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Method for overexpression of porcine circovirus type 2 nucleocapsid protein in cells of mammal

A technology for porcine circovirus and nucleocapsid protein, which can be applied in the biological field and can solve problems such as small homology

Active Publication Date: 2013-05-29
杭州贝尔塔生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The homology between PCV1 and PCV2 is more than 90%, but the homology between the two types is less than 80%

Method used

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  • Method for overexpression of porcine circovirus type 2 nucleocapsid protein in cells of mammal
  • Method for overexpression of porcine circovirus type 2 nucleocapsid protein in cells of mammal
  • Method for overexpression of porcine circovirus type 2 nucleocapsid protein in cells of mammal

Examples

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Embodiment 1

[0030] The present embodiment has described the whole experimental process of the cloning of porcine circovirus type 2 (PCV 2) SX04 strain whole genome sequence provided by the present invention such as figure 1 shown.

[0031] Weigh an appropriate amount of disease material (inguinal lymph nodes, hilar lymph nodes, and cervical lymph nodes, etc.), add an appropriate volume of tissue lysis buffer for homogenization; divide the tissue homogenate into 1.5ml eppendorf tubes, and centrifuge at 3000g for 10 minutes at room temperature; Take the supernatant and add proteinase K to a final concentration of 100μg / ml, lyse at 55°C for 3h; add an equal volume of phenol / chloroform, mix upside down, centrifuge at 12000g for 10min at 4°C; put the supernatant into a new 1.5ml eppendorf tube Medium; put the supernatant into a new 1.5ml eppendorf tube, add 2 times the volume of ice-cold absolute ethanol (containing 1 / 10 volume of 3M NaAc), mix up and down, -20°C, 40min, precipitate DNA; 4 Ce...

Embodiment 2

[0038] This example describes the construction of the eukaryotic expression plasmid containing porcine circovirus type 2 (PCV2) nucleocapsid protein gene provided by the present invention. Such as figure 2 shown

[0039] According to the genome sequence of the PCV2SX04 strain, specific primers (Table 2) were designed, and an HA tag was introduced at the C-terminus of the protein for protein detection.

[0040] Table 2 Primer sequences for porcine circovirus nucleocapsid protein gene amplification

[0041]

[0042] The PCR reaction was carried out according to the operation guide of the Roche Expand High Fidelity PCR System, using the extracted viral DNA as a template, and the PCR reaction parameters were pre-denaturation at 94°C for 3min, denaturation at 94°C for 15sec, annealing at 58°C for 45sec, and extension at 68°C for 2min; cycle 30 times, 72°C extension for 10min. After the reaction is completed, 1-21% agarose gel electrophoresis is used for detection, and a PCV2...

Embodiment 3

[0045] This example describes the transfection of pCI-PCVCapHA plasmid into porcine kidney cell PK15 provided by the present invention, and adding MG132 to the cell culture medium 24 hours after transfection. Such as image 3 shown.

[0046] According to the kit instructions, the pCI-PCVCapHA transfection-grade ultrapure plasmid was extracted with the QIAGEN Plasmid Plus Midi Kit. Under the mediation of liposome Lipofectamine 2000, pCI-PCVCapHA was used to transfect 80% confluent PK15 cells (No. 1 and No. 2 wells), and a control group (No. 3 well) which only transfected liposomes was set up. PK15 cells used for transfection and related experiments were cultured on 6-well plates. After 24 hours of transfection, replace the cell culture medium, and add MG132 (proteasome inhibitor, ), the concentration is 5nm; DMSO is added to the culture solution of No. 2 well, the concentration is 1ul / ml. Well 3 was a negative control. Continue culturing for 48 hours.

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Abstract

The invention discloses a method for overexpression of porcine circovirus type 2 nucleocapsid protein in cells of a mammal. According to the method disclosed by the invention, the stability is enhanced and the expression level of the virus protein in the cells of the mammal is further improved by inhibiting the ubiquitination of the porcine circovirus type 2 nucleocapsid protein. The method comprises the following steps: 1) cloning the genome sequence of a porcine circovirus type 2 SX04 strain; 2) constructing a eukaryotic expression plasmid containing the porcine circovirus type 2 (PCV2) nucleocapsid protein; 3) performing transfection on porcine kidney cells PK15 through the plasmid, and adding MG132 into a cell culture solution at 24 hours after transfection; and 4) culturing continuously for 48 hours, then collecting the cells and detecting the expression protein by immunoblotting. Compared with an ordinary eukaryotic expression method, the method has the advantage that the PCV2 nucleocapsid protein in the eukaryotic cells can be effectively stabilized. The detection by protein immunoblotting at 72 hours after transfection shows that, through the adoption of the method disclosed by the invention, the expression level of the PCV2 nucleocapsid protein can be improved by about 20 times to the greatest extent.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a method for highly expressing porcine circovirus type 2 nucleocapsid protein in mammalian cells. Background technique [0002] Porcine circovirus (PCV) was first discovered by Tischer in 1974 in a serially passaged PK15 cell line (PK15 ATCC CCL31). It is a new virus with no envelope and a diameter of 17nm. This circovirus widely exists in PK15 cells and does not cause clinical symptoms in pigs, so it is called porcine circovirus type I (PCV1). Postweaning multisystemic wasting syndrome (Postweaning multisystemic wasting syndrome, PMWS) was first discovered in western Canada in 1991, and has been widely prevalent in the country by 1994, and is now popular in many countries and regions in the Americas and Europe. India, Japan, South Korea, the Philippines, Taiwan Province of China, etc. also have reports of this disease. PMWS mainly infects 7-15 week-old pigs, and the affected pigs sh...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/66
Inventor 李龙于涟方立
Owner 杭州贝尔塔生物技术有限公司
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