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Pig breeding and respiratory syndrome recombined adenovirus and vaccine

A recombinant adenovirus and respiratory syndrome technology, applied in the direction of recombinant DNA technology, inactivation/attenuation, botanical equipment and methods, etc., to achieve broad application prospects and long-lasting protection

Inactive Publication Date: 2004-12-15
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

There is no report of recombinant adenovirus constructed into porcine reproductive and respiratory syndrome virus PRRSV

Method used

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  • Pig breeding and respiratory syndrome recombined adenovirus and vaccine
  • Pig breeding and respiratory syndrome recombined adenovirus and vaccine
  • Pig breeding and respiratory syndrome recombined adenovirus and vaccine

Examples

Experimental program
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Embodiment Construction

[0037] (1) Construction of Genetic Engineering Body—PRRSV Recombinant Adenovirus (rAd-GP5)

[0038] 1. Amplification, cloning, identification and sequencing analysis of PRRSV GP5 gene

[0039] 1.1 Design and synthesis of PCR primers According to the gene sequence of the PRRSV American standard strain ATCC VR-2332, a pair of specific primers for the GP5 gene of the PRRSV S1 strain (GeneBank accession number AF090173) were designed, and the 5' ends of the primers contained restriction endonuclease cutting site. Primers were synthesized by Shanghai Institute of Cell Biochemistry.

[0040] AdGP5.1: 5-GCC GGT ACC ACC ATG TTG GAG AAA TGC TTG AC-3

[0041] AdGP5.2: 5-GCA CTC GAG CTA AGG ACG ACC CCA TTG TTC-3.

[0042]1.2 S1 strain infects the extraction of total RNA of Marc-145 cells (commonly known and public) and inoculates the cytopathy of PRRSV strain S1 isolates reaching about 75% when freezing and thawing once to collect the cells, centrifuging at 4000rpm for 10min to precip...

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Abstract

The present invention relates to pig reproduction and respiratory syndrome virus recombined adenovirus and vaccine, and belongs to the field of biological high-tech. Through RT-PCR process to proliferate whole PRRSV GP5 sequence, cloning the gene sequence to the shuttle vector pShuttle-CMV of adenovirus carrier system, cotransforming colibacillus BJ5183 strain together with the skeleton vector of adenovirus carrier system to obtain recombinant plasmid, transfecting HEK293-A cell to obtain recombinant adenovirus and plaque purification, and RT-PCR and indirect immunofluorescence technique inspection, the recombinant adenovirus rAd-GP5 expressing PRRSV GP5 protein is constituted. The recombinant adenovirus can set ahead the expression of PRRSV GP5 protein and raise the expression amount to simulate the immune protecting reaction of body effectively.

Description

1. Technical field [0001] The porcine reproductive and respiratory syndrome virus (PRRSV) recombinant adenovirus and vaccine of the invention belong to the high-tech field of biotechnology, and are specially used to strengthen the immune protection of pigs against porcine reproductive and respiratory syndrome virus and reduce the economic loss of pig farms. 2. Technical background [0002] Recombinant adenovirus genetic engineering technology has been widely used in human gene therapy, and many recombinant adenoviruses used in human gene therapy have entered the third phase of clinical trials. Because the recombinant adenovirus has a wide range of hosts, can infect many types of cells, can express human and non-human proteins, and will not insert into the host chromosome, has a low reverse mutation rate, and can be replicated to a high titer. The GP5 protein is a glycoprotein of about 25 kD encoded by ORF5 of porcine reproductive and respiratory syndrome virus PRRSV, and the...

Claims

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Application Information

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IPC IPC(8): A61K39/235A61K48/00C12N7/08C12N15/09C12N15/33C12N15/85C12N15/861
Inventor 姜平李玉峰
Owner NANJING AGRICULTURAL UNIVERSITY
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