| Signal transducer and activator of transcription1(STAT1) is one of the members of the STAT family and is a signal molecule containing a combination with the tyrosine phosphorylation of Src homology region2(SH2), which can be activated under the stimulation of the external signal and be directly transferred to trigger the transcription of the corresponding target genes in the nucleus. The regulation range of STAT genes are wide, but each of STAT molecules has mutually specific functions, for example, STAT1and STAT2can promote the expression of interferon regulated genes; STAT3facilitates the expression of the acute phase response genes; STAT5mediated the expression of prolactin induced multiple genes. STAT1gene encodes two different mRNA molecules, P91(STAT-1α) and P84(STAT-1β). STAT-1β has less38amino acids than STAT-1α on its C-terminal, and the remaining structure is the same as STAT-1α, it can also be phosphorylated and combined to DNA, but does not have the function of activating gene transcription.Cells can produce type Ⅰ interferon (IFN-α,β) under the viruses stimulation; type Ⅰ IFN secretes to the outside of the host cell and binds to specific receptors on target cells surface; activates the JAK-STAT signaling pathway, thereby inducing interferon stimulated genes (ISGs) to generate a variety of anti-viral protein. In addition, in many types of host cells, IFN-y (type II) is the most important induction agent of MHCⅡ molecules expression, while STAT-1α is extremely important in IFN-y-induced CⅡTA expression and even the MHC Ⅱ molecules expression. Phosphorylated STAT-1α directly bound to CⅡ TA promoter, thereby stimulating the expression of CⅡ TA, and then initiated the expression of MHC Ⅱ molecules. Classical swine fever virus(CSFV) glycoprotein E2is positioned at the surface of infected cells and virus particles, binding to specific molecules on the cell surface and mediating the virus into cells. The E2protein of CSFV has rapid variation. All these are related to viral antigen presentation, which indicates that CSFV E2glycoprotein may be involved in the host immune escape mechanism.The purpose of this study is by means of prokaryotic expression to prepare pig STAT-1α polyclonal antibody, in order to lay the foundation for a further study interaction between STAT-1α and CSFV, which helps to clarify the pathogenic mechanism of CSFV, thus contributing to the countermeasures for antiviral immunity. Porcine STAT-1α gene was cloned by applying RT-PCR from pig kidney cells (PK-15)and subcloned into pET-32a vector. The recombinant plasmid pET-STAT1α was transformed into the host bacteria Rosetta (DE3), which was expressed finally through IPTG induction. The experimental results proved that the recombinant protein is expressed mainly in form of inclusion body. The inclusion body protein was purified, renatured, and used to immune mice with adjuvant. After the immunization of mice was carried out for four times, we achieved the porcine STAT-1α polyclonal antibody with high reactivity and specificity. Then we used the porcine STAT-la polyclonal antibody to verify the expression of STAT-la in CSFV infected and non-infected PK-15cells by IFA. Finally, we used western blot to analyze the expression changes of STAT-la protein in PK-15cells infected with CSFV. The results showed that the mouse anti-pig STAT-la polyclonal antibody not only can react with prokaryotic protein specifically, but also can react with STAT-1α protein of eukaryotic PK-15cells. Results showed that STAT-la protein is mainly expressed and located in the cytoplasm of PK-15cells, and cytoplasmic fluorescence intensity decreased after virus inoculation. Furthermore, CSFV can promote part of STAT-la protein into the nucleus. CSFV could slightly inhibit the STAT-1α protein expression, which inhibition degree was related with time course. |
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