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Fluorescently-labeled porcine reproductive and respiratory syndrome virus and building method and application thereof

A respiratory syndrome and fluorescent labeling technology, applied in the field of genetic engineering, can solve the problems of poor passaging ability, inconvenient observation, inconvenient PRRSV in-depth research, etc., and achieve the effect of complete structure and good stability

Active Publication Date: 2017-06-13
陕西嘉海和正生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the mechanism of PRRSV virulence enhancement is still unclear so far. Therefore, it is very necessary to establish an efficient and economical HP-PRRSV reverse genetics technology operation platform. The economic significance of PRRSV as a virus vaccine carrier is very huge
However, the existing PRRSV basic materials used for research are not easy to observe during the application process, and their transmission ability is poor, which brings inconvenience to the in-depth study of PRRSV

Method used

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  • Fluorescently-labeled porcine reproductive and respiratory syndrome virus and building method and application thereof
  • Fluorescently-labeled porcine reproductive and respiratory syndrome virus and building method and application thereof
  • Fluorescently-labeled porcine reproductive and respiratory syndrome virus and building method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0052] The construction method of the fluorescently labeled porcine reproductive and respiratory syndrome virus provided in this embodiment includes the following steps, and its construction strategy is as follows: figure 1 as shown ( figure 1 Middle: A is the structural model diagram of the fluorescent marker expression cassette, B is the structural model diagram of the transformation of the infectious cloning plasmid, C is the structural model diagram of the infectious cloning plasmid connected with the fluorescent marker expression cassette), and the regulatory sequence A fluorescent marker expression cassette that drives the expression of a fluorescent marker gene, introducing restriction sites (AsiS I and Mlu I endonucleases) at both ends of the fluorescent marker expression cassette, and then in the infectious cloning plasmid (pBAC-SD16 FL ) between the N protein of ) and the 3'-terminal non-coding sequence (3'-UTR) by introducing the same restriction site (AsiS I and Ml...

Embodiment 2

[0064] The method for constructing a fluorescently labeled porcine reproductive and respiratory syndrome virus provided in this example includes the following steps.

[0065] 2.1 Construction of fluorescent marker expression cassette

[0066] 2.1.1 Primer design: upstream primer (AsisI-TRS3-up):

[0067] 5'-GCGATCGCTAACCATAGTGTATAATA-3',

[0068] Downstream primer (TRS3-EGFP-lower):

[0069] 5'-CTCGCCCTTGCTCACCATTGCTGAAAATCATGAAGC-3',

[0070] Upstream primer (TRS3-EGFP-up):

[0071] 5'-GCTTCATGATTTTCAGCAATGGTGAGCAAGGGCGAG-3'. pBAC-SD16 FL As a template, use primers AsisI-TRS3-up and TRS3-EGFP-lower to amplify the gene sequence containing the first 18 bases of TRS3 sequence and EGFP gene; then use primers TRS3-EGFP-up and EGFP-MluI-Lower to amplify The target fragment containing the last 18 base sequences of TRS3 sequence and EGFP gene sequence. Finally, by nested PCR technology, using primers AsisI-TRS3-up and EGFP-MluI-Lower, using NEB Q5Hot Start High-Fidelity DNA Po...

Embodiment 3

[0077] The method for constructing a fluorescently labeled porcine reproductive and respiratory syndrome virus provided in this example includes the following steps.

[0078] 3.1 Construction of fluorescent marker expression cassette

[0079] 3.1.1 Primer design: upstream primer (AsisI-TRS4-up):

[0080] 5'-GCGATCGCTTGACCAGTGTCTACGCC-3',

[0081] Downstream primer (TRS4-EGFP-lower):

[0082] 5'-CTCGCCCTTGCTCACCATTCCAGGTGAAACCAATTG-3',

[0083] Upstream primer (TRS4-EGFP-up):

[0084] 5'-CAATTGGTTTCACCTGGAATGGTGAGCAAGGGCGAG-3'. pBAC-SD16 FL As a template, use primers AsisI-TRS4-up and TRS4-EGFP-lower to amplify the gene sequence containing the TRS4 sequence and the first 18 bases of the EGFP gene; then use primers TRS4-EGFP-up and EGFP-MluI-Lower to amplify The target fragment containing the last 18 base sequences of TRS4 sequence and EGFP gene sequence. Finally, by nested PCR technology, using primers AsisI-TRS4-up and EGFP-MluI-Lower, using NEB Q5Hot Start High-Fidelit...

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Abstract

The invention provides a fluorescently-labeled porcine reproductive and respiratory syndrome virus and a building method and application thereof and belongs to the technical field of gene engineering. The building method is characterized in that a fluorescence labeling expression box with a fluorescence labeling gene is connected to a connection site on an infectious clone plasmid to obtain the visual fluorescently-labeled porcine reproductive and respiratory syndrome virus, and the connection site is located between the downstream of an N protein gene sequence and the upstream of a 3' end noncoding sequence. The building method is low in operation difficulty, high in operability and capable of acquiring the fluorescently-labeled porcine reproductive and respiratory syndrome virus capable of stably expressing fluorescent protein and provides a powerful tool for further carrying out novel virus vaccine development and basic researches related to the porcine reproductive and respiratory syndrome virus.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a fluorescently labeled porcine reproductive and respiratory syndrome virus and its construction method and application. Background technique [0002] Porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV) belongs to Nidovirales (Nidovirales), Arteriviridae (Arteriviridae), Arterivirus (Arterivirus), is an enveloped single-stranded positive chain The RNA virus has a genome length of about 15.4kb, a poly(A) tail sequence at the 3' end of the genome, and a cap-like leader sequence at the 5' end. Its genome contains 9 overlapping open reading frames (openreading frame, ORF). Among the structural proteins of PRRSV, GP5, M, and N proteins are major structural proteins, while GP2a, 2b, GP3, and GP4 are minor structural proteins. [0003] Compared with classical genetic methods, reverse genetics technology has the a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/65C12N7/01C12Q1/70A61K39/12A61P31/14C12R1/93
CPCA61K31/12C12N7/00C12N15/65C12N15/85C12N2770/10021C12N2770/10034C12Q1/025
Inventor 王承宝
Owner 陕西嘉海和正生物技术有限公司
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