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Recombinant rhabditiform plasmid and application thereof to expression of PCV3 Cap protein and vaccine

A recombinant plasmid and rod-shaped technology, applied in the biological field, can solve the problems of difficult to estimate the impact of pig farming, no effective prevention and control measures for PCV3, etc., and achieve the effect of good antigenicity

Pending Publication Date: 2019-11-12
ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] PCV3 is a new type of circoviridae virus, its impact on the pig industry is difficult to estimate, but there are no effective prevention and control measures for PCV3

Method used

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  • Recombinant rhabditiform plasmid and application thereof to expression of PCV3 Cap protein and vaccine
  • Recombinant rhabditiform plasmid and application thereof to expression of PCV3 Cap protein and vaccine
  • Recombinant rhabditiform plasmid and application thereof to expression of PCV3 Cap protein and vaccine

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Embodiment 1 Amplification, cloning and identification analysis of PCV3 Cap gene

[0045] Using the plasmid containing the target gene PCV3 Cap as a template, two pairs of primers (Table 1) for amplifying the PCV3 Cap gene were used to amplify the target gene. 40s at ℃, 30s at 72℃, 5min at 72℃, 4℃∞, 35 cycles. PCV3-Cap2: 95°C for 5min, 95°C for 30s, 65°C for 25s, 72°C for 30s, 72°C for 5min, 4°C∞, 35 cycles. The target fragment was separated and recovered by 1% agarose gel electrophoresis, cloned into the pEASY-Blunt vector, and transformed into Trans5α competent cells, spread on a solid LB plate, cultured upside down at 37°C for 12 hours, and picked a single clone strain Shake culture in liquid LB for 12 hours, extract the plasmids and name them pEPC1 and pEPC2, and identify them by double-enzyme digestion with EcoRI and NotI, XhoI and KpnI respectively, and further confirm by Sanger sequencing.

[0046] Use primer shown in table 1 to carry out the amplification of P...

Embodiment 2

[0048] Construction and Identification of Embodiment 2 Recombinant Transfer Plasmid

[0049] The correctly sequenced pEPC1 and pEPC2 were digested with enzymes, respectively connected to the pFBD Dual vector, and named pFBD-P2C, the plasmids were extracted by conventional molecular biology methods, and identified by double digestion with EcoRI and NotI, XhoI and KpnI respectively.

[0050] The plasmids and expression vectors pFBD that were correctly identified by sequencing and containing PCV3-Cap1 and PCV3-Cap2 genes were digested with corresponding enzymes respectively, and connected to pFBD in sequence to construct a shuttle plasmid pFBD-P2C containing two PCV3Cap genes (such as Figure 4A shown). The enzyme digestion results were as follows: Figure 4B As shown, the plasmid pFBD-P2C can cut out a single gene fragment ( Figure 4B , lanes 2, 3) and a fragment containing two genes ( Figure 4A , lane 4), indicating that the plasmid was constructed successfully. DNA seque...

Embodiment 3

[0051] Example 3 Construction and Identification of Recombinant Bacmid-P2C

[0052] Transformation of plasmid pFBD-P2C into DH10Bac TM Competent cells, and spread on the blue-white spot screening plate containing tetracycline (10 μg / mL), kanamycin sulfate (50 μg / mL), gentamicin (7 μg / mL), IPTG / X-Gal, Inverted culture at 37°C for 48h. Pick the white colony and add it to the SOC medium, shake it for 3 hours, carry out PCR identification of the bacterial liquid according to the general primers shown in Table 1, and extract the correctly identified strain from the bacmid, and name it Bacmid-P2C.

[0053] Utilize the general primers in Table 1 to carry out PCR verification on the Bacmid-P2C bacterial solution, the results are as follows Figure 5 As shown, the primers Puc / M13-R and PHF were used to amplify to obtain a band of about 1400bp, and the primers Puc / M13-F and P10R were used to amplify to obtain a band of about 2400bp, which was consistent with the expected results, indi...

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Abstract

The invention relates to the technical field of biology, in particular to a recombinant rhabditiform plasmid and application thereof to expression of PCV3 Cap protein and a vaccine. A PCV3 Cap gene iscorrectly inserted into a baculovirus vector, a pFBD-P2C plasmid is constructed, a shuttle plasmid is obtained, the shuttle plasmid is transferred into SF9 insect cells by a lipofection method, recombinant baculovirus is obtained, the PCV3 Cap protein is successfully expressed by an insect expression system, and the molecular weight of the expressed protein is about 23 KDa; and indirect immunofluorescence tests and Western Blot show that Cap protein can be expressed correctly by a baculovirus expression system, and the Cap protein has good antigenicity.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant bacmid and its application in expressing PCV3 Cap protein and vaccine. Background technique [0002] Porcine circovirus (PCV) is a single-stranded circular non-enveloped DNA virus belonging to the genus Circovirus in the family Circoviridae and is one of the smallest animal viruses discovered so far. Before the discovery of porcine circovirus type 3 (PCV3) in 2016, PCV was divided into two types: porcine circovirus type 1 (PCV1) and porcine circovirus type 2 (PCV2). serotype. PCV1 was first discovered in PK15 cells in 1974; PCV2 is one of the main pathogens causing porcine multisystemic wasting syndrome (PMWS) in weaned piglets, and has strong pathogenicity. In 2016, Palinski et al. used metagenomic sequencing technology to discover a new virus from sick pigs in a commercial pig farm in the United States that had an outbreak of porcine dermatitis and nephropathy syndr...

Claims

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Application Information

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IPC IPC(8): C12N15/866C12N15/34
CPCC07K14/005C12N15/86C12N2710/14143C12N2750/10051C12N2800/105
Inventor 金宁一李昌许汪杜寿文姜宇航王茂鹏田明尧鲁会军李霄郝鹏飞宋利娜
Owner ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE
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