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Method for preparing porcine circovirus type 2 high-sensitivity cell line

A porcine circovirus, cell line technology, applied in the field of cell lines

Inactive Publication Date: 2014-01-08
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, related studies at home and abroad have used conventional PK-15 cells to obtain PCV2-sensitive cell lines after limited dilution, and have not yet been prepared by transgenic or gene silencing methods Report of PCV2 Highly Susceptible Cell Lines

Method used

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  • Method for preparing porcine circovirus type 2 high-sensitivity cell line
  • Method for preparing porcine circovirus type 2 high-sensitivity cell line
  • Method for preparing porcine circovirus type 2 high-sensitivity cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] According to the HMGCR gene sequence characteristics of pig PK-15 cells, the shRNA for HMGCR gene silencing was designed and synthesized, and its sequence is:

[0063] Sense:

[0064] 5'-caccgcagaaactgacacctcaagcttcaagagagcttgaggtgtcagtttctgcttttttg-3';

[0065] Antisense:

[0066] 5'-gatccaaaaaagcagaaactgacacctcaagctctcttgaagcttgaggtgtcagtttctgc-3';

[0067] At the same time, there is a negative control shRNA whose sequence is:

[0068] Control Sense:

[0069] 5'-caccgttctccgaacgtgtcacgtcaagagattacgtgacacgttcggagaattttttg-3';

[0070] Control Antisense:

[0071] 5'-gatccaaaaaattctccgaacgtgtcacgtaatctcttgacgtgacacgttcggagaac-3';

[0072] 2) Construction of HMGCR gene silencing vector: Digest the pGPH1 / GFP / Neo siRNA vector with BbsI and BamHI, recover the vector fragment, and synthesize the shRNA designed above according to the operation steps of the pGPH1-Neo siRNA expression vector kit manual of Shanghai Gemma Pharmaceutical Technology Co., Ltd. Sequences (Sense...

Embodiment 2

[0081] 1) Vector linearization: extract pGPH1 / GFP / Neo siRNA-HMGCR and pGPH1 / GFP / Neo siRNA-control plasmids, and digest pGPH1 / GFP / Neo siRNA-HMGCR and pGPH1 / GFP / Neo with restriction endonuclease BamHI, respectively siRNA-control vector with sterile ddH 2 O dissolved;

[0082] 2) Lipofectamine transfection: Transfect the above linearized carrier into PK-15 cells according to the instructions of the liposome transfection reagent Fugene HD, DMEM medium, 5% CO 2 , cultured at 37°C;

[0083] 3) Acquisition and identification of HMGCR gene silencing cells: the above-mentioned liposome-transfected cells were screened with G418 for 8-10 days to obtain resistant cells; conventional Western blotting methods and Relative quantitative real-time PCR were used to compare the obtained The cells were identified, and the HMGCR gene silenced cells were obtained, which were named PK-shHMG; the control cells transfected with the control plasmid pGPH1 / GFP / Neo siRNA-control were named PK-shNC-contr...

Embodiment 3

[0094] 3 Results After statistical analysis, there was no difference in the viability between PK-shHMG and PK-shNC-control cells ( Figure 4 ).

[0095] 4 Conclusion The activity of HMGCR gene silenced cell PK-shHMG and control cell PK-shNC-control is basically the same as that of normal cells, which can be used in the research of virus infection.

[0096] Example 4

[0097] Screening of Cells Highly Sensitive to Porcine Circovirus Type 2 (PCV2)

[0098] 1 cell HMGCR gene silenced cell PK-shHMG, and two control cells PK-shNC-control and PK-C1-control.

[0099] 2. Experimental process The above-mentioned HMGCR gene silenced and highly active cells and control cells were selected and inoculated with PCV2 virus. Relative quantitative real-time PCR was used to determine the viral genome copy number, and the IFA method was used to determine the virus titer.

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Abstract

The invention provides a cell line highly sensitive to a porcine circovirus type 2 (PCV2) and discloses a preparation method of the cell line. After the PCV2 is adopted to infect PK-15 cells, expression of 3-hydroxyl-3-HMG-CoAreductase (HMGCR) is obviously changed; and after HMGCR genes are subjected to silence, TCID50 (tissue culture infectious dose 50) of the PCV2 reaches 108.5 / ml. An HMGCR gene silence cell line can be obtained by RNA (ribonucleic acid) interfering technology, and the cell line highly sensitive to the PCV2 can be finally obtained through screening. By utilizing HMGCR gene silence cells prepared by the method to culture the PCV2, high-titer viruses can be obtained, and studying on pathogenesis of the PCV2, virus culturing and development and industrial production of totivirus inactivated vaccines are facilitated.

Description

Technical field: [0001] The invention provides a cell line highly sensitive to porcine circovirus type 2 (PCV2), and also discloses a preparation method of the cell line, which belongs to the technical field of bioengineering. Background technique: [0002] Multisystemic wasting syndrome in weaned piglets caused by porcine circovirus type 2 has caused huge economic losses to the swine industry. Currently, there is no effective treatment for this disease. In addition, although conventional PK-15 cells can be used to cultivate PCV2, PCV2 is difficult to cultivate and grows slowly, and the virus content of the whole virus culture can only reach 10% before inactivation. 5.5-6.0 TCID50 / mL severely restricts the production of PCV2 whole-virus inactivated vaccine, so it is necessary to study effective methods to obtain sufficient PCV2. At present, the main methods to increase the titer of PCV2 are: [0003] (1) Add chemical substances such as immune stimulators and aminodextr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C12N15/113C12Q1/68C12R1/91
Inventor 任林柱陈福旺杨鑫欧阳红生逄大欣曹宇航张明军董美辰
Owner JILIN UNIV
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