胃泌素调节激素（OXM）在双歧杆菌中的表达及其转基因双歧杆菌对肥胖小鼠食量、体重的影响
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摘要
【目的】构建oxyntomodulin基因的双歧杆菌表达载体,并用此表达载体转化长双歧杆菌,观察目的蛋白oxyntomodulin在双歧杆菌内的表达及其对小鼠摄食量和体重的影响。
【方法】以长双歧杆菌的基因组DNA为模板扩增复制子和多聚酶基因序列。PCR扩增来源于双歧杆菌的信号肽基因,保留克隆质粒pBAD-A的氨苄抗性基因和启动子基因,在多克隆位点Bst1107上插入双歧杆菌质粒复制子及聚合酶序列(BLP),并用来源于双歧杆菌的信号肽序列取代原质粒pBAD-A中基因Ⅲ序列。在BpiI和XbaI之间插入报告基因GFP基因序列(GFP)。BpiI和XbaI双酶切除大肠杆菌一双歧杆菌穿梭表达质粒载体pBADs-GFP中GFP基因序列,酶切产物与OXM目的片段进行连接反应,获得含OXM目的片段的大肠杆菌—双歧杆菌穿梭表达质粒载体PBBADs-OXM。以电穿孔法将此表达载体PBBADs-OXM转化B.longum NCC2705,于氨苄阳性MRS培养基中,37℃厌氧培养。做革兰氏染色,显微镜下观察转化后B.longumNCC2705形态。以转化后B.longum基因组DNA为模板,PCR扩增16s片段。以此双歧杆菌质粒为模板,扩增OXM片段。以终浓度为0.2%的阿拉伯糖诱导培养PBBADs-OXM转化的B.longum,于0、12、24、48小时分别留取菌体和菌液。提取菌体中蛋白。ELISA检测培养上清中OXM,以50ug总蛋白WESTERNBLOT。取断乳昆明小鼠,随机取8只小鼠给予正常饮食,其余小鼠给予高脂饮食,构建肥胖小鼠模型。取肥胖小鼠32只,随机分为模型组,阴性对照组,OXM组,阳性对照组,每组8只小鼠。阴性对照组小鼠给予阿拉伯糖(终浓度0.2%)诱导的B.longum NCC2705菌液0.9ml灌胃,OXM组小鼠,给予阿拉伯糖诱导的PBBADs-OXM转化B.longum菌液0.9ml灌胃,阳性对照组小鼠,给予赛尼可1g/kg灌胃,计各组小鼠每天食物摄取量,每周称量体重。30天后处死动物,检测血浆甘油三酯水平。另取2月龄昆明小鼠36只,随机分为3组,正常组给与生理盐水0.9ml灌胃;阴性对照组给与阿拉伯糖诱导后的B.longum NCC2705菌液0.9ml灌胃,OXM组给与阿拉伯糖诱导后的PBBADs-OXM转化双歧杆菌菌液0.9ml灌胃;每日下午,3组均给与13C标记的棕榈酸,六天后,处死动物。酶联免疫法检测小鼠血清中oxyntomodulin和Ghrelin表达浓度。质谱法检测13C/总碳比,观察各组小鼠脂肪代谢。
【结果】未携带来源于双歧杆菌质粒的复制子和聚合酶序列BLP的对照质粒pBS-GFP转化菌不能形成菌落,而携带BLP序列的重组质粒有菌落形成,并且此菌落中细菌保留了长型双歧杆菌的典型形态。PCR扩增在800bp和100bp处见条带出现。分别代表16s和OXM基因片段的形成。酶联免疫吸附试验结果显示Oxyntomodulin在PBBADs-OXM转化的B.longum NCC2705培养液上清和菌体沉淀中均有表达,且上清的表达水平高于菌体沉淀中的水平。WESTERNBLOTING显示:转化双歧杆菌在0.2%阿拉伯糖诱导后,在4KD-5KD之间可见蛋白表达条带。酶联免疫吸附试验结果显示Oxyntomodulin在PBBADs-OXM转化的B.longum NCC2705培养液上清和菌体沉淀中均有表达,且上清的表达水平高于菌体沉淀中的水平。动物实验观察到:经过口服阿拉伯糖诱导的pBBADs-OXM转化双歧杆菌菌液或赛尼可后,肥胖小鼠的食物摄取量与模型组小鼠比较显著减少。OXM组小鼠食量达正常小鼠水平。体重比较结果显示模型组小鼠体重显著高于正常组(P＜0.05),经过灌饲阿拉伯糖诱导的pBBADs-OXM转化双歧杆菌菌液,肥胖小鼠体重下降,显著低于模型组。