Difference Of Antibacterial And Lactoferrin Gene Expression At Different Ages And The Effects Of Weanling Age On Its Gene Expression Of Piglets

1, 14, 28, 42, 56 days piglets and piglets weaned at 21, 28 and 35 days respectively were used to study the difference of antibacterial peptide PR-39, Protegrin-1 and Lactoferrin gene expression and the effects of weanling ages on antibacterial peptide PR-39, Protegrin-1 and Lactoferrin gene expression by means of RT-PCR.The special primers for PR-39, Protegrin-1, Lactoferrin and p-actin were designed respectively according to the known porcine antibacterial peptide, lactoferrin and P-actin gene sequences. Then genome RNA was extracted from bone marrow and duodenum of pig and the aim gene mRNA amplified through RT-PCR with special primers. The DNA fragment of PR-39, Protegrin-1, lactoferrin and p-actin were obtained. The PCR product was cloned into vector and sequenced. Sequence analysis suggested that the gene homology of fragments obtained in this study were 100%, 99%, 99% and 91% comparable to that of the reported cDNA sequence (L23825 X79868 M81327 and BC067141) of pig in Genebank. Based on the antibacterial peptide and lactoferrin gene clone, the suitable anneal tempreture, MgCl2 concentration, and cycles in PCR system were discussed and the optimal semi-quantitative RT-PCR methods were constructed to detect the amounts of PR-39, Protegrin-1 and Lactoferrin mRNA expression in piglets.In experiment 1, total fifteen female 28 days-age weanling piglets (Duroc Landrace Jiaxing ) of five froups, each group of three pigs at 1, 14, 28, 42 and 56 days of age were slaughtered and sampled bone marrow and duodenum tissues, which were used to detect the difference of antimicrobial peptide and lactoferrin gene expression, In experiment 2, threegroup neonatal piglets (total eighteen female piglets) were weaned at 21, 28 and 35 days-age respectively. And at 21, 28 and 35 days-age, three piglets in each group were slaughtered, and bone marrow and duodenum tissue of piglets were samplinged. And then, three piglets were slaughtered and samplinged in 7 days after weaning in each group. The samples were collected for detecting the effect of weanling ages on the levels of antimicrobial peptide and lactoferrin gene expression.The results of experiment 1 showed that there had difference in levels of PR-39 and Protegrin-1 gene expression at different days-age. The trends of PR-39 and Protegrin-1 gene expression were coincident. Before weaning, the levels of PR-39 and Protegrin-1 gene expression increased gradually from 1 day to 14 days. But when the piglets were weaned at day 28, the levels of PR-39 and Protegrin-1 gene expression from 28 days to 42 days were decreaced markedly (p<0.05). From 42 days to 56 days, its expression levels were increased again. The lactoferrin gene had strong expression of piglet in duodenum at 1 days-age. Whereafter, the expression levels of lactoferrin were decreased from 1 day to 56 days and the decreasing level was significant (p<0.05) from 28 days to 42 days. The results of expreient 2 showed that the expression levels of PR-39, Protegrin-1 and lactoferrin were decreased significantly (p<0.05) from weanling days-age (21, 28 and 35 days-age respectively) to 7 days after weaning (28, 35 and 42 days-age respectively). Compared with 21 days-age, the expression levels of PR-39, Protegrin-1 and lactoferrin at 28 days-age were decreased by 38%, 34% and 77% (p<0.05) respectively when the piglets were weaned at 21 days-age; Compared with 28 days-age, the expression levels of PR-39, Protegrin-1 and taetoferrin at 35 days-age were decreased by 46%, 51% and 53% (p<0.05) repectively when the piglets were weaned at 28 days-age; Compared with 35 days-age, the expression levels of PR-39, Protegrin-1 and lactoferrin at 42 days-age were decreased by 50%, 49% and 59% (p<0.05) respectively when the piglets were weaned at 35 days-age.