Identification Of HO-1 Transcription Factor And Its Function Investigation Involvement In PRRSV Infection

PRRSV is widespread throughout the world,and from the economics perspective,PRRS is one of the most important infectious disease in pig production practice.As early as 1987 in the United States was first reported and in a few years in the Netherlands. The disease showed lots of clinical symptoms and serious reproductive barriers of the sow, the gilt(mainly for the third trimester miscarriage, mummy foetus, the increasing of the dead and the weak) and respiratory problemsof all the ages are two of the most main symptoms.Due to PRRSV antigen has many characteristics such as antigen high mutation respectively, ADE(antibody dependent enhancement effect), persistent infection and so on. These questions make it harder to control of PRRS, and current research of the disease and antigen has no breakthrough progress.This research firstly research the function and molecular mechanism of PRRSV infection from HO-1 promoter transcription,try to find out the key transcription factors, to establish the HO-1 as the new targets for prevention and control of PRRSV and provide sufficient scientific basis.Through the experiment of the study found that:1.Cloning, identification and preliminary analysis of theHO-1 promoter.Using a variety of software such as the Promoter Prediction 2.0 predicted HO-1 Promoter region sequence and design primers.With LA Taq polymerase and Infusion technology we construct recombinant plasmid containing the Promoter region length of 6553 bp, the Promoter region is obtained by sequencing sequence, and determined by testing luciferase activity with the Promoter activity.2.Filter HO-1 core promoter regionsBy building a series of sequential truncated promoter luciferase report vector, combined with fluorescein gene activity test report, we find the basis of HO-1 promoter region locating between(-440~-121), as long as the containing of this fragment will be able to start the transcription of HO-1;The enhanced HO- 1 promoter region identified in 44 bp range(-2065~-2021), under the condition of containing basic promoter segments of the luciferase activity is the foundation of the promoter region 7 times;Also found upstream existence transcriptional regulation region(-6553~-6316)238bp, this fragment to exist can lower the activity40% of the promoter region.3. A key prediction of transcription factor analysis and identification of Nrf2.Comprehensive utilization of software prediction for some transcription factors associated with transcriptional regulation HO-1, combined with the promoter fixed point mutation and luciferase reporter gene tests confirmed HO-1 gene promoter of Nrf2 binding sites really can regulate the transcription of HO-1, After Nrf2 binding site mutated, luciferase activity of this fragment was lost.4. The role of transcription factor Nrf2 in the regulation of HO-1 resisting PRRSV infection processUsing the specificity of the Nrf2 inducers, tBHQ,to induce expression, qPCR detection of Nrf2 and HO-1 mRNA level, with tBHQ the relationship between Nrf2 and HO-1 with the change of the concentration and time were positively correlated.Select the best effect of tBHQ time and concentration on Marc145 cell processed in advance, after the poison detection Nrf2, HO- 1 and N gene mRNA expression, preliminarily determine tBHQ through the induction of Nrf2 and regulating the HO-1 against PRRSV infection.To sum up, transcription factor Nrf2 through transcription regulation HO-1, in turn, to resist the effect of PRRSV infection, it offers a new way for prevention to PRRSV.