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

ctagctagcg tcgacgagct cgaattcttc atgtttggtc a 41

<210> 8

<211> 39

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

gccggggtac cttaatattg tctattacgg tttctaatc 39

<210> 9

<211> 24

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

ggaattcatc cggtatactg ggct 24

<210> 10

<211> 24

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

cggggtacct tactttccaa gtcg 24

<210> 11

<211> 37

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

gggcgcggat cctagttatt aatagtaatc aattacg 37

<210> 12

<211> 31

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

agatacaagc ttttactggg agccggagtg g 31

<210> 13

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 13

ctagctagca ccatggtgag caaggg 26

<210> 14

<211> 27

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 14

ccggaattcc ttgtacagct cgtccat 27

<210> 15

<211> 33

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 15

gaatccatgc ggctagcctg caaggaagat tac 33

<210> 16

<211> 38

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 16

gtcgacttac acgtccaggt caaaccagta ctgatcat 38

Claims (3)

1. a boar vaccine carrier, it is characterised in that: the preparation method of the carrier the following steps are included:

1) clone pig IgG Fc gene 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 carrier by Tn7 swivel base；

3) Bacmid liposome method transfection insect cell is recombinated, culture supernatant is collected, obtains recombinant baculovirus；

4) recombinate shape virus infection insect cell collects cell and does Western blot analysis 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 order is the SEQ ID NO:1 in sequence table in the step 1)；

E 2 gene of Classical Swine Fever sequence is the SEQ ID NO:2 in sequence table in the step 1)；

Control of the expression of E 2 gene of Classical Swine Fever by CMV promoter in the step 1)；

Baculoviral surface display vector is VSVG pseudotyping baculoviral 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. pig vaccine carrier according to claim 1, it is characterised in that: Bacmid carrier contains lucerne in the step 2) The literary californica nuclear polyhedrosis virus genome of Mu silver.

3. pig vaccine carrier according to claim 1, it is characterised in that: insect cell is meadow in the step 3) Noctuid gonad cell sf9.