Establishment Of MDBK Stable Cell Line Expressing ISG15Protein And A Preliminary Study Of The Interaction Between ISG15and Procine BVDV-2

Bovine Viral Diarrhea Virus (BVDV) is a member of the Pestivirus genus in Flavividae, which not only can infect cattle, but also make pig, sheep, deer and other wild animals ill. In recent years, BVDV is more prevalent in pig herds with mainly persistent infection-based. If pregnant animals infected with noncytopathic (NCP) BVDV between30-120days when the immune system of the fetus has not yet developed well, fetus can not identify the exogenous NCP BVDV as antigen, and this make fetus persistent infected with BVDV. It’s reported that only NCP BVDV can infect animal resulting in persistent infection. And escaping of the innate cellular immunity is the first setp of developing persistent infection. Exploring the mechanism of escaping the innate cellular immunity by procine BVDV will be helpful for studying the mechanism of persistent infection and taking an effective measure for the prevention and control of BVDV.Interferon-stimulated gene15(ISG15), the first identified ISG, is an important member of the Ubiquitin-like protein family. It’s one of the highest expressed ISG induced by interferon. ISG15can inhibit the replication of kinds of DNA and RNA viruses, and it’s function in the innate cellular immunity cannot be ignored. It has been demonstrated that ISG15plays an important role in the antiviral response after infection of human immunodeficiency virus, ebola virus, influenza B virus, etc.Bovine ISG15gene was cloned and expressed effectively in this study. And the anti-ISG15polyclonal antibody was prepared for the further study. Meanwhile a stable MDBK cell line expressing ISG15proteins was screened and established.On these bases, we explored the interaction between ISG15protein and procine BVDV-2isolate SH-28which lay the foundation for study of the antiviral mechanism of ISG15and the mechanism of escaping from the innate cellular immunity by procine BVDV-2. The main content of the study can be divided into the following three parts:1. Prokaryotic expression of ISG15protein and preparation of anti-ISGIS polyclonal antibodyExtracted the total RNA from the MDBK cells stimulated by recombinant human interferon IFN a-2A, the gene of interferon-stimulated gene15(ISG15) was amplified by RT-PCR. Then it was cloned into prokaryotic expression vector pCold-TF and transformed into E.coli BL21(DE3) with the recombinant plasmid pCold-ISG15. ISG15soluble recombinant protein about70KDa was expressed after induced by IPTG (isopropyl-β-d-thiogalactoside). The concertration of the purified ISG15protein was lmg/mL detected by BCA kit. Then the ICR mice were inolculated with the purified ISG15proteins for preparation of anti-ISG15polyclonal antibody. Collecting the blood of the mice after the fourth immunization, the antibody titer of the serum about1:102400was detected by indirect ELISA. And Western blot results indicated that the antiserum was specifically reacted well with the purified ISG15protein which meant that the anti-ISG15polyclonal antibody was effective and can be used for the following test.2. Establishment of MDBK stable cell line expressing ISG15proteinsTo probe into the interaction of ISG15and procine BVDV-2, a stable cell line expressing ISG15proteins was established. The ISG15gene, which amplified by RT-PCR, was cloned into the eukaryotic expression vector pEC129and then the positive recombinant eukaryotic expression plasmid pEC129-ISG15was developed. After extracted, the plasmid pEC129-ISG15was transfected into MDBK cells by Lipofectamine LTX and PlusTM Reagent kit and then screened by G418with the concertration of700ng/mL. The clone cell clusters were transferred into a new culture flask for enlarged cultivation after two weeks. Detected by indirect immunofluorescence assay (IFA) with the anti-ISG15polyclonal antibody, we found that the whole field of the tenth screened cell line were green fluorescence under microscopy. Besides, the result of Western blot indicated that the anti-ISG15polyclonal antibody was reacted well with the total proteins of the tenth screened cell line with a band about15KDa. It’s confirmed that a stable cell line expressing ISG15proteins was established successfully, named as E53, which lay a fundation for the following studies.3. Researches on the Interaction between ISG15protein and procine BVDV-2 To probe into the mechanism of procine BVDV-2escaping the innate cellular immunity, the interaction between ISG15protein and procine BVDV-2were studied. Infected with the procine BVDV-2isolate SH-28, the MDBK cells and E53cells were collected at different time point and their TCID50were detected, respectively. By drawing the growth kinetics of SH-28in different cell lines, we found that ISG15protein inhibited the replication of SH-28significantly. In the meantime, compared with the E53cells without infection, the expression of ISG15protein in E53cells infected with SH-28were up-regulated significantly by qRT-PCR. To explore the influence of ISG15protein on the inhibition of IFN-I induced by procine BVDV-2, the expression of IFN-I in MDBK cells and E53cells infected with SH-28after the treatment of poly Ⅰ:C were detected by real-time PCR, respectively. Results showed that after infected by SH-28, the expression of IFN-a and IFN-β in MDBK cells were reduced about2.49and3.31folds respectively, while they were reduced about6.29and9.71folds in E53cells. This meant that overexpression of ISG15proteins could strengthen the inhibition of IFN-I expression inducted by procine BVDV-2. However, the exact mechanisms involved are still to be unknown and remains to be further research. In Conclusion, this study laid the foundation for the further researches.