而长双歧杆菌B.longumNCC2705处理的小鼠其体重下降不显著,体重与模型组无差异(P＞0.05)。血浆甘油三酯检测结果:正常组血浆血脂:1.957±0.238mmol/L;模型组:5.468±0.759mmol/L;阴性对照组:2.021±0.177 mmol/L;OXM组:1.661±0.137 mmol/L;阳性对照组:1.692±0.486 mmol/L。模型组血浆甘油三酯显著高于正常组,OXM组甘油三酯水平显著低于模型组(P＜0.05)。ELISA方法检测显示小鼠血清中OXM水平分别为:NS组,36.183±3.876pg/mL;阴性对照组,38.233±4.139pg/mL;OXM组,37.044±4.870 pg/mL,无统计学差异(P＞0.05)。
各组小鼠血清中Ghrelin均值分别为:NS组,23.680±3.639pg/mL;阴性对照组32.145±1.571pg/mL;OXM组12.277±.580pg/mL。OXM组血清中Ghrelin与阴性对照组有统计学差异(P＜0.05)
质谱分析各组13C/总碳比比值分别为:NS组:-20.033±0.056;OXM组:-21.191±0.043;阴性对照组:-21.225±0.049。各组之间无差异。
【结论】克隆了人oxyntomodulin基因,构建了含该基因的穿梭质粒载体,筛选出了含有oxyntomodulin的转基因双歧杆菌。转基因双歧杆菌在体外经过诱导可以表达目的蛋白。转基因双歧杆菌能在体内表达oxyntomodulin,并发挥减轻体重,降低血浆甘油三酯的作用。OXM转化长双歧杆菌对肥胖小鼠脂肪代谢未见显著影响。
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.
【方法】以长双歧杆菌的基因组DNA为模板扩增复制子和多聚酶基因序列。PCR扩增来源于双歧杆菌的信号肽基因,保留克隆质粒pBAD-A的氨苄抗性基因和启动子基因,在多克隆位点Bst1107上插入双歧杆菌质粒复制子及聚合酶序列(BLP),并用来源于双歧杆菌的信号肽序列取代原质粒pBAD-A中基因Ⅲ序列。在BpiI和XbaI之间插入报告基因GFP基因序列(GFP)。BpiI和XbaI双酶切除大肠杆菌一双歧杆菌穿梭表达质粒载体pBADs-GFP中GFP基因序列,酶切产物与OXM目的片段进行连接反应,获得含OXM目的片段的大肠杆菌—双歧杆菌穿梭表达质粒载体PBBADs-OXM。以电穿孔法将此表达载体PBBADs-OXM转化B.longum NCC2705,于氨苄阳性MRS培养基中,37℃厌氧培养。做革兰氏染色,显微镜下观察转化后B.longumNCC2705形态。以转化后B.longum基因组DNA为模板,PCR扩增16s片段。以此双歧杆菌质粒为模板,扩增OXM片段。以终浓度为0.2%的阿拉伯糖诱导培养PBBADs-OXM转化的B.longum,于0、12、24、48小时分别留取菌体和菌液。提取菌体中蛋白。ELISA检测培养上清中OXM,以50ug总蛋白WESTERNBLOT。取断乳昆明小鼠,随机取8只小鼠给予正常饮食,其余小鼠给予高脂饮食,构建肥胖小鼠模型。取肥胖小鼠32只,随机分为模型组,阴性对照组,OXM组,阳性对照组,每组8只小鼠。阴性对照组小鼠给予阿拉伯糖(终浓度0.2%)诱导的B.longum NCC2705菌液0.9ml灌胃,OXM组小鼠,给予阿拉伯糖诱导的PBBADs-OXM转化B.longum菌液0.9ml灌胃,阳性对照组小鼠,给予赛尼可1g/kg灌胃,计各组小鼠每天食物摄取量,每周称量体重。30天后处死动物,检测血浆甘油三酯水平。另取2月龄昆明小鼠36只,随机分为3组,正常组给与生理盐水0.9ml灌胃;阴性对照组给与阿拉伯糖诱导后的B.longum NCC2705菌液0.9ml灌胃,OXM组给与阿拉伯糖诱导后的PBBADs-OXM转化双歧杆菌菌液0.9ml灌胃;每日下午,3组均给与13C标记的棕榈酸,六天后,处死动物。酶联免疫法检测小鼠血清中oxyntomodulin和Ghrelin表达浓度。质谱法检测13C/总碳比,观察各组小鼠脂肪代谢。
