| Classical swine fever(CSF) is highly pathogenic and contagious disease caused by classical swine fever virus(CSFV) which can lead to mass mortality of pig. Because of being a serious threat to the pig breeding industries, it has been declared as immediately being reported to World Organisation for Animal Health(OIE), and it is classified as the 1st animal disease in China. Although the C-strain vaccine developed out by China prevents and controls classical swine fever, the chronic and atypical swine fever keep transmission continuously in recent years which have caused huge economic losses. The study on CSFV will contribute to the prevention and control of swine fever. Protein disulphide isomerase(PDI) is a molecular chaperone, and has been proven on its important role in the process of enveloped virus invading in the host cells. Moreover PDI also plays a role in the ER stress. However, there is few research focused on the correlation between the expression of PDI and the infection of CSFV.This study established PDI quantitative PCR detection method, and detected the expression of PDI in host tissue and cellular which had infected with CSFV. Overexpressed PDI in macrophages, then we tried to analysis the interaction between CSFV and PDI in the host cells.(1)Designed PDI gene-specific amplification primers of pig tissues and cell lines and then it was amplified by RT-PCR.Ligated the PDI gene to pMD-19 T vector to construct correct standard plasmid. A SYBR Greenâ… real-time PCR method to detect PDI was established by optimizing the reaction conditions. The data showed this method had high specificity,sensibility and good repeatability. Use this method, we tested the expression of PDI in in the CSFV Shimen strain positive and CSFV Shimen strain negative tissues(heart, liver, spleen, lung, kidney and mesenteric lymph node tissue), results showed that the expression of PDI was downregulated.(2)The CSFV Shimen Strain infected macrophages and then detected the amount of mRNA and protein content of PDI at different time points of 0h, 12 h, 24 h, 48 h. Real-time PCR method was used to detect the PDI gene transcription change in macrophages, PDI protein expression change was detected by western blot and immunofluorescence. Compared with the control group, PDI transcription and protein expression is suppresse in CSFV Shimen positive macrophages.(3)Primers were designed according to the PDI gene coding region to amplify the PDI gene. The PDI gene was ligated to pCDH-CMV-MCS-EF1-GreenPuro vector to construct correct plasmid. The tests of PCR, enzyme digestion and sequencing proved that recombinant plasmid was successfully constructed. The constructed recombinant plasmid was transfected into macrophages and then infected with CSFV Shimen Strain. The results showed that there is a correlation between CSFV and PDI. CSFV can inhibit the expression of PDI, and further suppress the expression of ATF4 genes. Overexpression of PDI can inhibit the proliferation of CSFV in macrophages.In summary, this study established a PCR for PDI in pig tissues and cell lines. Moreover, CSFV infection in vivo and in vitro experiments obtained consistent results combined with overexpression of gene technology. CSFV infection can cause PDI expression down, thereby inhibiting gene expression of ATF4. Meanwhile, PDI also could downregulatated CSFV replication expression in vivo. |
