Different Alleles’ Identification And The Anti-Prrsv Effect Of Ifn-Induced Gene GBP1、GBP2in Tongcheng And Large White Pigs

Blue-ear disease, also known as porcine reproductive and respiratory syndrome (PRRS), is the most important worldwide diseases which infected in pigs. PRRS caused by PRRS virus (PRRSV), PRRSV infects mainly the PAMs. Symptoms of this disease are reproductive disorder in pregnancy and respiratory disorder in each age group. The infected pig presented typical clinical signs, e.g. fever, organic damage and anorexia. The best way dealing with PRRS are pathogen control, environmental control and immunization. Although these measures can play a role in disease control, they are not fundamentally eliminate the influence and prevalence of blue ear disease. Therefore, creating disease resistance breed likely become the most efficient way.Guanylate-binding proteins (GBPs) are kind of guanylic acid binding protein which are about65-67KD. These proteins can induced by interferon and have the GBPase activity, which are members of GTPasesdynamin family. Recent studies has shown that GBP2inhibits replication of Vesicular stomatitis virus (VSV) and Encephalomyo carditis virus(EMCV) in mice. Another study indicated that the tissues had obvious pathological damages after Infected by Mycobacterium bo vis BCG in Gbp1-/-mice. Even recently the structure of GBP protein has been reported, the specific function still not clearly understood. This experiment conducted based on previous research in Tongcheng piglets laboratory infected by HP-PRRSV. We indicated significantly more serious pathological tissue changes in large white piglets were significantly than Tongcheng piglets under the same conditions of infection under the same conditions of infection. We choose the differential expression genes (GBP1/GBP2) before and after infections in affymetrix microarrays in order to study the intervarietal SNP in GBP1/GBP2and it’s effect in Tongcheng and large White pigs.This research has been successfully sequenced the GBP1/GBP2CDS, creating vector expression of the pRK-flag-insert,and using Western blot to detected expression efficiency, Q-PCR to detect virus propagation efficiency.Results in this in this research are as follows:1. Sequencing methods were used to screen the SNPs in the CDS between Tongcheng pigs and Large White pigs, the intervarietal SNP were detected:There are4SNPs in GBP1,two changes the amino acids[c.8.A>C(K-G) and c.65.C>T(A-V),which located in β-1and α0of the GBP1protein]; There are5SNPs in GBPl,two changes the amino acids [c.538.T>C(Y-H) and c.1378.A>C(T-K), which located in a9and a10of the GBP2protein].2. Using the Clustalx to compare the GBP protein in pigs, human and mice. We found that GBP are highly conserved in different species (79%in human and pigs;82%in mice and pig). The most typical are GLYRTGKS,DTEG, TLRT and CAAX motif.3. Construct eukaryotic expression vector PRK-Flag-insert-GBP1/GBP2in Tongcheng and Large White pigs. After transfection in Marc-145and infection with PRRSV-WH2invitro, using semi-quantitative to detect successfulness of the transfection. Using western blot to detect the expression efficiency of pRK-Flag-insert-GBP1/GBP2, The results show that the PRK expression system had expressed protein stability and specificity.4. Quantitative RT-PCR to detect the Virus proliferation efficiency between two haploid type vector in Marc-145cell. Results has been indicated that overexpression of GBP1significantly able to inhibit PRRSV proliferation (P<0.01). These result in consistent with affymetrix microarrays. There is no significant inhibition between two haplotype in GBP2(p>0.01).