CN1242065C - Pig breeding and respiratory syndrome recombined adenovirus and vaccine - Google Patents

Abstract

The present invention relates to a porcine reproductive and respiratory syndrome virus (PRRSV) recombinant adenovirus and a vaccine, which belongs to the technical field of a new and high biotechnique. A PRRSV GP5 whole gene order is enlarged by a RT-PCR technique, and is cloned into a shuttle vector pShuttle-CMV of an adenovirus vector system; a recombinant plasmid is obtained by using the shuttle vector of the adenovirus vector system and a frame vector pAdEasy-1 of the adenovirus vector system to cotransform a colibacillus BJ5183 strain; a recombinant adenovirus is obtained by using the recombinant plasmid to transfect an HEK293-A cell, and plaque purification is carried out successfully; the recombinant adenovirus rAd-GP5 which expresses PRRSV GP5 protein successfully is constructed by using the RT-PCR technique and an indirect immunofluorescence technique. The expression of the PRRSV GP5 protein can be advanced and expression quantity can be raised in the recombinant adenovirus, which can effectively stimulate the immunologic and protective reaction of a body. The recombinant adenovirus vaccine has the advantages of security of a subunit vaccine, antigen proliferation ability of a low virulent vaccine and ability of stimulating the omnidirectional immunologic response of the body, and therefore, the recombinant adenovirus vaccine has a wide development and application prospect.

Description

Recombinant Adenoviruses and Vaccines of Porcine Reproductive and Respiratory Syndrome

1. Technical field

The porcine reproductive and respiratory syndrome virus (PRRSV) recombinant adenovirus and vaccine of the invention belong to the high-tech field of biotechnology, and are specially used to strengthen the immune protection of pigs against porcine reproductive and respiratory syndrome virus and reduce the economic loss of pig farms.

2. Technical background

Recombinant adenovirus genetic engineering technology has been widely used in human gene therapy, and many recombinant adenoviruses used in human gene therapy have entered the third phase of clinical trials. Because the recombinant adenovirus has a wide range of hosts, can infect many types of cells, can express human and non-human proteins, and will not insert into the host chromosome, has a low reverse mutation rate, and can be replicated to a high titer. The GP5 protein is a glycoprotein of about 25 kD encoded by ORF5 of porcine reproductive and respiratory syndrome virus PRRSV, and the antibody induced by it has neutralizing activity. The GP5 protein expressed by baculovirus can provide 50% protection to pregnant sows. In addition, the piglets born by immunized sows immunized with GP5 and GP3 expressed by baculovirus are PRRSV seronegative at weaning. After immunization of animals with GP5 plasmid under the control of cytomegalovirus promoter, specific neutralizing antibodies against GP5 were produced in pigs and mice. The monoclonal antibody test proves that the neutralizing activity of the monoclonal antibody against GP5 is stronger than that of the monoclonal antibody against GP4, and the GP5 protein can induce cell apoptosis. At present, there is no report of recombinant adenovirus constructed into porcine reproductive and respiratory syndrome virus PRRSV.

3. Contents of the invention

Technical Problems The purpose of this invention is to develop a PRRSV recombinant adenovirus and vaccine that can effectively control porcine reproductive and respiratory syndrome (PRRS), so as to make a new breakthrough in the current situation where PRRS is difficult to prevent and control.

Technical scheme Embodiment of the present invention is as follows:

The porcine reproductive and respiratory syndrome recombinant adenovirus is characterized in that the recombinant adenovirus rAd-GP5 belongs to: Adenoviridae (Adenoviridae), mammalian adenovirus (Mastadenovirus), human adenovirus species (Humanadenovirus), the The recombinant adenovirus can effectively express the target protein, and no clinical symptoms will appear after the animal is infected with the recombinant adenovirus, so it can be used to express the antigenic protein. The recombinant adenovirus has been preserved in Wuhan Institute of Virology, Chinese Academy of Sciences, with the preservation date: August 15, 2003, and the preservation number is IVCAS6.0200.

