Recombinant Porcine Epidermal Growth Factor Secreting Lactococcus Lactis Strain Promotes The Development Of Intestine In Early-weaned Piglets

The early weaning is the major traumatic events in the piglet growth process. Intestinal function disorder, intestinal microecological environmental imbalance and growth blocked were caused by the weaning stress and dietary changing, and the deficiency of milk-born growth factors are the main factor of intestinal impaired development. Epidermal growth factor (EGF) is an important growth factor in the regulation of cell proliferation, differentiation, metabolism, survival and apoptosis. Studies have shown that food-grade Lactococcus lactis (L. lactis) and NICE expression system have superior performance in the exogenous protein expression. This study constructs and expresses the functional proteins porcine EGF (pEGF), and uses of Lactococcus lactis as a vehicle for producing and delivering pEGF. Then we investigate the biological activity of recombinant pEGF and explore the applications feasibility of combination effects of Lactococcus lactis and specificity functional protein pEGF on early weaned piglets production. This study consists of three parts as follo wings:Exp.1Cloning and sequencing of the mature protein coding region of the porcine epidermal growth factorThis experiment, firstly, cloned coding sequence of the mature peptide of porcine egf gene, and formed the fusion fragment with USP45protein signal peptide coding sequence, through TA cloning obtained plasmid pMD19-SPUsp45-MPpEGF. The signal peptide coding sequence and the pegf gene mature peptide coding sequence were analyzed by bacteria liquid PCR, double restriction digestion and DNA sequencing.Results showed that we successfully cloned pegf gene encoding mature peptide sequence and constructed the pMD19-SPUsp45-MPpEGF plasmid. And it’s ready to construct the recombinant L. lactis secreting expression system.Exp.2Expression and secretion of recombinant pEGF in Lactococcus lactis NZ9000and the activity assay of pEGFFrom the double restriction enzyme digest of plasmid from experiment1, we obtained SPUsp45+MPpEGF fragment, and ligation with the backbone plasmid pNZ8148which containing the same restriction sites, then got the plasmid pNZ8148-SPUsp45-MPpEGF. The plasmid was transformed into E. coli MC1061 competent cells and the correctness of the sequence were identified by PCR analysis and DNA sequencing. Subsequently the pNZ8148-SPUsp45-MPpEGF plasmid from E. coli MC1061was transformed into L. lactis NZ9000, and obtained L. lactis NZ9000/pNZ8148-SPUsp45-MPpEGF secretion expression system from identification. Through the1ng/ml final concentration of nisin induced the expression of exogenous pEGF, and detected pEGF expression from western blot. Using mouse fibroblasts CCL-1L929culture and adding recombinant pEGF form the induced L. lactis NZ9000/pNZ8148-SPUsp45-MPpEGF recombinant strain, to investgate the biological activity of recombinant pEGF stimulate the fibroblast proliferation and differentiation. The results showed that:1. Successfully constructed L. lactis secretion expression system L. lactis NZ9000/pNZ8148-SPUsp45-MPpEGF,.2. pEGF was detected fron L. lactis NZ9000/pNZ8148-SPUsp45-MPpEGF which induced by nisin in both of the culture medium and the bacterial cells, whereas no pEGF in non-induced bacterial culture. Induced by nisin, along with the extension of time pEGF secretion showed an increasing trend, induced12h secretion stabilized, induced24h the concentration of pEGF up to1000ng/ml.3. The density of mouse fibroblasts CCL-1L929cells increased when add nisin induced L. lactis NZ9000/pNZ8148-SPUsp45-MPpEGF (LL-pEGF) supernatant. Cell count results showed that different supernatant treated for24h, the cell number are higher in recombinant human EGF treatment (rhEGF,10ng/ml) and LL-pEGF (20ul) treatment than in the vector control (LL-EV,20ul)(P<0.05). The number of cell in LL-pEGF (40ul) is extremely significantly higher than in the LL-EV (20ul) treatment (P<0.01). While the number of cells in rhEGF, LL-pEGF (20ul) and LL-pEGF (40ul) treatment group is not significantly different (P>0.05), but the LL-pEGF has the trendcy to promote cell proliferation than the rhEGF treatment. These indicating that recombinant pEGF effectively stimulate mouse CCL-1L929cell proliferation like rhEGF. The results