Application of porcine circovirus type 2 double-copy infectious clone to indirect ELISA detection method

A technology of infectious cloning and porcine circovirus, applied in the fields of application, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems of genetic instability and low efficiency of virus rescue, achieve sustained and stable genetics, improve Amplification efficiency and accuracy, effect of improving efficiency

Inactive Publication Date: 2017-05-24
广东永顺生物制药股份有限公司
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  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The present invention aims to overcome the disadvantages of low PCV2 virus rescue efficiency and genetic instability, improve virus culture

Method used

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  • Application of porcine circovirus type 2 double-copy infectious clone to indirect ELISA detection method
  • Application of porcine circovirus type 2 double-copy infectious clone to indirect ELISA detection method
  • Application of porcine circovirus type 2 double-copy infectious clone to indirect ELISA detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0068] Embodiment: Carry out virus rescue to porcine circovirus type 2 double-copy infectious clone according to the following steps:

[0069] Step 1: Construction of double-copy infectious clone pEGFP-N1-BD:

[0070] (1) Extraction of PCV2 DNA

[0071] a. Isolate the PCV2 strain from the clinical PMWS symptom pig group, and carry out gene comparison and sequence analysis to it; take 200 μL of the cell culture containing the PCV2-GD strain, add 20 μL Proteinase K solution, and mix;

[0072] b. Add 200 μL of buffer solution GB, mix thoroughly by inversion, place at 70°C for 10 minutes, wait for the solution to become clear and centrifuge briefly to remove the water droplets on the inner wall of the tube cap, add 200 μL absolute ethanol, fully shake and mix for 15 seconds, and briefly centrifuge to remove Water droplets on the inner wall of the pipe wall cover;

[0073] c. Add the solution obtained in the previous step to the adsorption column CB3 provided with the kit, centri...

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Abstract

The invention relates to a porcine circovirus type 2 (PCV2) double-copy infectious clone virus rescue method and application thereof to an indirect ELISA detection method. PCV2 genome DNA is obtained on the basis of clinical separation by adopting a proper gene engineering technology method, specific primers are designed, full-length genes are obtained through polymerase chain reaction (PCR), two copied PCV2 full-length genes are in end-to-end connection to be connected on a pEGFP-N1 vector in series to form PCV2 double-copy whole-gene recombinant plasmid, ST cells are transfected, blind passage is conducted continuously for 60 generations, the infectivity on the ST cells and the virus reproduction stability are authenticated by virtue of a PCR detection method and an indirect immunofluorescence assay (IFA) technology, and foundation is laid for further development of the pathogenesis of a circovirus and research of a diagnosis technology.

Description

technical field [0001] The technology relates to a construction method of a recombinant porcine circovirus type 2 (PCV2) strain and its application in an indirect ELISA detection method, belonging to the field of agricultural biotechnology, specifically the field of a recombinant porcine circovirus type 2 strain. Background technique [0002] Porcine circovirus type 2 (PCV2) is the main pathogen that causes multisystemic wasting syndrome (Postweaning multisystemic wasting syndrome, PMWS) in weaned piglets. Clinically, PMWS is characterized by shortness of breath, diarrhea, anemia, interstitial pneumonia, progressive emaciation and obvious lymph node lesions in weaned piglets. PCV2 mainly affects piglets and fattening pigs. Diseases related to this virus include: congenital tremor (CT) of neonatal piglets, diarrheal disease of neonatal piglets, proliferative necrotizing pneumonia (PNP), and fattening porcine dermatitis and nephritic syndrome (PDNS). ), respiratory disease sy...

Claims

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Application Information

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IPC IPC(8): C12N15/34C12N15/85C12Q1/70C12Q1/68G01N33/577G01N33/569
Inventor 唐志玲林旭埜林德锐齐冬梅胡美容周燕芬谢少霞陈丹
Owner 广东永顺生物制药股份有限公司
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