Studies On Pulmonary Immune Defence Related Gene-Surfactant Protein A In Pigs

Porcine Respiratory Disease (PRD) is regarded as the first disease which affects the global porcine production. It's important to study and excavate on pulmonary immune defence related genes, which will improve the porcine ability of anti-PRD in heredity. Pulmonary immune defence related gene- porcine Surfactant Protein A (SP-A) was taken as the candidate gene which had immune defence function in porcine respiratory system in this research. These main results were as follows:①Full length cDNA of porcine SP-A was isolated by combination with RT-PCR and RACE, and the complete cDNA sequence was 1 910 bp including a 747 bp open reading frame (ORF) encoding a 248-amino-acid protein. It was found the porcine SP-A had a signal peptide in the N-terminal segment which was composition of 20 amino acids residues, and had four structural domains: a short N-terminal segment, a collagen-like region (CLR), a neck region, and a C-terminal carbohydrate recognition domain (CRD) by bioinformatics. Then the full-length DNA sequences of porcine SP-A was obtained by comparative genomics and primer walking. The results showed SP-A gene spanned about 4 229 bp which composed of 4 exons and 3 introns.②The experiment group 1 was composition of 361 Large White pigs (LW) and 132 Songliao Black pigs (SB) which were reared in the same conditions and period. Thirty-three single nucleotide polymorphisms (SNPs) were found by PCR-SSCP. There were five SNPs in exon, three of them were same-sense mutation, and two of them were missense mutation resulted in alanine changed to threonine, and phenylalanine changed to tyrosine. The results showed that the SNPs which we found in LW and SB were whole linkage disequilibrium, and there wasⅠhaplotype,Ⅱhaplotype andⅢhaplotype. A chi-square analysis suggested that the allele frequencies and genotype frequencies of SP-A were in Hardy-Weinberg equilibrium (P>0.05) in LW, but were in disequilibrium (P<0.01) in SB. And the SP-A gene had intermediate polymorphism in LW and SB.The frequencies of the SP-A haplotypes in different sex were not statistiatics difference (P>0.05) in LW and SB. The pigs of SP-AⅡhaplotype had the highest incidence of PRD which was 33.67%, and there was exceeding significant difference between incidence of PRD and different haplotypes (P<0.01) in LW. The pigs of SP-AⅡhaplotype had the highest incidence of PRD which was 10.53%, and there was significant difference between the incidence of PRD and different haplotypes (P<0.05) in SB. It indicated there was a certain association extent between haplotype and the incidence of PRD. And the SP-AⅠhaplotype was anti-PRD haplotype. It's predicted the pigs of SP-AⅠhaplotype had higher pulmonary immune defence function.③It was successful to establish the model of acute lung injure (ALI) in pigs via intravenous injection of 0.05 ml/kg avoirdupois oleic acid (OA) by a few each time and more times. It contributes to the further study on ALI and innate immune of the lung.④The experiment group 2 was composition of 54 pigs which containedⅠ,ⅡandⅢhaplotypes. They were established the model of OA-ALI in 50±1 day-old, and the level of lung water content was taken as marker of the degree of lung injury. The SP-A mRNA expression quantity in lung tissue and the SP-A content in BALF were determined with FQ-PCR and western blot method, respectively. The results showed: with the lung water content increasing, the SP-A mRNA expression quantity and the SP-A content decreased at 1h time point. Comparing to the 1h, 3h and 6h, the lung water content increased to the highest, and the SP-A mRNA expression quantity and the SP-A content decreased to the lowest at 3h time point. Comparing to the 1h time point, the lung water content decreased, the SP-A mRNA expression quantity exceeding significant increased (P<0.01), and the SP-A content significant increased (P<0.05) at 6h time point. Moreover, comparing to theⅡandⅢhaplotype, the lung water content of SP-AⅠhaplotype heightened smallest, the SP-A content of SP-AⅠhaplotype was higher, and was closed to the control group; at 1h and 3h time point, SP-A mRNA expression quantity of SP-AⅠhaplotype was also higher, and was also closed to the control group, and at 6h time point, mRNA expression quantity of SP-AⅠhaplotype was higher thanⅡandⅢhaplotype significantly. These results indicated: the SP-A mRNA expression quantity in lung tissue and the SP-A content in BALF had the reverse trend with the level of lung water content, and the same with the degree of lung injury. In the condition of the same etiological agent damage, pigs of SP-AⅠhaplotype could recover SP-A of pigs with SP-AⅠhaplotype in shorter time, which was profit to relieving etiological agent damage to the lung as soon as possible。So, it's predicted pigs with SP-AⅠhaplotype had higher ability to coping etiological agent damage.The above findings indicated, the same to human and mouse, porcine SP-A also had function of pulmonary immune defence. There were association between haplotypes and the incidence of PRD. The pigs of SP-AⅠhaplotype had higer ability of pulmonary immune defence, which could make the degree and time of injury by etiological agent the lowest, and made the possibility of secondary infection decrease, so the incidence of PRD was lower in pigs of SP-AⅠhaplotype.Results of this study revealed the function of pulmonary immune defence of porcine SP-A in all-around for first time. Furthermore, this work contributes to judgement the degree of lung injury during the PRD, and clinical diagnosis of PRD and exploitation of newtype medicine of prevention and cure PRD, and selection the pigs which had higher pulmonary immune defence.