| New bone,20 kg,40 kg,60 kg,90 kg of pigs and piglets weaned at 28 days respectively were used to study the developmental gene expression of antimicrobial peptide PMAP,Prophenin, PR-39 and Protegrin-1 and the effect of lactoferrin on their gene expression in vivo and in vitro by means of semi-quantitative and quantitative PCR. Results are as follow:1.The special primers for PMAP, Prophenin, PR-39 and Protegrin-1 were designed respectively according to the known porcine antimicrobial peptide gene sequences.Then genome RNA was extracted from bone marrow of pig and the aim gene mRNA amplified through RT-PCR with special primers.The DNA fragments of antimicrobial peptides 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 98%, 98%,100%, and 99% comparable to that of the reported cDNA sequence (L39641, X89202,L23825 and X79868) of pig in Genbank. Based on the antimicrobial peptide gene clone, the optimal semi-quantitative and quantitative PCR methods were constructed to detect the amounts of antimicrobial peptides gene expression in pigs.2.Total fifteen female pigs (Duroc×Landrace×Yorkshine) of five groups, each group of three pigs from new bone,20,40,60 and 90 kg of avoirdupois were slaughtered and sampled bone marrow, which were used to detect the developmental gene expression of antimicrobial peptide.The results showed that there was difference in levels of antimicrobial peptides gene expression at different avoirdupois.The trends of PMAP, Prophenin, PR-39 and Protegrin-1 gene expression were coincident. The levels of antimicrobial peptide gene expression increased gradually from new bone to 20 kg, decreased from 20 kg to 40 kg, increased markedly (P<0.05) from 40 kg to 60 kg, and reduce again at the stage of 60 kg to 90 kg. 3.Ninety piglets (Duroc×Landrace×Yorkshine) were randomly divided into three groups, each of which was replicated three times.The groups received the same basal diet, supplemented with 0.125% and 0.25% lactoferrin respectively.After the feeding trails, three piglets in each treatment with the similar live weight were slaughtered and sampled bone marrow, which were used to detect the effects of lactoferrin on antimicrobial peptide gene expression using semi-quantitative and quantitative PCR. The results of semi-quantitative PCR showed that adding lactoferrin in the diet could improve the gene expression of antimicrobial peptide. Compared with control group, supplementation with 0.125% and 0.25% lactoferrin enhanced PMAP gene expression by 111.1% (P<0.01)and 70.3% (P<0.05);Prophenin gene expression by 62.9%(P>0.05) and 67.5% (P>0.05); PR-39 gene expression by 63.09% (P<0.05) and 22.91% (P>0.05);Protegrin-1 212.6% (P<0.05) and 106.3% (P>0.05),respectively.4.We evaluated gene expression of PMAP, Prophenin, PR-39 and Protegrin-1 after induced by lactoferrin (0,10,100,1000μg/ml) in bone marrow cell for 3 h,6 h,12 h and 24 h, respectively.The results showed that lactoferrin increased antimicrobial peptide mRNA expression at 3 h and 6 h, and decreased at 12 h and 24 h. Lactoferrin at 10 and 100μg/ml significantly (P<0.05) increased PMAP and Prophenin mRNA expression at 6 h, respectively.At 24 h,10μg/ml lactoferrin reduced PMAP gene expression (P<0.05). Incubation with 100 or 1000μg/ml lactoferrin reduced Prophenin gene expression (P<0.05) at 24 h, respectively. Lactoferrin at 100 and 1000μg/ml significantly (P<0.05) increased PR-39 mRNA expression at 3 h, respectively. Incubation with 10 or 100μg/ml lactoferrin reduced PR-39 gene expression (P<0.05)at 24 h, respectively.It was concluded that:gene expression of antimicrobial peptide has biology development at different avoirdupois.Lactoferrin could improve the gene expression of antimicrobial peptide in vivo.In vitro,different concentration of lactoferrin could increase the gene expression of antimicrobial peptide also, and different culturing time has different effect.
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