【结果】未携带来源于双歧杆菌质粒的复制子和聚合酶序列BLP的对照质粒pBS-GFP转化菌不能形成菌落,而携带BLP序列的重组质粒有菌落形成,并且此菌落中细菌保留了长型双歧杆菌的典型形态。PCR扩增在800bp和100bp处见条带出现。分别代表16s和OXM基因片段的形成。酶联免疫吸附试验结果显示Oxyntomodulin在PBBADs-OXM转化的B.longum NCC2705培养液上清和菌体沉淀中均有表达,且上清的表达水平高于菌体沉淀中的水平。WESTERNBLOTING显示:转化双歧杆菌在0.2%阿拉伯糖诱导后,在4KD-5KD之间可见蛋白表达条带。酶联免疫吸附试验结果显示Oxyntomodulin在PBBADs-OXM转化的B.longum NCC2705培养液上清和菌体沉淀中均有表达,且上清的表达水平高于菌体沉淀中的水平。动物实验观察到:经过口服阿拉伯糖诱导的pBBADs-OXM转化双歧杆菌菌液或赛尼可后,肥胖小鼠的食物摄取量与模型组小鼠比较显著减少。OXM组小鼠食量达正常小鼠水平。体重比较结果显示模型组小鼠体重显著高于正常组(P＜0.05),经过灌饲阿拉伯糖诱导的pBBADs-OXM转化双歧杆菌菌液,肥胖小鼠体重下降,显著低于模型组。而长双歧杆菌B.longumNCC2705处理的小鼠其体重下降不显著,体重与模型组无差异(P＞0.05)。血浆甘油三酯检测结果:正常组血浆血脂:1.957±0.238mmol/L;模型组:5.468±0.759mmol/L;阴性对照组:2.021±0.177 mmol/L;OXM组:1.661±0.137 mmol/L;阳性对照组:1.692±0.486 mmol/L。模型组血浆甘油三酯显著高于正常组,OXM组甘油三酯水平显著低于模型组(P＜0.05)。ELISA方法检测显示小鼠血清中OXM水平分别为:NS组,36.183±3.876pg/mL;阴性对照组,38.233±4.139pg/mL;OXM组,37.044±4.870 pg/mL,无统计学差异(P＞0.05)。
各组小鼠血清中Ghrelin均值分别为:NS组,23.680±3.639pg/mL;阴性对照组32.145±1.571pg/mL;OXM组12.277±.580pg/mL。OXM组血清中Ghrelin与阴性对照组有统计学差异(P＜0.05)
质谱分析各组13C/总碳比比值分别为:NS组:-20.033±0.056;OXM组:-21.191±0.043;阴性对照组:-21.225±0.049。各组之间无差异。
【结论】克隆了人oxyntomodulin基因,构建了含该基因的穿梭质粒载体,筛选出了含有oxyntomodulin的转基因双歧杆菌。转基因双歧杆菌在体外经过诱导可以表达目的蛋白。转基因双歧杆菌能在体内表达oxyntomodulin,并发挥减轻体重,降低血浆甘油三酯的作用。OXM转化长双歧杆菌对肥胖小鼠脂肪代谢未见显著影响。
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.
引文
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[1]Katie Wynne and Stephen R Bloom.The role of oxyntomodulin and peptide tyrosine-tyrosine(PYY) in appetite control.Nature Clinical Practice Endocrinology & Metabolism.November 2006.Vol2.No11;612-620.
