| Diarrhea and edema disease are two major infectious diseases which cause the death of post-weaning piglets, and the diseases that lead to huge economic loss in the swine business. The major pathogen of these two diseases is enterotoxigenic E. coli F18(ETEC F18). In the long-term, breeding for disease resistance during the selection process is a kind of feasible and efficient method, improved resistance to agent from heredity character, and combined with immunizations controlled diseases in pigs. BPI has a series of biological functions such as an opsonic function, promoting complement activation, opsonization for increased phagocytosis, and it plays a very important role in the natural defense in animal body. As a kind of super antibiotic, BPI become popular and was considerd to be a potential antibiotic and medicine in GNB infection. We study BPI gene from the level of group, DNA, mRNA, cell and protein aiming at analyzing its relationship with Escherichia coli F18-resistant and fuction and mechanism of immunity response ability. Moreover, it will provide the fundation for further research in the breeding of ETEC resistant pigs in the future. The main results were showed as following:1. PCR-RFLP method was used to study the genetic variation within exon10of BPI gene in10Chinese native and commercial pig breeds and wild boars with the analysis of restricted enzyme Hpa II digestion. Two alleles and three genotypes were detected, in which AA genotype in wild boars. Tibetan pig, Rongchang pig and Huai pig was the dominant genotype, and the allele A was the dominant gene. Among all commercial breeds, the higher frequency of genotype AA was present in Duroc pigs. AA genotype is the same with the GenBank sequence. BB genotype was synonymous mutations in A27G and missense mutation in T118G respectively. An unrooted consensus tree was constructed using UPGMA method based on allele frequency which divided the11pig populations into two clusters. One cluster included wild boars, Huai pig, Tibetan pig, Rongchang pig and Duroc pigs. The other contained Large White, Landrace, Fengjing pig, Meishang pig, Erhualian pig and Sutai pig. The study demonstrated that there were differences in normal disease-resistance ability in11pig populations and these differences revealed no significant correlation with body size and types of production. We studied3genotypes on exon10of BPI gene by HpaⅡ restriction site in F18resistant and susceptible groups, the results showed that the frequency of ECF18-resistant individuals in the AA genotype was90%, which was significantly higher than those for the AB and BB genotypes. We can conclude that AA genotype in exon10of BPI gene is a beneficial genotype in combating diarrhea and edema disease that is caused by ECF18in weaned piglets.2. Based on the established resource populations of Sutai pig, the expression of BPI gene was assayed by Real-time PCR to detect the tissue expression and analyze the differential expression between Escherichia coli F18resistant and sensitive piglets, to study BPI gene of Sutai piglets ranging from newborn to post-weaning day aged8,18,30and35days respectively. Besides BPI gene expression was also investigated in post-weaning piglets of Large White, Sutai(Resource Population of resistance to E.coli F18) and Meishan. This study aimed at providing a theoretical foundation for further research on the role of BPI gene in hostimmunity and resistance to E. coli F18. The results showed that the expression of BPI gene was extremely low or undetectable in tissues including heart, liver, spleen, lung, kidney, stomach, muscle, thymus, and lymph nodes, which was in a stark contrast to the significantly high levels in duodenum and jejunum. In the tissues of both jejunum and duodenum, the mRNA expression of BPI gene in resistant individuals was significantly higher than that in the sensitive individuals (P<0.05). The results showed that BPI was found to be highly expressed consistently at the four time points in jejunum and duodenum. The expression of BPI gene in35-day was significantly higher than in18-day in jejunum (P<0.05), and there was no difference among8-day,18-day and30-day in jejunum (P>0.05). Besides, there was significantly higher expression in35-day than other different development stages in duodenum (P<0.05), while there was no difference among8-day,18-day,30-day in duodenum (P>0.05). The expression of BPI gene in Post-weaning Piglets of Large White, Sutai and Meishan breeds showed that there were highly expressed in jejunum and duodenum in three breeds, and the jejunum and duodenum expression of BPI gene in Sutai resistant populations was significantly higher than Large White and Meishan (P<0.05). The results suggested that BPI gene was likely to be related to the intestinal infection caused by E. coli F18. It is possible that the increased expression of BPI gene in intestinal is in connection with the resistance to E. coli F18.3. The expression level of BPI gene in3genotypes in exon10by Hpa Ⅱ site was defected here. We found that the expression of genetype AA was significantly higher than genetype AB and BB in jejunum and duodenum (P<0.05). This result demonstrates that the genotype AA of BPI gene plays a direct role in preventing ETEC F18-induced diarrhea and edema disease in weaned piglets, and that individual resistance to ETEC F18is related to the upregulation of BPI gene expression in the gut. Genetype AA in exon10of BPI gene is expected to become a genetic marker for future breeding against E. coli and gram-negative bacteria diseases.4. Correlation annlysis of part immune indicators of BPI gene in Sutai pig was measured with ELISA kit in the study. The results showed that BPI gene AA genotype was significantly lower than AB genotype in Sutai pig of IL-12(P<0.05) and lower than BB genotype(P>0.05), which was no significant difference. There was no difference in other cytokines. We can deduce that AA genotype might have strong immune response and can be feasibility for future breeding for disease-resistance against E. coli F18.5. In order to study the BPI protein expression and antibacterial function in vitro, the yeast expression vector, the prokaryotic expression vector and mammal expression vector were successfully constructed. By optimization of expression conditions. there was no recombinant protein detected in the yeast or the prokaryotic, which might be caused by stonger bacteriostatic action of the BPI protein. The recombinant protein expression was found in the293-6E cell culture supernatant finally. Bioinformatics was used to study the fuction of BPI protein, the results showed that BPI physical and chemical properties, protein secondary structure and protein level3structure analysis was in accordance with what we expected. BPI consisted of extracellular region, intracellular region and cross membrane area. BPI belongs to secreted protein and has biological functions mainly in Mitochondria and Golgi. The success of the BPI recombinant protein expression in vitro will provide basic theory for the further research on the biological function and antibacterial test of the BPI protein. |
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