The above-mentioned porcine reproductive and respiratory syndrome recombinant adenovirus is constructed by the following method:

(1) Amplification and cloning of the GP5 gene A pair of specific primers for the GP5 gene of the PRRSV domestically isolated S1 strain (GeneBank accession number AF090173) was designed according to the gene sequence of the PRRSV American standard strain ATCC VR-2332, and the primer sequence as follows:

AdGP5.1: 5-GCC GGT ACC ACC ATG TTG GAG AAA TGC TTG AC-3

AdGP5.2: 5-GCA CTC GAG CTA AGG ACG ACC CCA TTG TTC-3

Using the cDNA product of the S1 strain as a template, the whole GP5 gene of the PRRSV domestic isolate S1 was amplified by the primers. The GP5 gene was cloned into the shuttle vector pShuttle-CMV, and then identified by enzyme digestion and PCR to obtain the shuttle vector pShuttle-CMV-GP5 containing the GP5 gene;

(2) Homologous recombination of the shuttle vector pShuttle-CMV-GP5 containing the GP5 gene and the adenovirus backbone vector pAdEasy-1 The identified shuttle vector pShuttle-CMV-GP5 containing the GP5 gene was digested and linearized with the adenovirus backbone vector pAdEasy-1 was transformed into Escherichia coli strain BJ5183 by electroporation method, and under the action of Escherichia coli recombinase, homologous recombination occurred between the shuttle vector and the backbone vector, realizing the transfer of foreign genes into the adenovirus backbone vector, The recombinant adenovirus plasmid containing exogenous gene was obtained through kanamycin and enzyme digestion identification and screening;

(3) Obtaining recombinant adenovirus The identified recombinant adenovirus plasmid was transfected into HEK293-A cells (purchased from the company) by cationic liposome method, and the recombinant adenovirus plasmid was packaged into a complete virus in 293 cells.

Vaccines made with recombinant adenoviruses of porcine reproductive and respiratory syndrome described above:

The purified recombinant adenovirus was continuously passaged on HEK293-A cells for 30 times, and the transcription and expression of porcine reproductive and respiratory syndrome GP5 gene were detected, which proved that the expression of PRRSV GP5 protein was stable. The toxicity of the recombinant adenovirus was stable at 10 ^4.33 TCID ₅₀ /1.0ml.

During the expanded culture process, the purified recombinant adenovirus with preserved PRRSV was used to inoculate HEK293-A cells growing into a single layer at 10 ⁴ TCID ₅₀ . When the cell lesion reaches 70-90%, the virus is collected, and the PRRSV recombinant adenovirus vaccine can be obtained after repeated freezing and thawing once.

Beneficial effect

The present invention proposes for the first time the use of adenovirus vectors to construct PRRSV recombinant adenoviruses, and the recombinant adenoviruses break through the limitations of conventional PRRSV inactivated vaccines and attenuated vaccines. The recombinant vaccine has the replication characteristics of the attenuated vaccine and the safety of the inactivated vaccine, and enables the expression of PRRSV GP5 protein in advance, which changes the characteristics of low and slow production of neutralizing antibodies after PRRSV infection in pigs. Because the recombinant adenovirus vaccine uses human adenovirus serotype 5 carrier, it has no pathogenicity to humans, pigs and other animals, and it is easy to pass the safety evaluation.

Tests have proved that the PRRSV recombinant adenovirus constructed by the present invention has stable expression of foreign proteins, and its potency is stable, and the potency of the seed virus is stable at 10 ^4.33 TCID ₅₀ /1.0ml. The recombinant adenovirus produced neutralizing antibodies against PRRSV through mouse immunization test and neutralization test, and the neutralizing antibody titer was 1:28.

Recombinant adenovirus is not pathogenic to animals, it can be transmitted through a variety of ways, and can infect cohabiting pigs, which is very important in recombinant vaccines. This characteristic can make pigs that have not been immunized or missed immunization also obtain immunization, so that the antibody level of the group is more consistent, and can better resist the attack of exogenous virus. The mice were immunized with the recombinant adenovirus to obtain specific neutralizing antibodies against PRRSV, and the antibodies could neutralize PRRSV in vitro.

Compared with the attenuated and inactivated vaccines of PRRSV currently in use, the virus can replicate and proliferate in pigs and continuously express GP5 protein, so that the body is continuously stimulated by GP5 protein and continuously produces neutralizing antibodies against GP5 protein, so that It can provide long-lasting protection to the pig body, so it has broad application prospects in the prevention and treatment of PRRS.