[2]Le Quellec A,Kervran A,Blache P et al.Oxyntomodulinlike immunoreactivity:diurnal profile of a new potential enterogastrone.J Clin Endocrinol Metab 1992;74:1405-9.
[3]Jens Juul Holst.Glucagon-like Peptide 1(GLP-1):An Intestinal Hormone,Signalling Nutritional Abundance,with an Unusual Therapeutic Potential.Jens Juul Holst.Trends Endocrinol Metab.1999 Aug;10(6):229-235.
[4]Holst,J.2005 Glucagon-like peptide-1:physiology and therapeutic potential.Curr.Opin.Endocrinol.Diab.12,56-62.
[5]Imery(u|¨)z N,Ye(?)en BC,Bozkurt A,Coskun T,Villanueva-Pe(?)acarriilo ML,Ulusoy NB.Glucagon-like peptide-1 inhibits gastric emptying via vagal afferent-mediated central mechanisms.Am J Physiol.1997 Oct;273(4 Pt 1):G920-7.
[6]Wettergren A,Wφjdemann M,Meisner S,Stadil F,Holst JJ.The inhibitory effect of glucagon-like peptide-1(GLP-1) 7-36 amide on gastric acid secretion in humans depends on an intact vagal innervation.Gut.1997 May;40(5):597-601.
[7]Tesfaye Tolessa,Mark Gutniak,Jens Juul Holst,Suad Efendic,and Per M.Hellstr(o|¨)m.Inhibitory Effect of Glucagon-like Peptide-1 on Small Bowel Motility.Fasting but not Fed Motility Inhibited via Nitric Oxide Independently of Insulin and Somatostatin.Denmark.J Clin Invest.1998 Aug 15;102(4):764-74.
[8]R(u|¨)ttimann EB,Arnold M,Hillebrand J,Geary N,Langhans W.Intrameal Hepatic-Portal and Intraperitoneal Infusions of Glucagon-Like Peptide-1(GLP-1) Reduce Spontaneous Meal Size in the Rat via Different Mechanisms.Endocrinology.2008 Oct 23.
[9]Murphy KG,Dhillo WS,Bloom SR.Gut peptides in the regulation of food intake and energy homeostasis.Endocr Rev.2006 Dec;27(7):719-27.
[10]HS Besterman,GC Cook,DL Saron,ND Christofides,MG Bryant,M Gregor,S R Bloom.Gut hormones in tropical malabsorption.November1979 British Medical Journal 17,1252-1255.
[11]Dakin,C.L.,Small,C.J.,Batterham,R.L.,Neary,N.M.,Cohen,M.A.,Patterson,M., Ghatei,M.A.& Bloom,S.R.2004 Peripheral oxyntomodulin reduces food intake and body weight gain in rats.Endocrinology 145,2687-2695.(doi:10.1210/en.2003-1338).
[12]14..Dakin,C.L.,Small,CJ.,Park,A.J.,Seth,A.,Ghatei,M.A.&Bloom,S.R.2002 Repeated ICV administration of oxyntomodulin causes a greater reduction in body weight gain than in pair-fed rats.Am.J.Physiol.Endocrinol.Metab.283, E1173-E1177.
[13]Mark A.Cohen,Sandra M.Ellis,Carel W.Le Roux,Rachel L.Batterham,Adrian Park,Michael Patterson,Gary S.Frost,Mohammad A.Ghatei and Stephen R.Bloom Oxyntomodulin Suppresses Appetite and Reduces Food Intake in Humans.J Clin Endocrinol Metab.2003 Oct;88(10):4696-701.
[14]S.Pellissier,K.Sadaki,D.Le-Nguyen,D.Batalle,C.Jarrousse.Oxyntomodulin and glicentin are potent inhibitors of the fed motility pattern in small intestine.Neurogastroenterol Motil (2004) 16,455-463.
[15]C.L.Dakin,I.Gunn,C.J.Small,C.M.B.Edwards,D.L.Hay,D.M.Smithl,M.A.Ghatei and S.R.Bloom.Oxyntomodulin Inhibits Food Intake in the Rat.Endocrinology.2001 Oct;142(10):4244-50.
