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PCV2 (porcine circovirus 2) recombinant baculovirus-like particle subunit vaccine and preparation method

A recombinant baculovirus and subunit vaccine technology, applied in the field of bioengineering, can solve problems such as lack of highly immunogenic PCV2 vaccine, and achieve the effect of reducing vaccine impurities, high content, and mild animal reactions

Inactive Publication Date: 2018-05-04
北京生科基因科技有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] One of the technical problems to be solved by the present invention is to solve the problem of lack of highly immunogenic PCV2 vaccine in China, and provide a domestically produced PCV2 recombinant baculovirus-like particle subunit vaccine with high immunity, good safety and industrial production Preparation

Method used

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  • PCV2 (porcine circovirus 2) recombinant baculovirus-like particle subunit vaccine and preparation method
  • PCV2 (porcine circovirus 2) recombinant baculovirus-like particle subunit vaccine and preparation method
  • PCV2 (porcine circovirus 2) recombinant baculovirus-like particle subunit vaccine and preparation method

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Experimental program
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Embodiment 1

[0038] The acquisition of embodiment 1PCV2ORF2 recombinant baculovirus

[0039] The plasmid pUC57-PCV2 (synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.) containing the codon PCV2 recombinant baculovirus ORF2 nucleotide sequence was artificially synthesized. The nucleotide sequence of PCV2 ORF2 is GenBank AIT11738.1 sequence. According to the multiple cloning site sequence of the shuttle vector pAcGP67-A, EcoR1 and BglII restriction sites were added to the 5' and 3' ends of the ORF2 sequence, respectively. The PCV2 recombinant baculovirus ORF2 gene synthesized in the present invention has a full length of 713bp, and its specific sequence is shown in SEQ ID NO.1.

[0040] The recombinant plasmid pUC57-PCV2 was digested with EcoR1 / BglII, and the target fragment was purified and recovered. The recovered target fragment was cloned into the shuttle vector pAcGP67-A (BD BiosciencesPharmingen, San Diego, CA, such as figure 1 shown), the recombinant shuttle vector pAcGP67-...

Embodiment 2

[0044] Example 2 Preparation of PCV2 recombinant baculovirus-like particles

[0045] 1. Cell preparation

[0046] Take the frozen Sf9 working cell bank cells and disperse the cells to 75cm 2 In the cell flask, add EX-CELL TM 420 serum-free medium. Cultivate at 27°C for 24 hours, transfer the cells to the culture flask at a dispersion ratio of 1:5, add new serum-free medium, continue culturing at 27°C, and collect the cells. Passage in the same way, and expand the culture until the required number of cells is expanded. to 4×10 5 The cells / ml cell density is connected to the cell bioreactor for full suspension serum-free culture, and cultured at 27°C until the cell density is 2×10 6 cells / ml, it is used for poisoning.

[0047] 2. Virus inoculation, cultivation and harvesting

[0048] Inoculate the Sf9 working bank cells with a density of 2×106 cells / ml in the bioreactor with the virus seed for production at an inoculation amount of MOI=1, and culture the virus in full sus...

Embodiment 3

[0051] Example 3 Preparation of PCV2 Recombinant Baculovirus-like Particle Subunit Vaccine

[0052] 1. Obtaining the virus stock solution

[0053] The Sf9 working bank cells were cultured in full suspension serum-free, when the cell density reached 1.5×10 6 The cells / ml were infected with the recombinant baculovirus, and the virus inoculation volume was MOI=2. Continue the full suspension serum-free culture for 6 days, and directly harvest the cell culture medium, which is the virus stock solution.

[0054] 2. Filtration of virus liquid

[0055] The harvested cell culture supernatant was filtered and sterilized through a 1.0 μm membrane filter system and a 0.22 μm membrane filter system in sequence, and the filtrate was stored at 2-8°C.

[0056] 3. Virus inactivation

[0057] In order to ensure the safety of the PCV2 recombinant baculovirus-like particle subunit vaccine, we inactivated the recombinant baculovirus in the virus fluid. Diethyleneimine BEI was used as inactiv...

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Abstract

The invention discloses a PCV2 (porcine circovirus 2) recombinant baculovirus-like particle subunit vaccine and a preparation method thereof. According to the vaccine, an insect taken as a host cell is transfected with a eukaryotic expression vector containing a PCV2 recombinant baculovirus ORF2 gene fragment. The nucleotide sequence of the PCV2 recombinant baculovirus ORF2 gene fragment is as shown in SEQ ID NO. 1, and the eukaryotic expression vector is pAcGP67-A-PCV2. According to the method, the recombinant baculovirus containing the highly immunogenic PCV2 ORF2 nucleotide sequence is constructed by a molecular biology means, and PCV2 capsid protein can be efficiently expressed by the recombinant virus in the Sf9 cell line and assembled into PCV2 virus-like particles with PCV2 surfaceantigens in a cytoplasm. A harvested virus stock solution is processed by a filtration system and inactivated, and supplemented with an adjuvant, and the PCV2 recombinant baculovirus-like particle subunit vaccine with high immunity and good safety is prepared.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a PCV2 recombinant baculovirus-like particle subunit vaccine and a preparation method thereof. Background technique [0002] Porcine circovirus (Porcine circovirus, PCV) belongs to Circoviridae (Circoviridae) genus Circovirus (Circovirus), non-enveloped single-stranded circular DNA virus, is currently the smallest DNA virus found. According to genetic composition and viral antigenicity, PCV is divided into two serotypes, PCV1 and PCV2. Among them, PCV1 is the non-pathogenic type, and PCV2 is the pathogenic type. [0003] Porcine circovirus disease (PCVD) is an infectious disease of pigs caused by PCV2 virus. It is an important immunosuppressive disease of pigs. The disease occurs seriously in pigs and has caused huge economic losses to the global pig industry. PCV2 is considered to be the main pathogen causing weaned piglet multisystemic wasting syndrome (PWM...

Claims

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Application Information

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IPC IPC(8): A61K39/12C12N15/866C12N15/34A61P31/20
CPCA61K39/12A61K2039/5258C07K14/005C12N15/86C12N2710/14022C12N2710/14043C12N2750/10034
Inventor 任红涛刘健鹏邹权
Owner 北京生科基因科技有限公司
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