4. Description of drawings

Figure 1 Schematic diagram of cloning PRRSV GP5 gene into the shuttle vector

Figure 2 Schematic diagram of the homologous recombination of the shuttle vector containing the GP5 gene and the backbone vector of the adenovirus and obtaining the recombinant adenovirus

Figure 3 PRRSV-S1 GP5 gene RT-PCR product

1: 100bp DNA Ladder 2: PCR fragment of GP5 gene of PRRSV-S1 strain

Figure 4 PCR identification results of GP5 gene recombinant plasmid pShuttle-CMV-GP5

1: 100bpDNA Ladder 2-4: PCR products of 3 different recombinant colony plasmids

Figure 5 GP5 gene recombinant plasmid KpnI/HindIII enzyme digestion identification results

1: λDNA/EcoRI+HindI II Marker 2-4: Cloning of three different recombinant plasmids

Figure 6 PCR identification of GP5 gene in pAd-GP5 plasmid

1: 100bp DNA Ladder 2-3: PCR to amplify the GP5 gene fragment from the pAd-GP5 plasmid of different clones

Figure 7 PacI digestion identification of recombinant plasmid

1: λDNA/EcoRI+HindI II Marker 2: PacI digestion of No. 1 recombinant plasmid pAd-GP5 3: PacI digestion of No. 2 recombinant plasmid pAd-GP5

Figure 8 Detection of GP5 protein mRNA expressed by recombinant adenovirus

1: RT-PCR of total RNA extracted from 100bp DNA Ladder 2 293-A 3: RT-PCR of total RNA extracted from 293-A infected with recombinant virus

Figure 9 Indirect immunofluorescence antibody staining results of recombinant adenovirus-infected HEK293-A cells

A: 293-A cells not infected with recombinant adenovirus, no fluorescence B: 293-A cells infected with recombinant adenovirus, with obvious fluorescence

5. Specific implementation methods:

(1) Construction of genetic engineering body-PRRSV recombinant adenovirus (rAd-GP5)

1. Amplification, cloning, identification and sequencing analysis of PRRSV CP5 gene

1.1 Design and synthesis of PGR primers According to the gene sequence of the PRRSV American standard strain ATCC VR-2332, a pair of specific primers for the GP5 gene of the PRRSV S1 strain (GeneBank accession number AF090173) were designed. endonuclease cutting site. Primers were synthesized by Shanghai Institute of Cell Biochemistry.

AdGP5.1: 5-GCC GGT ACC ACC ATG TTG GAG AAA TGC TTG AC-3

AdGP5.2: 5-GCA CTC GAG CTA AGG ACG ACC CCATTG TTC-3.

1.2 Extraction of total RNA from Marc-145 cells (commonly known and public) infected by S1 strain Inoculation of PRRSV strain S1 isolates with cytopathic changes of about 75% and freezing and thawing once to collect cells, centrifuging at 4000rpm for 10min to precipitate cells, discarding supernatant; Add 1.0ml TRIZOL (Invitrogen) reagent to the cell pellet in the bottle, and vortex to mix. The following operations were carried out according to the instructions of the kit.

1.3 Reverse transcription Take 1.0 μl of Oligod(T) _12-18 , 14.0 μl of total cellular RNA, 0.5 μl of 10mM dNTPs, 4.0 μl of 5×reverse transcriptase buffer, and make up to 19.5 μl with DEPC water. After fully mixing the contents, act in a water bath at 65°C for 10 minutes, add 0.5 μl of mouse-derived reverse transcriptase M-MLV (purchased from Biological Company) after cooling, act at 37°C for 1.0 h after thorough mixing, and then act at 95°C for 5 minutes, then Cool quickly on ice to serve as template for PCR reactions.

1.4 Take cDNA 2.0μl, AdGP5.1 1.0μl, AdGP5.2 1.0μl, 10mM dNTPs 1.0μl, Mg ^++- free buffer 5.0μl, 25mM Mg ⁺⁺ 2.0μl, rTaq DNA polymerase 1.0μl, use sterile Make up to a total volume of 50.0 μl with double distilled water. Perform the reaction on a PCR machine. The cycle parameters were pre-denaturation at 94°C for 3 min; denaturation at 94°C for 45 s, annealing at 58°C for 45 s, and extension at 72°C for 60 s, for a total of 35 cycles; and extension at 72°C for 10 min. PCR products were subjected to 1% (g/ml) agarose gel electrophoresis.