of effect of I. lactis secretion of recombinant pEGF on the BrdU labeling mitosis of CCL-1L929found that the number of BrdU positive cells in rhEGF (10ng/ml)(20.16±2.59%), LL-pEGF (20ul)(25.01±2.98%) and LL-pEGF (40ul)(25.37±1.26%) was significantly higher than in LL-EV (20ul)(11.57±1.19%) treatment group (P<0.01). There’s no difference of the number of BrdU positive cells in rhEGF, LL-pEGF (20ul) and LL-pEGF (40ul) treatment group (P>0.05), but the LL-pEGF has the trendcy to promote cell proliferation than the rhEGF treatment (P>0.05).These results suggested that recombinant L. lactis induced by nisin produce and efficiently secrete pEGF, and the pEGF is a fully biologically active protein in this study. Exp.3Effcets of recombinant pEGF secreting Lactococcus lactis on the early weaned pigletsFor this experiment through intragastric perfusion the culture medium of recombinant L. lactis/pEGF by nisin-A induced (LL-pEGF), to investgate the growth performance of early weaning piglets, intestinal development, including morphology, gastrointestinal enzyme activity and microbial flora. While designed the negative control group, antibiotic control group, empty vector-L. lactis culture fluid control group (LL-EV), to explore the applications feasibility of antibiotic-free diet with recombinant pEGF-L. lactis peplacing of antibiotics. Experimental results show that:1. The LL-pEGF treatment increased the average daily gain (ADG) of weaned piglets in the first week and overall experiment period (P<0.05). The average daily feed intake (ADFI) in each group had no significant difference (P>0.05). LL-pEGF had the tendency to decrease the ratio of ADFI/ADG (P>0.05).2. LL-pEGF treatment increased mean villous height in duodenum, jejunum, and ileum compare with control group (P<0.05). Ileum villus height in LL-pEGF treatment was higher than in LL-EV treatment (P<0.05). Ileum villus height in antibiotic treatment group was significantly higher than in control group (P<0.05). In terms of crypt depth, there was no significant difference among the treatment groups at the duodenum, jejunum and ileum (P>0.05).3. No significant difference was observed in lactase activity from the three segments of intestine of each treatment group (P>0.05). The LL-pEGF group compared with the control group significantly increased invertase activity (P<0.05) in the duodenum, jejunum and ileum. LL-pEGF treatment group increased ileal maltase activity (P<0.05) than in the group of antibiotics and LL-EV. The activity of APA, APN and DPPIV were found increasing in the duodenum compare with control and the antibiotic control (P<0.05). The activity of APA also increased in the LL-pEGF treatment than the control and LL-EV treatment in the jejunum (P<0.05).4. Ileal E. coli in LL-pEGF group and antibiotic group was significantly lower than in the control and LL-EV group (P<0.05). LL-pEGF and antibiotic treatment had lower ileum number of Enterococci, but the difference was not significant (P>0.05). Ileal Bifidobacteria in each treatment was not difference (P>0.05). Lactobacillus in the ileal digesta was higher in the LL-pEGF group than in the control and antibiotics group(P<0.05). For the microflora in the cecum, compared with the control and LL-VE treatments, antibiotic treatment decreased the number of E. coli (P<0.05). LL-pEGF treatment has the tendency to reduce the number of E. coli in the cecum, but the difference was not significant (P>0.05). No significant difference was observed in Enterococcus and Bifidobacterium among the treatments in the cecum (P>0.05). LL-pEGF treatment increased the amount of Lactobacillus in the cecum compared with the control and antibiotic treatments (P<0.01).In summary, this study successfully constructed genetic engineering bacteria L. lactis NZ9000/pNZ8148-SPUsp45-MPpEGF which efficiently secretion exogenous expression pEGF protein. The secreted pEGF is a fully biologically active protein, as demonstrated by its capacity to stimulate L929mouse fibroblast cell line proliferation. The in vivo trial in the early weaned piglets showed that the recombenant pEGF L. lactis NZ9000promote intestinal epithelial cells development, improve the secretion of intestinal epithelial digestive enzymes, regulate intestinal micro-ecological balance, thus improve the health of weaned piglets and increase the production performance.