| [Background]As one of milk growth factors, EGF can stimulate the DNA synthesis and cell proliferation of intestine, promote intestine development, and improve gut flora to increase the production performance and immune function in early-weaned animals.[Objective]In order to develop the more suitable expression forms applied to livestock science and clinical medicine, the objective of this experiment was to compare the biological activity of intracellularly expressed EGF(IE-EGF), extracellularly expressed EGF(EE-EGF), and tagged EGF(T-EGF) from recombinant S.cerevisiae encompassing INVSc1-IE(+), INVSc1-EE(+), and INVSc1-TE(-)both in vivo and in vitro, resepectively.[Methods](1) In this study, sp EGF(TAA+), Mfα-sp EGF(TAA+), and sp EGF(TAA-) genes were originated from that synthetic optimized EGF from breast tissue of Neijiang pig(p EGF), and then were cloned into p YES2/CT. Recombinant S.cerevisiae INVSc1 expressing EGF protein was generated by transforming the plasmid into S.cerevisiae INVSc1. The target EGF protein including IE-EGF, EE-EGF, and T-EGF were determined by Tricine-SDS-PAGE electrophoresis and Western blotting.(2) The biological activities were determined in vivo and in vitro(early-weaned SD rats). Strains of S.cerevisiae expressing IE-EGF, EE-EGF, and T-EGF were designated INVSc1-IE(+), INVSc1-EE(+), and INVSc1-TE(-), respectively. In vitro, the proliferation of rat enterocyte was also measured using the methods of cell counting. In vivo, a total of 60 early-weaned SD rats are assigned to 6 treatment groups, as follows: a basic diet(the control group), empty-vector-expressing S. cerevisiae [the INVSc1(EV) group], T-EGF-expressing S. cerevisiae [the INVSc1-TE(-) group], EE-EGF-expressing S. cerevisiae [the INVSc1-EE(+) group], IE-EGF-expressing S. cerevisiae [the INVSc1-IE(+) group], and positive group [therh-EGF group]. All of the rats were killed after 21 d to determine their production performance(e.g., ADG, ADFI, and F/G), intestinal development(e.g., mean villous height, crypt depth, and the content of total protein, DNA, and RNA), physio-biochemical indexes(e.g., CK and LDH), and immunological function(e.g., Ig A, Ig G, and Ig M).(3) The biological activities were further determined in vivo and in vitro(early-weaned piglets).In vitro, the proliferation of piglets enterocyte was also measured using the methods of cell counting. In vitro, we assigned 24 pigs to each of 5 groups that were provided a basic diet(the control group), empty-vector-expressing S. cerevisiae [the INVSc1(EV) group], T-EGF-expressing S. cerevisiae [the INVSc1-TE(-) group], EE-EGF-expressing S. cerevisiae [the INVSc1-EE(+) group], and IE-EGF-expressing S. cerevisiae [the INVSc1-IE(+) group], all of which were delivered as 60 μg/kg body weight(BW) EGF per day. All of the piglets were killed after 21 d to determine their their production performance(e.g., ADG, ADFI, and F/G), intestinal development(e.g., mean villous height, crypt depth, IVR, and the content of total protein, DNA, and RNA), physio-biochemical indexes(e.g., CK, LDH, ALP, and Sucrase), immunological function(e.g., Ig A, Ig G, and Ig M), and proliferating cell nuclear antigen(PCNA).(4) In 21 days piglets experiment, the fences from different treatments including: Control, INVSc1(EV), INVSc1-TE(-), INVSc1-EE(+), INVSc1-IE(+) group were collected in 0 day, 7days, 14 days, and 21 days, respectively. The digestive metabolism(e.g., crude protein, crude fat, crude fiber, and energe) was also studied by means of collection full excrements analysis method. In addition, the diversity and stability of the fecal bacterial microbiota in weaning piglets were studied using the DGGE techniques based on genes encoding 16 S r DNA V3.(5) In addition, enzyme activities of alkaline phosphatase(ALP), creatine kinase(CK), Lactate dehydrogenase(LDH), Sucrase were assayed by their enzyme activity detection kits, respectively; In addition, m RNA expression level of genes encompassing ALP, CK, LDH, Surcase, and EGF-Receptor were also measured using Fluorogenic Quantitative PCR.