| Porcine reproductive and respiratory syndrome(PRRS), which caused by porcine respiratory syndrome virus(PRRSV), had given rise to great financial losses worldwide and affected the development of swine industry seriously. Host would produce interferon(IFN) to resist against PRRSV invasion and infection, while virus has evolved a series of strategies to escape immune response of the host and there is no exact evidence about its immune suppression mechanism. PRRSV nonstructural protein 11(Nsp11) has been recognized to play a key role in the IFN suppression, but its specific function is still unknown. This study expressed and purified PRRSV Nsp11 which had been reported to be toxic when it express in E.coli and Nsp11 monoclonal antibody was prepared. i TRAQ proteomics technology identified PRRSV Nsp11 could regulate host 434 proteins differentially expressed at 1.2 times. And flow cytometry and Western Blot experiments demonstrate Nsp11 could cause endoplasmic reticulum stress of PAM 3D4/21 cells then induce cell apoptosis. In order to reveal the PRRSV Nsp11 biology function, our study has carried out the following work:1. Expression and purification of PRRSV Nsp11.In order to optimize the conditions of PRRSV Nsp11 expression, the p ET-30a-Nsp11 prokaryotic expression plasmid was transformed into E.coli BL21(DE3). Sampling test was at different time points and with different induction concentration of IPTG. The condition in 37 ℃, 30 min 0.2 m M IPTG inducing expression effect was optimal and recombinant protein mainly exists in the form of soluble with the molecular weight about 32 k Da. Recombinant protein was obtained with the concentration of 0.6mg/m L after purification through Ni-NTA column. 2. Generation of monoclonal antibody against PRRSV Nsp116 weeks of female BALB/C mice was immuned with the purifed Nsp11. Then through cell fusion ELISA screen and cell clones, we obtained 8 strains producted stable antibody against PRRSV Nsp11(1A4、1A9、1D1、4G9、5D8、5G1、1C10、5B11) and 6 strains among these had well reaction specificity verifed by IFA and Western Blot(1A4、1A9、1D1、4G9、5D8、5G1). 3. Cellular localization of PRRSV Nsp11After PRRSV infected Marc-145 cells, at different time points(6h, 12 h, 24 h, 36 h, 48h) cell samples were collected. With PRRSV Nsp11 monoclonal antibody as primary antibodies and FITC labled goat-anti-mouse lg G as second antibodies, IFA results showed that with the increasion of infected cells, Nsp11 presented cytoplasmic localization. 4. Data analysis of PRRSV Nsp11 proteomicsProteomics can analyze by the high-throughput detection at the level of the whole organism. In this study, using Nsp11 recombinant lentivirus to infected PAM 3D4/21 cells, we proceeded the i TRAQ proteomic analysis. In total, we obtained information for 5023 proteins and identified 434 proteins differentially expressed at different ratio more than 1.2 including 184 up-regulated proteins and 250 down-regulated proteins. Analysis the abundance of CAPN2, AAMP(up-regulated) and CD63(down-regulated) by Western Blot was in accordance with the data of proteomics, that declared the reliability of proteomics data.5. PRRSV Nsp11 arouses endoplasmic reticulum stress and apoptosisCaspase3 is a key molecules to cause apoptosis. Proteomics and Western Blot had verified PRRSV Nsp11 could increase the expression and activation of caspase3. Flow cytometry experiment shows that PRRSV Nsp11 can induce apoptosis. The experiment of dual-luciferase suggested that Nsp11 can lead to the upregulation of key apoptosis molecules CHOP by endoplasmic reticulum stress. Western Blot detected PRRSV Nsp11 promoted molecular chaperone expression of GRP78 and GRP94 and e IF2α phosphorylation, thus PRRSV could cause PAM 3D4/21 endoplasmic reticulum stress. PRRSV could activate the JNK signaling pathway to cause apoptosis during the process of infected host cell. Western Blot results showed PRRSV Nsp11 could promote the phosphorylation of JNK, thus PRRSV Nsp11 leading to endoplasmic reticulum stress and apoptosis. |
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