1.5 Cloning of RT-PCR products

1.5.1 Recovery and purification of PCR products

After the PCR product was subjected to agarose gel electrophoresis, the agarose block containing the target band was cut out under ultraviolet light, and the target segment in the gel was recovered with a DNA rapid recovery and purification kit. The specific operation steps were carried out according to the instructions of the Huashun Gel Recovery Kit.

1.5.2 Digestion and purification of PCR products The total volume of the digestion reaction is 50 μl. 10×M buffer 5.0 μl, PCR recovered fragment 20.0 μl, KpnI 1.0 μl, XhoI 1.0 μl, make up to a total volume of 50.0 μl with sterile double distilled water. 37°C for 3.0h, and then perform agarose gel electrophoresis to determine whether the digestion is complete. After the enzyme digestion is complete, use the agarose gel recovery kit to recover the digested product, and the method is the same as above.

1.5.3 pShuttle-CMV digestion and purification The total volume of the digestion reaction is 50 μl. 10×M buffer 5.0 μl, pShuttle-CMV plasmid 20.0 μl, KpnI 1.0 μl, XhoI 1.0 μl, make up to a total volume of 50.0 μl with sterile double distilled water. Simultaneously set the KpnI and XhoI single enzyme digestion control of the shuttle vector. 37°C for 3.0h, and then perform agarose gel electrophoresis to determine whether the digestion is complete. After the enzyme digestion is complete, use the agarose gel recovery kit to recover the digested product, and the method is the same as above.

1.5.4 Ligation reaction between target fragment and pShuttle-CMV Enzymatic digestion and recovery of 2.0 μl of PCR product, 4.0 μl of vector pShuttle-CMV digestion and recovery of product, 1.0 μl of 10× ligase buffer, 1.0 μl of T4 DNA ligase, and sterile Make up the total volume to 10.0 μl with double distilled water. After mixing the products, they were ligated overnight at 14°C, and the vector self-ligation control was set at the same time. The ligation product was transformed into DH5α.

1.5.5 Preparation and transformation of Escherichia coli DH5α competent bacteria

For the preparation of DH5α-competent bacteria, see molecular cloning.

1.5.6 Extraction and identification of recombinant plasmids

Plasmid extraction uses conventional alkaline lysis method. The extracted recombinant plasmid is identified by PCR and double enzyme digestion, and the target band we need can be seen in both PCR and double enzyme digestion identification.

1.6 cDNA sequence analysis

According to the sequencing primer sequence of pShuttle-CMV vector, a pair of sequencing primers were synthesized by Shanghai Institute of Cell Biochemistry. The primer sequence was Adseq1: 5-GGT ATA TAT AAG CAG AGC TG-3; Adseq2: 5-GTG GTA TGG CTG ATT ATGATC AG-3. The sequencing work was assisted by Bao Biological Engineering (Dalian) Co., Ltd. Bidirectional sequencing was performed on a DNA sequencer (ABIPRISMTM377XL DNA Sequencer). And use NCBI BLAST to compare the sequence measured and its deduced amino acid sequence with the nucleotide and protein of the PRRSV GP5 gene in the GeneBank database for homology comparison, and then use Vector NTI and DNAstar software to carry out gene restriction endonuclease and reading frames. It has been proved that the homology between the cloned gene and the S1 strain is very high, and the cloned gene fully complies with the requirement of the vector reading frame.

2 Homologous recombination between the shuttle vector (pShuttle-CMV-GP5) containing the GP5 gene and the adenovirus backbone vector (pAdEasy-1)

2.1 For linearization and purification of pShuttle-CMV-GP5 PmeI, use 5.0 μl of 10×NE buffer4, 0.5 μl of 100×BSA, 30.0 μl of pShuttle-CMV-GP5, 1.0 μl of PmeI, and make up to a total volume of 50.0 μl with double distilled water. After 3.0 hours at 37°C, 2.0 μl was taken for 1% (g/ml) agarose gel electrophoresis. Recovery and purification were carried out using the Mini Gel Recovery Kit. The purification method is the same as above.