[Results](1) As shown in the results of RT-PCR and sequencing, the recombinant plasmidsincluding p YES2-sp EGF(TAA+), p YES2-Mfα-sp EGF(TAA+), and p YES2-sp EGF(TAA-) were constructed successfully, and these EGF protein-expressed recombinant S.cerevisiae were also received successfully. The target protein encompassing IE-EGF, EE-EGF, and T-EGF were identified by the methods of Tricine-SDS-PAGE electrophoresis and Western blot.(2) In the in vitro experiment, the proliferation of enterocyte was significantly stimulated by both IE-EGF and EE-EGF compared with T-EGF(P <0.05). In the in vivo experiment, the production performance(e.g., body weight), intestinal development(e.g., mean villous height, crypt depth, total protein, DNA, and RNA), the physio-biochemical indexes(e.g., CK and LDH), and immunological function(e.g., Ig A, Ig G, and Ig M) of the rats were significantly enhanced in the recombinant S.cerevisiae groups including INVSc1-IE(+), INVSc1-EE(+), and INVSc1-TE(-) group compared with the control and INVSc1(EV) group(P <0.05). Our data further compare the biological activities of different expression forms EGF protein, the ADG, intestinal development, and immunological function were significantly improved in the INVSc1-IE(+) group when compared with the INVSc1-EE(+) and INVSc1-TE(-) group(P <0.05).(3) In the in vitro piglets experiment, the proliferation of enterocyte was significantly stimulated by both IE-EGF and EE-EGF compared with T-EGF(P <0.05). In the in vivo piglets experiment, recombinant S. cerevisiae survived throughout the intestinal tract. The production performance(e.g., ADG, ADFI, F/G), intestinal development(e.g., mean villous height, crypt depth, IVR, and the contents of total protein, DNA, and RNA), the physio-biochemical indexes(e.g., ALP, Sucrase, CK, and LDH), and immunological function(e.g., Ig A, Ig G, and Ig M) of the piglets were significantly enhanced in the recombinant S.cerevisiae groups including INVSc1-IE(+), INVSc1-EE(+), and INVSc1-TE(-) group when compared with the control and INVSc1(EV) group(P <0.01). In addition, our data further compare the biological activities of different expression forms EGF protein, the production performance, intestinal development, the physio-biochemical indexes, and immunological function were significantly improved in the INVSc1-IE(+) group when compared with the INVSc1-EE(+) and INVSc1-TE(-) group(P <0.05).(4) Accroding to the V3 region 16 S r DNA gene patterns using DGGE banding profiles, the populations and composition of fecal bacterial were more steady in recombinant S.cerevisiae groups including INVSc1-IE(+), INVSc1-EE(+), and INVSc1-TE(-) group when compared with the control and INVSc1(EV) group. In addition, the digestive metabolism efficiency(e.g., crude protein, crude fat, crude fiber, and energy) were higher in the INVSc1-IE(+), INVSc1-EE(+), and INVSc1-TE(-) group than the control and INVSc1(EV) group. Futhermore, the digestive metabolism efficiency and the populations and composition of fecal bacterial were improved in the INVSc1-IE(+) group when compared with the INVSc1-EE(+) and INVSc1-TE(-) group.(5) Enzyme activities(e.g., ALP, CK, LDH, Surcase) and m RNA expression level of genes encompassing ALP, CK, LDH, Sucrase, and EGF-Receptor of duodenum, jejunum, ileum were significantly increased in the INVSc1-IE(+), INVSc1-EE(+), and INVSc1-TE(-) group than the control and INVSc1(EV) group(P <0.01). Futhermore, the enzyme activities and m RNA expression level of genes of intestine in the INVSc1-IE(+) group were also higher in the INVSc1-EE(+) and INVSc1-TE(-) group(P <0.05).[Conclusions]Our data further demonstrate various forms EGF-expressed recombinant S.cerevisiae including INVSc1-IE(+), INVSc1-EE(+), and INVSc1-TE(+), have the high biological activities, and their concentration of target protein are about 30 mg.L-1. In fact, IE-EGF is more suitable than EE-EGF or T-EGF for applications in livestock production and clinical research. |
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