[16]Catherine L.Dakin,Caroline J.Small,Rachel L.Batterham,Nicola M.Neary,Mark A.Cohen,Michael Patterson,Mohammad A.Ghatei and Stephen R.Bloom Peripheral Oxyntomodulin Reduces Food Intake and Body Weight Gain in Rats.Endocrinology,doi:10.1210/en.2003-1338.EndocrinologyVol.145,No.6,2687-2695.
[17]Owais B.Chaudhri a,James R.C.Parkinson a,Yu-Ting Kuo b,c,Maralyn R.Druce a,Amy H.Herlihy d,Jimmy D.Bell b,Waljit S.Dhillo a,Sarah A.Stanley a,Mohammad A.Ghatei a,Stephen R.Bloom a.Differential hypothalamic neuronal activation following peripheral injection of GLP-1 and oxyntomodulin in mice detected by manganese-enhanced magnetic resonance imaging.Biochemical and Biophysical Research Communications 350(2006)298-306.
[18]Fernandez MF,Boris S,Barbes C.Probiotic properties of human lactobacilli strains to be used in the gastrointestinal tract.Journal of Applied Microbiology 2003,94,449-455.
[19]Katie Wynne,Adrian J.Park,Caroline J.Small,Michael Patterson,Sandra M.Ellis,2Kevin G.Murphy,Alison M.Wren,Gary S.Frost,Karim Meeran, Mohammad A.Ghatei,and Stephen R.Bloom.Subcutaneous Oxyntomodulin Reduces Body Weight in Overweight and Obese Subjects A Double-Blind, Randomized,ControlledTrial.Diabetes2005;54:2390-2395.
[20]K Wynne 1,A J Parkl,C J Small,K Meeran 1,M A Ghateil,G S Frost and S R Blooml.Oxyntomodulin increases energy expenditure in addition to decreasing energy intake in overweight and obese humans:a randomised controlled trial.International Journal of Obesity advance online publication 18 April 2006;doi:10.1038/sj.ijo.0803344.
[21]A.Le Quellec,.Kervran,.Blache,A.J.Ciurana,and D.ataille.Oxyntomodulin-Like Immunoreactivity:Diurnal Profile of a New Potential Enterogastrone.Journal of Clinical Endocrinology and Metabolism.Vol.74,No.6,1405-1409.
[22]Caroline J.Small and Stephen R.Bloom.Gut hormones and the control of appetite.Trends in Endocrinology and Metabolism Vol.15 No.6 August 2004;259-263.
[23]Tang-Christensen M,Vrang N,Larsen PJ 2001 Glucagon-like peptide containing pathways in the regulation of feeding behaviour.Int J Obes Relat Metab Disord 25(Suppl 5):S42-S47.
[24]Jorgensen R,Kubale V,Vrecl M,Schwartz TW,EUing CE.Oxyntomodulin differentially affects glucagon-like peptide-1 receptor beta-arrestin recruitment and signaling through Galpha(s).J Pharmacol Exp Ther.2007 Jul;322(1): 148-54.Epub 2007 Mar 29.
[25]Catherine L.Dakin,Carouline J.Small, Rachel L.Batterham,Nicola M.Neary,Mark A.Cohen, Michael Patterson, Mohammad A.Ghatei, and Serphen R. Bloom.Peripheral Oxyntomodulin Reduces Food Intake and Body Weight Gain in Rats.2004.Endocrinology 145(6):2687-2695.
[26]van der Werf MJ,Venema K.Bifidobacteria:Genetic Modification and the Study of Their Role in the Colon.J Agric Food Chem,2001,49(1):378-383.
[27]Woodmansey EJ,McMurdo ME,Macfarlane GT,Macfarlane S.Comparison of compositions and metabolic activities of fecal microbiotas in young adults and in antibiotic-treated and non-antibiotic-treated elderly subjects.Appl Environ Microbiol,2004,70(10):6113-6122.
[28]M.F.Ferna'ndez,S.Boris and C.Barbe's.Probiotic properties of human lactobacilli strains to be used in the gastrointestinal tract.J Appl Microbiol.2003;94(3):449-55.
[29]H.Annuk,J.Shchepetova,T.Kullisaar,E- Songisepp,M.Zilmer and M.Mikelsaar.Characterization of intestinal lactobacilli as putative probiotic candidates.Journal of Applied Microbiology 2003,94,403-412.
