Cloning And Expression Of Porcine Lactoferrin Gene And Effect Of Expression Products On Growth,Immunity And Gene Expression Of Antimicrobial Peptide In Weanling Pigs

Porcine Lactoferrin (PLF) gene was cloned from mammary gland cells of lactating sow by RT-PCR, expressed it in silkworm pupae with bacoluvirus expression system. In vitro antimicrobial trial, effect of recombinant expression products (rPLF) to growth performance, immune functions, intestinal microflora, configuration of small intestine, expression of antimicrobial peptides of weaned piglets was studied. Results are as bellow:1. Two segments of porcine lactoferrin gene were cloned from mammary gland cells of 15-day lactating sow by RT-PCR method. Full length porcine lactoferrin gene was cloned successfully by the two segments cloned respectively and linked together. Results of agarose gel electrophoresis showed that molecular weight of porcine lactoferrin gene is approximately 2.1 kb which was consistent to the molecular weight estimated according to its primary structure. Sequence results showed that its primary structure was consistent to the PLF gene sequence reported in Genebank (L778S7). These results indicated that PLF gene was cloned correctly.2. Recombinant baculovirus transfer vector pBlueBacHisC-PLF was constructed. Recombinant baculoviurs were constructed by homologous recombination. The recombinant baculovirus were propagated in Sf9 insect cells and inoculated silkworm pupae. SDS-PAGE results of silkworm pupae cell lysate showed that there was a distinct band which molecular weight was approximately 80kD. Further detection by western blot showed that there was a band at the right place of the distinct band in SDS-PAGE, which molecular weight was consistent to the molecular weight of recombinant porcine lactoferrin. These results indicted that porcine lactoferrin was expressed successfully in silkworm pupae.3. The antimicrobial activities of test silkworm pupa containing rPLF and their hydrolysates generated by pepsin were determined at Ih, 2h, 4h, 8h, 16h and 24h after exposure to bacteria using time-kill method. No significant inhibitory effect of the control silkworm pupa (no rPLF) was found on test bacteria until 16h. However, thetest silkworm pupa demonstrated bacteriostatic activity at 2h, and the activity was higher than control ones at 4h, 8h, 16h and 24h (P<0.01). After hydrolyzing by pepsin, the control silkworm pupa didn't exhibit more potent activity than intact ones. On the other hand, the antimicrobial potency of proteolytic test silkworm pupa was greater than undigested ones (P<0.01).4, Ninety Duroc X Landrace X Jiaxin pigs (45 males,45 females) of 28 days of weanling age which average body weight was 6.13kg were fed for 30 days. Results of the feeding trial showed that rPLF significantly decreased diarrhea ratio and feed gain ratio (F/G) ,and increased average daily gain (ADG) in weanling pigs .In contrast to the control group ,rPLF treated group decreased the rate of diarrhea by 24.58%(P<0.01),increased the ADG by 34.04%(P<0.01),and decreased the F/G by 12.83%(P<0.05); ninety Duroc X Landrace X Yorkshire weanling pigs(45 males,45 females) of 28 days of weanling age which average body weight was 7.05kg were fed for 30 days in earlier stage(l-15 day of post weaning) and later stage (16-30day of post weaning ), and performance was determined at 15and 30 days respectively. In contrast to the control group, rPLF treated group decreased the rate of diarrhea by 66.25%(P<0.01),increased the ADG by 41.84%(P<0.01),and decreased the F/G by 14.78%(P<0.05) in the earlier stage; and rPLF treated group decreased the rate of diarrhea by 75.20%(P<0.01),increased the ADG by 15.19%(P>0.05),and decreased the F/G by 12.74%(P>0.05) in the later stage. These two trial experiments revealed that rPLF can effectively prevent and control diarrhea ,and the effect of rPLF in the earlier stage on ADG and F/G was much more potent than in the later stage.5. 90 weanling piglets (Duroc X Landrace X Jia) were fed for thirty days, After feeding trial, three gilts which is close by weight in each treatment were slaughtered. Blood sample were collected, and serum was separat...