Expression Of Oxyntomodulin In Bifidobacterium And The Effect On Foodintake And Bodyweitht Of Fat Mouse

OBJECTIVE To express OXM in B.longum and in vivo by constructing E.coli-B.longum Shuttle-expression plasmid vector and observe its effection on foodintake and bodyweight of fat mouse.METHOD Replicon and polymerase gene(BLP) were amplified from B.longum isolated from feces and were inserted into pBAD-A.The XynF signal peptide (XynFs)fragment was amplified.With reserving the ampicillin resistance gene and promotor(BADara) of vector pBAD-A constructed previously,XynFs fragment substituted the geneⅢsequence.E.coli-B.longum Shuttle-expression plasmid vector pBBADs-GFP was constructed by inserting GFP in BpiI and XbaI sites and inserting BLP simultaneously at Bst1107 site.So the E.coli-B.longum Shuttle-expression plasmid vector pBBADs-GFP was constructed and then was verified by sequencing.OXM B.longum expression vector was constructed by inserting human oxm gene into pBBADs-GFP to substitute the GFP sequence at BpiI and XbaI site.B.longum was transformed with pBBADs-OXM by method of electroporation on the conditions that 2.5kv,25μF,200Ω,then was cultured anaerobically in plate with MRS nutrient medium of agar(15g/liter)and ampicillin(60μg/ml) at 37℃.The genome DNA of B.longum was extracted with benzyl chloride as previously described in these procedure.The 16s fragment and the OXM sequence of the transformed B.longum were detected by PCR.The milky colony with round-integrity-smooth fringe was inoculated into ampicillin(60μg/ml) positive liquid MRS nutrient medium with 0.5g/liter cysteine and was cultured at 37℃anaerobically.When the bacterial density reached to 6×10~6(OD＝0.6),a small quantity of liquid culture was sucked for Gram staining,then germs were observed by microscope.OXM expression in B.longum was induced by arabinose(final concentration 0.2%).Supernatant of 3ml culture fluid were collected before inducing and after inducing 12hours,24hours and 48hours respectively and.were divided into 20μl and stored at -70℃.Precip was resuspensed in TES solution [10mmol/LTris-HCI(PH8.0),1 mmol/1EDTA(PH8.0),0.9%(wt/vol)NaCI],adding lysozyme(20mg/ml).Germs were broken by ultrasonic treatment,boiled for 5 minutes at 100℃,then were centrifuged on condition of 10000×g/minute for 20 minutes.The sediment was dissolved in 100μl sample loading buffer and divided to 20μl for storage at -70℃.20μl supernatant and 10μl precip lysateing were used to detected the OXM by euzymelinked immunosorbent assay.The whole procedures were performed following steps stated as ELISA kit.Proteins Sample of 10μl supernatant and precip were subjected to SDS-polyacrylamide gel electrophoresis(PAGE) with a 5%(wt/vol) stacking and a 15%(wt/vol) separating gel and were detected by Rabbit Anti-Oxyntomodulin IgG..In animal experiment,36 KUNMING mouse of 2 months age(Laboratory Animal Center,Southern Medical University)were divided into 3 groups in random.All animals accessed to food and water freely with room temperature 21℃-23℃and illumination of 12 hours of light and 12 hours of darkness.Each group was treated through intragastric administration.Every morning,mouse of normal group were fed with 0.9ml saline,mouse of B.longum group were fed with B.longum NCC2705 0.9ml(bacterial density 6×10~6) induced by arabinose(terminal concentration0.2%),and the group of OXM was treated with PBADs-OXM transformed B.longum 0.9ml(bacterial density 6×10~6) induced by arabinose(terminal concentration0.2%).After 6 days,animals were sacrificed and blood was collected to isolate for serum.The OXM and GHR in serum were detected by euzymelinked immunosorbent assay,and 13 carbon/total carbon was evaluated by method of mass spectrum.Another number of KUNMING mouse of 21 days age accessed to food and water freely with room temperature 21℃-23℃and illumination of 12 hours of light and 12 hours of darkness.8 of them were fed with normal food,the others were fed with high fat diet.Mouse gaining 20%body weight of normal mouse were regard as obesity model.Chose 32 mouse of obesity model and divided them randomly into model group,B.longum group,OXM group,orlistat group.Every morning,mouse of normal group were fed with 0.9ml saline,mouse of,B.longum group were fed with B.longumNCC2705 0.9ml(bacterial density 6×10~6) induced by arabinose(terminal concentration0.2%),and the group of OXM was treated with pBBADs-OXM transformed B.longum 0.9ml(bacterial density 6×10~6) induced by arabinose(terminal concentration0.2%).Mouse of orlistat group were treated with orlistat 1g/kg(dissolved in 0.9ml saline),once a day.Measured the body weight weekly and count the foodintake everyday.Animals were sacrificed the 30 days later and blood were remained.The plasma triglyceride.was detected.RESULTS The pBBADs-OXM transformed B.longum could form colonies,and the germs kept the typical morphous of B.longum,while there was no colony formed in plates for B.longum transformed by pBS-GFP without BLP.Straps at 800bp and 100bp were observed by 2%agar gel electrophoresis,which accorded with the 16s and OXM fragment respectively.OXM was detected not only in supernatant but also in precip by ELISA and the OXM concentration in supernatant seemed to be higher,than that in precip.It showed:The pBBADs-OXM transformed B.longum expressed protein of 4kd to 5kd both in supernatant and precip when they were induced by arabinose(terminal concentration0.2%).The concentration of OXM in serum of the NS group,B.longum group,OXM group is 36.183±3.876pg/mL;38.233±4.139 pg/mL;37.044±4.870 pg/mL(P＞0.05) respectively.Comparison among each group was no significant difference(P＞0.05).While the concentration of GHR in serum of the 3 groups was:group of NS:23.680±3.639pg/mL,group of B.longum:32.145±1.571 pg/mL,group of OXM:12.277±.580pg/mL.The difference was significant when compared the GHR concentration of OXM group with that of B.longum group(P＜0.05).The foodintake of fat mouse fed with PBBADs-OXM transformed B.longum was reduced to normal level,the difference with model mouse was significant(P＝0.001).And the weight of OXM group lower than mouse of model group significantly(P＜0.05).While the weight change of mouse of the B.longum group wans not evidently.The plasma triglyceride of fat mouse was cut down significantly to 1.661±0.137g after PBBADs-OXM transformed B.longum by oral administration.The difference between OXM group and normal group was not significant.Experiment shew:PBBADs-OXM transformed B.longum can reduce the foodintake of mouse evidently to the level of normal mouse(P＞0.05).The differences of bodyweight are significant between the model group and normal group,and OXM group as well(P＜0.05).CONCLUSION BLP conduces to the duplication and existing stably for plasmid in B.longurn.The pBBADs-OXM transformed B.longum can inform typical colony and the bacterium keeps the typical morphous of B.longum,this illustrates:BLP from B.longum is necessary for plasmid to duplicate and be present stably in B.longum. Our experiment displayed a decrease of GHR in serum,but no phenomenon appeared for OXM compared with OXM groups.This may because that,the OXM experssed by the pBBADs-OXM transformed B.longum performs its function to decrease the food intake and bodyweight of mouse by interfering the secretion of GHR at the local gastrointestinal tract possibly after oral administration.What we have done makes it possible to express OXM in vivo and there is no need to purify protein,these may contribute to decreasing the by-effect on central nervous system and cutting down the cost so that obesity patients can follow the treatment continuously.On the other hand,it may be profit to the industrialization of bifidobacterium.