[30]Liam 0'Mahony,Jane McCarthy,Peter Kelly,George Hurley,Fangyi Luo, Kersang Chen, Gerald C.O'Sullivan,Barry Kiely,J.Kevin Collins,Fergus Shanahan and Eamonn M.Quigley.Lactobacillus and bifidobacterium in irritable bowel syndrome:Symptom responses and relationship to cytokine profiles.Gastroenterology,Volume 128,Issue 3,March2005,Pages541-551.
[31]Hyeyoung Kim,Kubum Kwack,Dae-Young Kim,Geun Eog Ji.Oral probiotic bacterial administration suppressed allergic.responses in an ovalbumin-induced allergy mouse model.FEMS Immunology and Medical Microbiology 45 (2005)259-267.
[32]Hoarau C,Lagaraine C,Martin L,Velge-Roussel F,Lebranchu Y Supernatant of Bifidobacterium breve induces dendritic cell maturation,activation,and survival through a Toll-like receptor 2 pathway.J Allergy Clin Immunol.2006Mar;117(3):696-702.Epub 2006 Jan 27.
[33]Michael de Vresea,Petra Winklera,Peter Rautenbergb,Timm Harderc,Christian Noahb,Christiane Lauea,d,Stephan Otte,Jochen Hampee,Stefan Schreibere, Knut Hellerf,Ju"rgen .chrezenmeira.Effect of Lactobacillus gasseri PA 16/8,Bifidobacterium longum SP 07/3,B.bifidum MF 20/5 on common cold episodes:A double blind,randomized,controlled trial.Clinical Nutrition(2005)24,481-491.
[34]Limdi JK,O'Neill C,McLaughlin J.Do probiotics have a therapeutic role in gastroenterology?World J Gastroenterol,2006,12(34):5447-5457.
[35]成虹,胡伏莲.微生态调节剂的临床应用.中国新药杂志,1999,8(4):276-278.
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[40]兰景刚,胡宏.双歧杆菌抗衰老作用的初步研究.中国微生态学杂志,1995,7(6):8-10
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[2]Le Quellec A,Kervran A,Blache P et al.Oxyntomodulinlike immunoreactivity:diurnal profile of a new potential enterogastrone.J Clin Endocrinol Metab 1992;74:1405-9.
[3]Jens Juul Holst.Glucagon-like Peptide 1(GLP-1):An Intestinal Hormone,Signalling Nutritional Abundance,with an Unusual Therapeutic Potential.Jens Juul Holst.Trends Endocrinol Metab.1999 Aug;10(6):229-235.
[4]Holst,J.2005 Glucagon-like peptide-1:physiology and therapeutic potential.Curr.Opin.Endocrinol.Diab.12,56-62.
[5]Imery(u|¨)z N,Ye(?)en BC,Bozkurt A,Coskun T,Villanueva-Pe(?)acarrillo ML,Ulusoy NB.Glucagon-like peptide-1 inhibits gastric emptying via vagal afferent-mediated central mechanisms.Am J Physiol.1997 Oct;273(4Pt 1):G920-7.
[6]Wettergren A,Wφjdemann M,Meisner S,Stadil F,Holst JJ.The inhibitory effect of glucagon-like peptide-1(GLP-1)7-36 amide on gastric acid secretion in humans depends on an intact vagal innervation.Gut.1997 May;40(5):597-601.
[7]Tesfaye Tolessa,Mark Gutniak,Jens Juul Holst,Suad Efendic,and Per M.Hellstr(o|¨)m.Inhibitory Effect of Glucagon-like Peptide-1 on Small Bowel Motility.Fasting but not Fed Motility Inhibited via Nitric Oxide Independently of Insulin and Somatostatin.Denmark.J Clin Invest.1998,5;102(4):764-74.
[8]Tolessa T,Gutniak M,Holst JJ,Efendic S,Hellstr(o|¨)m PM.lucagon-like peptide-1retards gastric emptying and small bowel transit in the rat:effect mediated through central or enteric nervous mechanisms.Dig Dis Sci.1998Oct;43(10):2284-90.
