Expression Of Gallus Epidermal Growth Factor In Lactococcus Lactis And Its Preliminary Application In Broilers

Epidermal growth factor?EGF?is a small polypeptide that can promote cell proliferation and differentiation,which plays an important role in a series of physiological processes.Currently,EGF from mammals have been well studied and evidence shows that it can affect growth performance,immune function and intestinal development of animals.However,there is few research on chicken epidermal growth factor?gallus epidermal growth factor,gEGF?and the growth and development of young commercial broilers are easy to be affected by bad conditions for their weak resistance.Thus,the aim of this research is to express the mature gEGF with Non-antibiotic screening NICE system and to investigate its biological function through in vitro and animal experiment.The synthesized fusion gene fragment of SP_usp45 and gEGF was cloned into the expression vector pNZ8149,constructing the recombinant vector pNZ8149-gEGF,which was then transformed into Lactococcus lactis NZ3900 to obtain the recombinant strain LL-pNZ8149-gEGF,which could secrete gEGF.The recombinant bacteria was then induced with nisin and the expressed protein was detected by weistern blot and SDS-PAGE and the gEGF protein was purified from the supernatant by HisTrap column..gEGF of different concentration was added to the medium after the UMNSAH/DF-1cells starved for 12 hrs before they reached 60%of the full monolayer cells to investigate its effect on the proliferation of UMNSAH/DF-1 cells.The results showed that 1-10?g/mL gEGF could significantly promote the proliferation of UMNSAH/DF-1 cells,and5?g/mL gEGF exerted the strongest effect.5-day-old 817 broilers was divided into 3 groups,each was orally administered daily with 1 mL M17 medium,supernatant of LL-pNZ8149 fermentation which was induced with Nisin,supernatant of LL-pNZ8149-gEGF fermentation which was induced with Nisin respectively,continuously for 2 weeks and daily feed intake and weight were recorded to calculate the average daily feed intake,average daily gain and feed conversion ratio?FCR?in 1 and 2 weeks,which were used to evaluate the effect of gEGF on the growth performance of broilers.The results showed that gEGF significantly increased the final weight,average daily gain,average daily feed intake and FCR during2 weeks;the chickens were killed after two weeks and spleen,thymus and bursa of Fabricius were taken out to calculate the immune organ index.Blood was collected from wing vein to determine the levels of serum IgG and IgA and the mucosa of duodenum ulcer,jejunum and ileum were scraped from each tissue to measure the level of sIgA.These changes of immune organ index and levels of those immunoglobulins after gEGF treatment were used to evaluate the effect of gEGF on the immune function of the broilers.The results showed that gEGF significantly increased index of thymus and spleen,serum IgG and IgA as well as mucosa sIgA level of duodenum.In addition,a section of the duodenum,the jejunum and the ileum were used for tissue slice,and the villus height and crypt depth were determined,and changes of villus height,crypt depth and VH/CD were used to evaluate the effect of gEGF on the intestinal development.The results showed that gEGF can significantly increase villus height of duodenum and jejunum and decrease the depth of duodenum.The expression of gEGF in the non-antibiotic screening and expression system of Lactococcus lactis has been successfully achieved,and the expressed product could promote the proliferation of UMNSAH/DF-1 cells.Animal experiment illustrated that gEGF could increase the growth performance,improve the immune function and promote the intestinal development tract of the broilers.