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

[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...

[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...

[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.