[9]R(u|¨)ttimann EB,Arnold M,Hillebrand JJ,Geary N,Langhans W.Intrameal Hepatic-Portal and Intraperitoneal Infusions of Glucagon-Like Peptide-1 (GLP-1) Reduce Spontaneous Meal Size in the Rat via Different Mechanisms.Endocrinology.2008 Oct 23.
[10]Murphy KG,Dhillo WS,Bloom SR.Gut peptides in the regulation of food intake and energy homeostasis.Endocr Rev.2006 Dec;27(7):719-27.
[11]HS Besterman,GC Cook, DL Sarson,ND Christofides,MG Bryant,M Gregor,SR Bloom.Gut hormones in tropical malabsorption.November1979 British Medical Journal 17,1252-1255.
[12]Dakin,C.L.,Small,C.J.,Batterham,R.L.,Neary,N.M.,Cohen,M.A.,Patterson,M., Ghatei,M.A.& Bloom,S.R.2004 Peripheral oxyntomodulin reduces food intake and body weight gain in rats.Endocrinology 145,2687-2695.(doi:10.1210/en.2003-1338).
[13]Dakin,C.L.,Small,C.J.,Park,A.J.,Seth,A.,Ghatei,M.A.&Bloom,S.R.2002 Repeated ICV administration of oxyntomodulin causes a greater reduction in body weight gain than in pair-fed rats.Am.J.Physiol.Endocrinol.Metab.283,E1173-E1177.
[14]Mark A.Cohen,Sandra M.Ellis,Carel W.Le Roux,Rachel L.Batterham,Adrian Park,Michael Patterson, ary S.Frost,Mohammad A.Ghatei and Stephen R.Bloom Oxyntomodulin Suppresses Appetite and Reduces Food Intake in Humans.J Clin Endocrinol Metab.2003 Oct;88(10):4696-701.
[15]S.Pellissier,D.Le-nguyen,D.Bataille,CJarrousse.Oxyntomodulin and glicentin are potent inhibitors of the fed motility pattern in small intestme.NeurogastroenterolMotil(2004) 16,45 5-463.
[16]C.L.Dakin,I.Gunn,C.J.Small,C.M.B.Edwards,D.L.Hay,D.M.Smithl,M.A.Ghatei and S.R.Bloom.Oxyntomodulin Inhibits Food Intake in the Rat.Endocrinology.2001 Oct;142(10):4244-50.
[17]Catherine L.Dakin,Caroline J.Small,Rachel L.Batterham,Nicola M.Neary,Mark A.Cohen,Michael Patterson,Mohammad A.Ghatei and Stephen R.Bloom Peripheral Oxyntomodulin Reduces Food Intake and Body Weight Gain in Rats.Endocrinology,doi: 10.1210/en.2003-1338.Endocrinology Vol.145,No.6.2687-2695.
[18]Owais B.Chaudhri a,James R.C.Parkinson a,Yu-Ting Kuo b,c,Maralyn R.Druce a,Arny H.Herlihy d,Jimmy D.Bell b.Waljit S.Dhillo a,Sarah A.Stanley a,Mohammad A.Ghatei a,Stephen R.Bloom a.Differential hypothalamic neuronal activation following peripheral injection of GLP-1 and oxyntomodulin in mice detected by manganese-enhanced magnetic resonance imaging.Biochemical and Biophysical Research Communications350(2006)298-306.
[19]Fernandez MF,Boris S,Barbes C.Probiotic properties of human lactobacilli strains to be used in the gastrointestinal tract.Journal of Applied Microbiology 2003,94,449-455.
[20]Katie Wynne, Adrian J.Park,Caroline J.Small,Michael Patterson,Sandra M.Ellis,2Kevin G.Murphy, Alison M.Wren,Gary S.Frost,Karim Meeran, Mohammad A.Ghatei,and Stephen R.Bloom.Subcutaneous Oxyntomodulin Reduces Body Weight in Overweight and Obese Subjects A Double-Blind,Randomized,ControlledTrial.Diabetes2005;54:2390-2395.
[21]K Wynne 1,A JParkl.C J SmalLK Meeran 1,M AGhateil,G S Frost and S R Bloom 1.Oxyntomodulin increases energy expenditure in addition to decreasing energy intake in overweight and obese humans:a randomised controlled trial.International Journal of Obesity advance online publication 18 April 2006;doi: 10.1038/sj.ijo.0803344.
