Expression And Bioactivity Detection Of Human Epidermal Growth Factor In Lactobacillus Casei

Intestinal microbiota play important roles in animal health by regulating nutrient absorption,inhibiting pathogen bacteria population and stimulating mucosal immunization.As the dominant groups of microorganisms in human and mammalian gastrointestinal tract,Lactobacilli are effective at preventing intestinal disease in both humans and animals due to their ability to inhibit pathogen adhesion to the intestinal surface and prevent inflammatory processes.Epidermal Growth Factor(EGF)is a kind of growth factor that plays important role in regulating cell growth,proliferation and differentiation,and it functions in traumatic treatment and in adjuvant intestinal infection recovery.Researches have shown that Lactobacillus casei(L.casei)can prevent intestinal associated diarrhea and infection.Previously,the plasmid expression system of L.casei was constructed,which can successfully express heterologous proteins.This research is aimed at constructing a recombinant L.casei to express hEGF,to testing if L.casei has the potential to be an excellent live vehicle for protein delivery and intestinal protein expression in situ.The bioactivity of recombinant protein was confirmed through cell proliferation assay and mice experiment.Results were listed as follows:Based on the sequence on Genbank,the hEGF gene was synthesized through Splicing overlapping extension PCR(SOE PCR)and cloned into three L.casei expression plasmids: intracellular expression vector pELX1;Secretory expression vector pELSH,which used the Spusp45 signal peptide;Surface exhibition vector pELWH,which used the Slp A protein as the signal for the secretion of the fusion protein,yielding recombinant L.casei which can constitutively express hEGF protein.Transformants were then analyzed by Western blot assay.Result had shown only in L.casei /pELWH-hEGF that Slp A-hEGF fusion protein was detected both on the cell surface and in the supernatant.The molecular weight of the Slp A-hEGF fusion protein was estimated as to be approximately 55 kDa.NIH/3T3 cell was cultured in vitro,compared with L.casei and L.casei /pELWH,the supernatant of L.casei /pELWH-hEGF was added to detect the proliferation activity of Slp A-hEGF.Results had shown that the recombinant protein did work in cell proliferation.The proliferative effect of ten-fold dilution of L.casei/pELWH-hEGF supernatant were equal with the standard hEGF at concentration 10 ng/mL.Interestingly,a significant increase in the cell proliferation of NIH/3T3 was observed both in the supernatant of L.casei and L.casei/pELWH.To investigate the effect of recombinant L.casei on mouse intestinal morphology,SPF female mice with the weight range of 18-22 g were selected.It was designed to feed the mice with the culture medium of L.casei/pELWH-hEGF,at the same time with the the CK treatment?PBS treatment?MRS treatment?L.casei treatment and L.casei/ pELWH treatment as contrast.Results showed that duodenum villous height of MRS treatment was significantly higher than PBS treatment(P < 0.01).L.casei treatment significantly increased the mean villous height in duodenum compared with MRS treatment(P < 0.01).pELWH-hEGF treatment significantly increase the mean villous height in duodenum compared with L.casei treatment and pELWH treatment(P < 0.01).In terms of villous height in jejunum and ileum or the crypt depth of Duodenum ?jejunum and ileum,there was no significant difference.This research lays the foundation that L.casei afford the potential to be an excellent live vehicle for protein delivery.