Porcine circovirus II-type recombinant baculovirus as well as preparation method and application thereof

A technology for recombining baculovirus and porcine circovirus, applied in the fields of biotechnology and virology, can solve the problems of little prospect and low value-added titer

Active Publication Date: 2013-05-29
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The value-added titer of PCV2 on cells is very low, and the prospect of developing traditional inactivated vaccines and attenuated vaccines is not great

Method used

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  • Porcine circovirus II-type recombinant baculovirus as well as preparation method and application thereof
  • Porcine circovirus II-type recombinant baculovirus as well as preparation method and application thereof
  • Porcine circovirus II-type recombinant baculovirus as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Construction of a recombinant baculovirus QP-Ac-PCV3 ORF2 expressing the immunogenic gene ORF2 gene of artificially modified synthetic PCV2

[0063] (1) Construction of recombinant baculovirus QP-Ac-PCV3ORF2

[0064] 1. Artificial modification and synthesis of the main immunogenic gene ORF2 of PCV2

[0065] Referring to the ORF2 gene sequence of the PCV2b isolate (GenBank No.GU450327), the PCV2 ORF2 gene was artificially synthesized according to the codon preference, and its nucleotide sequence is shown in SEQ ID NO.1.

[0066] 2. Construction of baculovirus transfer vector pFBDPHm3ORF2

[0067] Using the pFBDPHmHNM1P10eGFP plasmid (Qian Ping, a recombinant baculovirus expressing the HA-NA-M1 gene of the artificially modified and synthesized influenza A H1N1 virus, patent publication number CN101624580A, patent number ZL200910063217.6) as the backbone, through NcoI and KpnI enzymes pFBDPHmHNM1P10eGFP was cut, and the large fragment was recovered to obtain pFB...

Embodiment 2

[0090] Embodiment 2: the preparation of recombinant baculovirus QP-Ac-PCV3ORF2 inactivated vaccine

[0091] 1. Inactivated vaccine configuration

[0092] The recombinant baculovirus obtained in Example 1 was inoculated with a multiplicity of infection of 3-5pfu / cell (the insect cell High Five of suspension culture TM , purchased from Invitrogen) sf9 cells, collected cells and supernatant after 72 hours, processed by a cell disruptor, centrifuged to remove cell debris, inactivated by 7mM binary ethyleneimine (BEI) for 36-48 hours, added an equal amount of sulfur Sodium sulfite was neutralized and emulsified with oil adjuvant to prepare inactivated vaccine, which was stored at 4°C for later use.

[0093] 2. Inspection of technical indicators of recombinant baculovirus QP-Ac-PCV3ORF2 inactivated vaccine

[0094] (1) Physical shape

[0095] Appearance This product is milky white homogeneous emulsion.

[0096] The dosage form is oil emulsion. Take a clean straw to suck up a sma...

Embodiment 3

[0104] Example 3: Safety and immune efficacy experiments of recombinant baculovirus QP-Ac-PCV3ORF2 inactivated vaccine

[0105] (1) Safety test of recombinant baculovirus QP-Ac-PCV3ORF2 inactivated vaccine of the present invention

[0106] 1. Newborn piglet safety test

[0107] Take 4 batches of inactivated vaccines prepared in the laboratory, take 3 bottles from each batch, mix them evenly, select healthy 1-day-old newborn piglets, and inoculate 10 pigs in each batch, and inject 2 times the dose of 4mL circovirus oil emulsion into each muscle Inactivated vaccine, another 4 pigs were used as blank control. Before the vaccination and 1 week after the vaccination, the body temperature of all piglets was measured twice a day and the growth status, spirit and appetite of the piglets were observed for 14 consecutive days. The body temperature rose by 0.2-0.5°C on the day of vaccination, and returned to normal the next day, with normal spirit and appetite, and no adverse reaction...

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Abstract

The invention discloses porcine circovirus II-type recombinant baculovirus as well as a preparation method and application thereof. ORF2 gene is artificially synthesized by referring to a PCV2b isolated strain ORF2 gene sequence; the synthesized ORF2 gene is connected to pFBDPHmHNM1P10eGFP plasmid by adopting the plasmid as a framework vector, so that a baculovirus transfer vector pFBDPHm 30RF2 is obtained. The baculovirus transfer vector pFBDPHm30RF2 is mixed with DH10Bac escherichia coli competent cells, and the positive bacterial colony is selected to obtain a recombinant rod granule rBac-PVR30RF2; the rod granule is transferred with a sf9 cell to obtain the recombinant baculovirus QP-Ac-30RF2. The recombinant baculovirus can be used for efficiently expressing the PCV20RF2 protein and forming virus-like particles. The VLP which is expressed and packaged by the recombinant baculovirus disclosed by the invention is used for preparing inactivated vaccine, and the organism is induced to generate specific immunity response after a 28-day-aged piglet is immunized, and the pig body can be completely protected from virulent attacks of the porcine circovirus.

Description

technical field [0001] The invention relates to the fields of biotechnology and virology. Specifically relate to a kind of porcine circovirus type 2 recombinant baculovirus, also relate to a kind of preparation method of porcine circovirus type 2 recombinant baculovirus, also relate to a kind of porcine circovirus type 2 recombinant baculovirus in the preparation of porcine circovirus type 2 recombinant baculovirus Application in inactivated vaccines for viral infections. The recombinant virus of the invention is used to express the artificially modified porcine circovirus type 2 ORF2 gene and assemble into porcine circovirus type 2-like virus particle VLP, which can be used to prepare a safe circovirus vaccine. Background technique [0002] Porcine circovirus type 2 (porcine circovirus 2, PCV2) is a non-enveloped single-stranded negative-sense DNA virus discovered in the early 1990s, belonging to the genus Circovirus. PCV2 can cause a variety of diseases such as piglet we...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/34C12N15/866A61K48/00A61P31/20
Inventor 钱平李祥敏彭波陈焕春
Owner HUAZHONG AGRI UNIV
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