[22]A.le Qullec,A.Kervran,P.Blache,A.J.Ciurana,and D.Bataille.Oxyntomodulin-like Immunoreactivity: Diurnal Profile of a New Potential Enterogastrone.Journal of Clinical Endocrinology and Metabolism.Vol.74,No.6.1405-1409.
[23]Caroline J.Small and Stephen R.Bloom.Gut hormones and the control of appetite.TRENDS in Endocrinology and Metabolism Vol.15 No.6 August 2004;259-263.
[24]Tang-Christensen M,Vrang N,Larsen PJ 2001 Glucagon-like peptide containing pathways in the regulation of feeding behaviour.Int J Obes Relat Metab Disord 25(Suppl 5):S42-S47.
[25]Jorgensen R,Kubale V,Vrecl M,Schwartz TW,Elling CE.Oxyntomodulin differentially affects glucagon-like peptide-1 receptor beta-arrestin recruitment and signaling through Galpha(s).J Pharmacol Exp Ther.2007 Jul;322(1):148-54.Epub 2007 Mar 29.
[26]Catherine L.Dakin,Caroline J.Small,Rachel L.Batterham.Peripheral Oxyntomodulin Reduces Food Intake and Body Weight Gain in Rats.2004.Endocrinology145(6):2687-2695.
[27]van der Werf MJ,Venema K.Bifidobacteria:Genetic Modification and the Study of Their Role in the Colon.J Agric Food Chem,2001,49(1):378-383.
[28]Woodmansey EJ,McMurdo ME,Macfarlane GT,Macfarlane S.Comparison of compositions and metabolic activities of fecal microbiotas in young adults and in antibiotic-treated and non-antibiotic-treated elderly subjects.Appl Environ Microbiol,2004,70(10):6113-6122.
[29]M.F.Ferna'ndez,S.Boris and C.Barbe's.Probiotic properties of human lactobacilli strains to be used in the gastrointestinal tract.J Appl Microbiol.2003;94(3):449-55.
[30]H.Annuk,J.Shchepetova,T.Kullisaar,E-Songisepp,M.Zilmer and M.Mikelsaar.Characterization of intestinal lactobacilli as putative probiotic candidates.Journal of Applied Microbiology 2003,94,403-42.
[31]Liam 0'Mahony,Jane McCarthy,Peter Kelly,George Hurley,Fangyi Luo, Kersang Chen,Gerald C.O'Sullivan,Barry Kiely,J.Kevin Collins,Fergus Shanahan and Eamonn M.Quigley.Lactobacillus and bifidobacterium in irritable bowel syndrome:Symptom responses and relationship to cytokine profiles.Gastroenterology,Volume128,IXynFsue 3,March 2005,Pages 541-551.
[32]Hyeyoung Kim,Kubum Kwack,Dae-Young Kim,Geun Eog Ji.Oral probiotic bacterial administration suppreXynFsed allergic.responses in an ovalbumin-induced allergy mouse model.FEMS Immunology and Medical Microbiology 45(2005)259-267.
[33]Hoarau C,Lagaraine C,Martin L,Velge-Roussel F,Lebranchu Y.Supematant of Bifidobacterium breve induces dendritic cell maturation,activation,and survival through a Toll-like receptor 2 pathway.J Allergy Clin Immunol.2006 Mar;117(3):696-702.Epub 2006 Jan 27.
[34]Michael de Vresea,Petra Winklera,Peter Rautenbergb,Timm Harderc,Christian Noahb,Christiane Lauea,d,Stephan Otte,Jochen Hampee,Stefan Schreibere,Knut Hellerf,Ju¨rgen.chrezenmeira.Effect of Lactobacillus gasseri PA 16/8,Bifidobacterium longum SP 07/3,B.bifidum MF 20/5 on common cold episodes:A double blind,randomized,controlled trial.Clinical Nutrition(2005)24,481-491.
[35]Limdi JK,O'Neill C,McLaughlin J.Do probiotics have a therapeutic role in gastroenterology? World J Gastroenterol,2006,12(34):5447-5457.
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