2.2 Preparation of Escherichia coli BJ5183 electrotransformation competent bacteria Pick a fresh BJ5183 colony on a streptomycin (30μg/ml) resistance plate and inoculate it in 10ml LB medium containing 30μg/ml streptomycin, 37°C 200rpm Shake culture overnight; the next day, inoculate a new LB medium containing streptomycin at 1/1000 volume, shake culture to make A550≈0.8, put the culture on ice bath for 1.0h; centrifuge at 3000rpm at 4°C for 10min; Resuspend the cells with the sterile ice-cold WB solution of the initial bacterial volume; (WB=10% ultrapure glycerol, 90% distilled water v/v); centrifuge at 3000rpm for 30min; resuspend the cells with the initial volume of WB solution again Centrifuge; centrifuge at 3000rpm for 30min, pour off the excess supernatant, resuspend the bacteria in the remaining 1/500 volume of WB solution, aliquot 40μl per tube, and store at -70°C.

2.3 Co-transform BJ5183 bacteria with pShuttle-CMV-GP5 and pAdEasy-1 Take out two tubes of BJ5183 electrotransformation competent bacteria stored at -70°C, and slowly melt them in an ice bath; take the purified pAdEasy-1 vector and linearized pShuttle-CMV -GP5 plasmids were mixed in a sterilized 500 μl centrifuge tube; the mixture of linearized pShuttle-CMV-GP5 plasmid and pAdEasy-1 vector and linearized pShuttle-CMV-GP5 were added to two tubes of BJ5183 electrotransformation competent bacteria as Control; transfer the BJ5183 bacteria after adding the plasmid into the ice-cold electrode cup, so that the droplet is suspended in the center of the metal plate of the electrode cup; perform electrotransformation immediately, and the parameters of the electrotransformer are set to: 25kV, 25μF, 200Ω; Immediately add 1.0ml of room temperature LB solution to one tube, and fully resuspend the bacteria; shake the resuspended bacteria at 37°C for 1.0h; spread the recombinant bacteria on three kanamycin-resistant plates respectively, and control the bacteria Spread one plate and incubate overnight.

2.4 Selection of recombinant bacteria and plasmid extraction After at least 24 hours of cultivation, pick the smallest colony on the kanamycin-resistant plate and inoculate it in 3.0ml LB medium containing kanamycin, and cultivate overnight; extract the plasmid and dissolve it in 20μl In sterile double distilled water, then carry out 0.8% (g/ml) agarose gel electrophoresis; the suspicious recombinant plasmid is transformed into DH5α competent bacteria, and is coated with kanamycin plate; /ml) agarose gel electrophoresis.

2.5 Identification of recombinant plasmids

2.5.1 PCR of recombinant plasmid The total reaction volume is 25.0 μl, and the extracted plasmid is used as a template. Recombinant plasmid 0.25μl, AdGP5.10.5μl, AdGP5.20.5μl, 2.5mM dNTPs 2.0μl, 25mM Mg ⁺⁺ 1.0μl, 10×Mg ⁺⁺ freebuffer 2.5μl, rTaq enzyme 0.25μl, add water to a final volume of 25.0μl. The PCR cycle parameters are the same as the amplification parameters of the GP5 gene fragment.

2.5.2 Single enzyme digestion of recombinant plasmid: 10×NE Buffer1 2.0μl, 100×BSA 0.2μl, PacI 0.2μl, recombinant plasmid 10.0μl, make up to a total volume of 20.0μl with sterile double distilled water, digest at 37℃ for 10.0h , and then carry out 0.8% (g/ml) agarose gel electrophoresis.

2.6 Purification of recombinant plasmid The identified recombinant plasmid pAdEasy-1-GP5 was transformed into DH5α and spread on the kanamycin resistance plate; pick the colony on the kanamycin plate and inoculate it in 3.0ml containing 50μg/ml kanamycin Shake overnight culture in the plain LB medium; Inoculate the overnight culture into 5.0ml LB medium according to the volume of 2%, shake culture, make OD600≌1.0-1.5; ml sterilized centrifuge tube, discard the supernatant completely. The plasmid purification method was carried out according to the instructions of the Qiagen company plasmid purification kit.

