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Construction and application of recombinant peste des petits ruminants virus expressing fused exogenous epitope N protein

A technology of Peste des petits ruminants and viruses, applied in antiviral agents, viruses/phages, medical preparations containing active ingredients, etc., can solve the problem of inability to distinguish naturally infected animals from immunized animals

Inactive Publication Date: 2013-07-31
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The commonly used PPRV vaccine strain Nigeria 75 / 1 is obtained by multiple passages and attenuated on Vero cells. It is a safe and economical vaccine. However, the defect of this vaccine is that it cannot distinguish naturally infected animals from immunized animals[5]

Method used

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  • Construction and application of recombinant peste des petits ruminants virus expressing fused exogenous epitope N protein
  • Construction and application of recombinant peste des petits ruminants virus expressing fused exogenous epitope N protein
  • Construction and application of recombinant peste des petits ruminants virus expressing fused exogenous epitope N protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Construction of recombinant virus fused with foreign epitope

[0034] The plasmid pCAGGS-N was used as a template, and Epitope-F and HA-R were used as primers for PCR amplification. The PCR product was double digested with SacI and KpnI and cloned into pCAGGS between the SacI and KpnI restriction sites to construct pCAGGS- N HA Plasmid and sequenced; using plasmid pCAGGS-N as template, Epitope-F and FLAG-R as primers for PCR amplification, the PCR product was double digested with SacI and KpnI and then cloned into pCAGGS between the SacI and KpnI restriction sites , Construct pCAGGS-N FLAG Plasmid and sequenced; using pCAGGS-N as template, Epitope-F and 5B19-R1 as primers for PCR amplification, the PCR product was digested with SacI and cloned into pBlueScrip II KS(+) vector (purchased from Stratagene) Between SacI and EcoR V sites, construct pBlue-5B19 1 Plasmid, then pBlue-5B19 1 The plasmid was used as a template, and Epitope-F and 5B19-R2 were used as prime...

Embodiment 2

[0035] Example 2: Rescue of recombinant virus expressing fusion foreign epitope N protein

[0036] The infectious clone plasmid pNg75 / 1-GFP / N HA Vero cells were co-transfected with the helper plasmids pCAGGS-N, pCAGGS-P, and pCAGGS-L to rescue the virus. The specific test method refers to the literature [6], and the rescued virus is named rPPRV / GFP / N HA (The coding nucleic acid sequence is shown at positions 1116-17936 in SEQ ID No. 1). The same method rescued recombinant virus rPPRV / GFP / N FLAG (The coding nucleic acid sequence is shown in position 1116-17933 in SEQ ID No. 2) and rPPRV / GFP / N 5B19 (The coding nucleic acid sequence is shown at positions 1116-17957 in SEQ ID No. 3). In order to identify the effects of three foreign epitopes on the function of N protein, the virus rescue efficiency was used to verify whether the function of N protein was affected. The expression of GFP was observed 4-6 days after virus rescue, the result ( figure 2 ) Shows: First, use the helper pl...

Embodiment 3

[0037] Example 3. RT-PCR identification of recombinant virus

[0038] The infectious clone plasmid pNg75 / 1-GFP / N HA Co-transfected Vero cells with the helper plasmids pCAGGS-N, pCAGGS-P, and pCAGGS-L to rescue the virus. Refer to the literature for specific test methods [6]. Rescue the recombinant virus rPPRV / GFP / N fused with the HA epitope HA . In order to determine whether the HA epitope was successfully introduced into the carboxyl end of the N gene, the harvested rPPRV / GFP / N HA RNA was extracted from virus liquid and rPPRV / GFP virus liquid, and RT-PCR was performed with NP-rtpcr-F and NP-rtpcr-R as primers. The amplified fragment was the same size as the fragment amplified by RT-PCR of the control rPPRV / GFP with the same primers. Further sequencing analysis showed that the HA epitope had been successful at the carboxyl end of the N protein. The same method detected that FLAG and 5B19 epitopes were successfully introduced into the carboxyl end of the N protein.

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Abstract

The invention relates to the field of immune marking vaccines and in particular relates to construction and an application of recombinant peste des petits ruminants virus expressing fused exogenous epitope N protein.

Description

Technical field [0001] The invention relates to the field of immunolabeled vaccines. Specifically, the present invention relates to the construction and application of a recombinant Peste des ruminants virus expressing a fused foreign epitope N protein. Background technique [0002] Peste des petits ruminants (PPR) is an acute and severe infectious disease caused by Peste des petits ruminants virus (PPRV) of the measles virus genus of the Paramyxoviridae family; it is clinically characterized by fever, stomatitis, and diarrhea It is characterized by pneumonia. Serious harm to small ruminants such as goats and sheep, causing significant economic losses to the animal husbandry [1]. PPR was introduced into my country's western border areas in 2007, causing a local epidemic and posing a serious threat to my country's animal husbandry industry. Peste des petits ruminants virus genome is a single-stranded negative-stranded RNA with no segmentation, with a genome length of 15948bp. ...

Claims

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Application Information

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IPC IPC(8): C12N7/01A61K39/155A61P31/14C12R1/93
Inventor 步志高陈伟业
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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