2.7 Linearization of pAdEasy-1-GP5 recombinant plasmid 10×NE buffer1 20.0 μl, 100×BSA 2.0 μl, PacI 1.0 μl, recombinant plasmid 150.0 μl, and then add double distilled water to a total volume of 200.0 μl. Act at 37°C for 12.0 hours, and then perform 0.8% (g/ml) agarose gel electrophoresis. The digested product was extracted with phenol: chloroform, precipitated with ethanol, and then dissolved in sterile double distilled water under sterile conditions to make the plasmid concentration 0.1-1.0 μg/μl.

2.8 Recovery, culture and cryopreservation of HEK293-A cells Gently shake and melt a tube of cells in a 37°C water bath; carefully wash the outer wall with 70% alcohol and put it into an ultra-clean workbench; transfer the cells into a sterile 15ml Add 10% serum DMEM to the centrifuge tube, centrifuge at 600g for 5min to pellet the cells; discard the supernatant, resuspend the cells with 10.0ml 10% newborn calf serum DMEM, and then add it to a 100ml cell bottle; 37 Cultivate in a 5% carbon dioxide incubator. Digest the cells with trypsin at room temperature for 1-3 minutes to separate the cells until a single layer is formed; tap the cell bottle wall to move the cells, and use an inverted microscope to observe whether the cells are rounded and separated; use new nutrient solution to separate the cells as needed bottle. In order to preserve the specific phenotype of a cell subtype, the cell density at which the cell bank is established must be below 50%. Completely digest the healthy growing cells in the logarithmic phase; centrifuge to pellet the cells; use up a small amount of 10% serum DMEM to resuspend the cells; use a hemocytometer to count; use 5% serum DMEM+10%DMSO+40 % newborn bovine serum to make the density of cells per milliliter reach 1×106; put 1.0ml of cells in a 1.8ml cryopreservation tube; put the cryopreservation tube in a low-temperature refrigerator built into a foam box for 24 hours; quickly put the cells in the cryogenic refrigerator Transfer to liquid nitrogen.

2.9 HEK-293A cells transfected with pAdEasy-1-GP5 recombinant plasmid

Transfection was performed on a 24-well plate. On the day before transfection, 5.0× ¹⁰⁴ HEK293-A cells were inoculated in each well of the 24-well plate, and 1.0ml of nutrient solution was used for each well, and the nutrient solution contained 10% newborn bovine serum and 1% penicillin and streptomycin; one day before transfection, take out a tube of newly ordered TransFastTM Transfection Reagent (purchased from the company) to bring it to room temperature, and then add 400.0 μl of room temperature nuclease-free Resuspend the liposome membrane in pure water, vortex for 10s, and store at -20°C; 4 hours before transfection, replace the nutrient solution in the 24-well plate with a new nutrient solution; take out the transfection reagent stored at -20°C, and use After reaching room temperature and vortexing, if the solution is in the upper part of the tube, it can be centrifuged to sink it to the bottom of the tube; first add 1979 μl of OPTI-MEM serum-free medium to a sterilized glass bottle, and linearize pAdEasy-l-GP515 .0μl (about 5μg), then add 6.0μl TransFastTM Transfection reagent and vortex immediately; incubate the mixture at room temperature for 10-15min; remove the cell plate from the carbon dioxide incubator, discard the supernatant; vortex the mixture quickly again, Add 2.0ml of the mixture into 12 wells of a 24-well plate, shake and mix well, then immediately put the cell plate into a carbon dioxide incubator; after incubating for 1.0h, gently add 1.0ml of complete growth solution at room temperature, and put the cells Put them in a carbon dioxide incubator and observe the cell changes every day.

2.10 Plaque purification of recombinant virus

Inoculate 3.0×105 cells in each well of a 6-well cell plate and culture statically in a 5% carbon dioxide incubator; Inoculate the virus with 1/5 volume (600.0 μl) of the added nutrient solution, absorb at 37°C for 1.5h; add 2.0ml DMEM with 2% newborn bovine serum for 2.0h; completely discard the supernatant, and wash with preheated sterile PBS Twice; 2% agarose after high pressure is placed in a 50°C water bath, 2×DMEM containing 2% penicillin, streptomycin and 4% serum is preheated to 37°C; in a sterile 100.0ml glass bottle Add 10.0ml of 2×DMEM and 10.0ml of 2% agarose to the medium, mix thoroughly and place in a 37°C water bath; add 3.0ml of covering agar to each well of a 6-well plate, spread the plate, and observe plaques; Pick up the agarose block containing plaques with the tip of a sterile pipette and put them into a sterile centrifuge tube, add 200.0 μl of sterile PBS, mash the agar block and freeze-thaw it three times; centrifuge at 12000rpm for 5min; inoculate the harvested virus in a new in a 24-well cell plate.

2.11 Determination of TCID ₅₀ of the recombinant virus According to the method introduced in the virology operation manual, the virus was serially diluted 10 times with the maintenance solution containing 2% serum, 1% penicillin and streptomycin. Then inoculated in 96-well cell culture plates covered with monolayer of HEK293-A cells, inoculated 8 wells for each dilution, and inoculated 100 μl in each well. At the same time, two rows of wells were set to use maintenance solution as a control. Cultivate in a 5% CO2 incubator at 37°C, and observe cell changes every day. And calculate virus TCID50 according to Reed-Muech method. The potency value was 10 ^4.33 TCID ₅₀ /1.0ml.

2.12 RT-PCR detection of transcription of recombinant adenovirus GP5 gene Take out a bottle of HEK293-A cells inoculated with recombinant adenovirus simultaneously and HEK293-A cells not inoculated with recombinant adenovirus, use TRIZOL reagent to extract total RNA according to the product instructions, and press RT-PCR was used to detect GP5 protein mRNA in the above method. It is proved that the recombinant adenovirus has successfully transcribed the PRRV GP5 gene.

2.13 Indirect immunofluorescence experiment HEK293-A cells were inoculated on a 96-well plate; the recombinant adenovirus was diluted with maintenance solution, and the cells were inoculated at two dilutions of 10 ^-2 and 10 ^-3 respectively, and a blank control was set at the same time; Incubate in a 5% carbon dioxide incubator at 37°C for 48-72h; discard the nutrient solution, wash the cells with PBS, then discard the PBS; add 150 μl of 4°C cold acetone (80% acetone, 20% double distilled water) to the 96-well plate In each well of each well, place the cell plate at 4°C for 30 min; discard the acetone, and dry the 96-well plate at room temperature; dilute the serum, and take three dilutions of monoclonal antibody: 1:200, 1:300, and 1:400. The sera of mice immunized with eukaryotic expression plasmids of GP5 were taken at three dilutions of 1:20, 1:40, and 1:80, and each dilution was inoculated into two wells; 50 μl of serum was added to each well; After covering the lid, incubate at 37°C for 30 min in a wet box; remove the diluted serum, invert it on a paper towel to dry the cell plate, wash 6 times with 200 μl of PBS; add 50 μl of 1:100 diluted fluorescently labeled goat antibody Add mouse IgG to each well and incubate at 37°C for 30 min in a wet box; remove the liquid in the well, dry the cell plate on a paper towel, wash 4 times with PBS; observe under a fluorescent microscope.

The results ( FIG. 9 ) showed that: Group B was inoculated with the virus, and the 293 cytoplasm infected with the recombinant adenovirus showed obvious fluorescence, while the control group A was not inoculated with the virus, and no fluorescence was seen without the recombinant adenovirus.

2.14 Mouse Immunization Test of Recombinant Adenovirus

Twenty 8-week-old healthy clean mice were randomly divided into four groups, 5 in each group. One group was set as the control, and the other three groups were given oral, subcutaneous and intramuscular injections. The immunization dose was 10 ⁴ TCID ₅₀ /0.2ml, and the immunization interval was 2 weeks. After four consecutive immunizations, the serum of mice was collected for virus neutralization test. There was no significant difference in neutralizing antibody titers among the three immunization methods, and the neutralizing antibody titer was 1:28.

2.15 Stability test of recombinant adenovirus

The recombinant adenovirus rAd-GP5 was continuously passaged on 293 cells for 30 generations, and the transcription and expression of GP5 protein in rAd-GP5 were detected every 5 generations, and the half-infection amount of the tissue cells was measured at the same time. The results showed that the expression of GP5 protein was stable during the continuous passage of the recombinant adenovirus rAd-GP5, and the half infection amount of the tissue cells did not change.

To sum up, the purified recombinant adenovirus rAd-GP5 was continuously passaged on HEK293-A cells for 30 times, and the transcription and expression of the recombinant adenovirus to GP5 protein gene were examined every 5 passages, and its TCID ₅₀ was determined at the same time. The results proved that the expression of GP5 protein gene by the recombinant adenovirus was stable, and half of the tissue cells were infected stably, and the cytopathic changes appeared regularly after inoculation. The valency of the seed poison was stable at 10 ^4.33 TCID ₅₀ /1.0ml.

The vaccine was prepared by recombinant adenovirus of porcine reproductive and respiratory syndrome, and the purified recombinant adenovirus was continuously passaged on HEK293-A cells for 30 times, and the transcription and expression of porcine reproductive and respiratory syndrome GP5 gene were detected to prove the expression of PRRSV GP5 protein Stablize. The toxicity of the recombinant adenovirus was stable at 10 ^4.33 TCID ₅₀ /1.0ml.

During the expanded culture process, the purified and preserved PRRSV recombinant adenovirus was used to inoculate HEK293-A cells growing into a single layer at 104 TCID50. When the cell lesion reaches 70-90%, the virus is collected, and the PRRSV recombinant adenovirus vaccine can be obtained after repeated freezing and thawing once.

Claims (2)

1. porcine reproductive and respiratory syndrome recombinant adenovirus makes up by the following method and forms:

(1) amplification of GP5 gene, clone have designed a pair of Auele Specific Primer at the domestic strain isolated S1 of PRRSV strain GP5 gene according to the gene order of PRRSV America standard strain ATCC VR-2332, and primer sequence is as follows: AdGP5.1:5-GCC GGT ACC ACC ATG TTG GAG AAA TGC TTG AC-3AdGP5.2:5-GCA CTC GAG CTA AGG ACG ACC CCA TTG TTC-3

CDNA product with the Marc-145 cell of S1 virus strain infection is a template, use the full gene of GP5 that the RT-PCR technology has amplified the domestic strain isolated S1 of PRRSV strain, 5 ' end at primer contains restriction enzyme digestion sites respectively, by these two restriction enzyme sites, the GP5 gene clone is gone among the shuttle vectors pShuttle-CMV, cut with PCR by enzyme then and identify, obtain to contain the shuttle vectors pShuttle-CMV-GP5 of GP5 gene;

(2) containing the shuttle vectors pShuttle-CMV-GP5 of GP5 gene and the shuttle vectors pShuttle-CMV-GP5 that contains the GP5 gene that adenovirus skeleton carrier pAdEasy-1 homologous recombination is identified is transformed among the coli strain BJ5183 by electric method for transformation with adenovirus skeleton carrier pAdEasy-1 behind linearization for enzyme restriction, under the effect of intestinal bacteria recombinase, homologous recombination takes place between shuttle vectors and skeleton carrier, realized foreign gene is changed over to the adenovirus skeleton carrier, obtained to contain the recombinant adenovirus plasmid of foreign gene through the screening of 50g/ml kantlex;

(3) recombinant adenovirus plasmid identified of the acquisition of recombinant adenovirus is by cationic-liposome method transfection HEK293-A cell, and recombinant adenovirus plasmid becomes complete recombinant adenovirus rAd-GP5 in 293 cell internal packings.

2. use the vaccine of the described porcine reproductive and respiratory syndrome recombinant adenovirus of claim 1 rAd-GP5 preparation, comprise: with the recombinant adenovirus rAd-GP5 of purifying continuous passage 30 times on the HEK293-A cell, per 5 generations inspection recombinant adenovirus is transcribed and expression the GP5 protein gene, measures its TCID simultaneously ₅₀, prove that PRRSV GP5 protein expression is stable, connect poison back cytopathy occurrence law, the malicious valency of planting poison is stabilized in 10 ^4.33TCID ₅₀/ 1.0ml;

In enlarging the virus culture process, take the PRRSV recombinant adenovirus of cryopreservation by 10 ⁴TCID ₅₀Inoculation grows up to the HEK293-A cell of individual layer, receives poison when cytopathy reaches 70-90%, once can obtain PRRSV recombinant adenovirus rAd-GP5 vaccine through multigelation.

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