稳定表达长效胰高血糖素样肽-1重组酿酒酵母的构建及其应用研究
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摘要
由于肠道内吸收不充分,目前大多数肽类及蛋白质药物仍然通过非肠道途径给药。治疗用肽类和蛋白质通常用于治疗慢性疾病,长期注射给药会产生明显的不良结果。相对于这种不方便且存在潜在问题的给药方法,口服途径具有患者自我给药优势,可大大提高患者接受性和依从性,同时可避免注射给药带来的疼痛、不适和感染的可能。因此,开发有效而成功的口服给药系统以使这类药物发挥最大治疗潜力成为制药研究的一个重要课题。将表达肽类和蛋白药物的重组微生物用作药物释放系统是一个创新性的途径。细菌和酵母都可成为胃肠道中释放药物的新型载体。
胰高血糖素样肽-1(glucagon-like peptide-1,GLP-1)称为肠促胰岛素,是一种分泌自肠道细胞的肽激素,具有血糖依赖性促胰岛素分泌、刺激β-细胞增殖等多种生理功能。在2型糖尿病的临床治疗中具有较好的应用前景。但由于体内二肽基肽酶-Ⅳ(DPP-Ⅳ)的快速降解和肾脏代谢作用,导致其半衰期很短,使其临床应用受到了很大限制。因而,开发长效药用GLP-1类似物已成为受关注的一个热点。
酿酒酵母(面包酵母)本身富含维生素、消化酶等,我国台湾、香港等地素有饮用酵母茶或酵母饮料的悠久历史。若使其胞内再能稳定表达多肽类糖尿病治疗药物,以酿酒酵母细胞作为药物载体——缓释“胶囊”,研发治疗性功能酵母饮品、食品或生物药物,使人们在品尝美味饮品或食品的同时,起到预防和治疗糖尿病等慢性疾病的作用,前景十分诱人。
本研究旨在构建一种胞内稳定表达多肽类糖尿病治疗药物的酿酒酵母,为研发治疗性功能酵母饮品、食品或生物药物奠定基础。首先从构建以质粒pUC18作为骨架的整合型载体入手,以酿酒酵母染色体中多拷贝串联排列的rDNA序列作为外源基因多拷贝整合的位点,设计两对引物扩增rDNA序列中位于25S和5S rDNA之间的一段2.2kb的同源重组热点区域,得到AB和CD两条rDNA片段。将AB、CD分别连接到pUC18的SacI/EcoRI和PstI位点,得到pUC18-Sr。以质粒pYX212作为模板,设计两对引物U1/U2和T1/T1,分别扩增得到营养标记基因URA3和包括组成型磷酸丙糖异构酶(triose-phosphate isomerase,TPI)启动子在内的表达盒(TPI cassette)片段,将URA3基因和表达盒依次连接到pUC18-Sr的SalI/XbaI和XbaI/KpnI位点,得到载体pNK1。此外,将表达盒和来自于质粒pYEX的选择标记Leu2-d基因片段依次连入载体pUC18-Sr的XbaI/KpnI和平端化的XbaI位点,得到载体pNK2。
为检验载体pNK1和pNK2的功能,将来自质粒pEGFP-N1的绿色荧光蛋白基因(GFP)的BamHI/HpaI片段连接到pNK1和pNK2的BamHI/XmaI位点,构建表达载体pNK1-GFP和pNK2-GFP。将两个载体线性化后转化酿酒酵母,在Ura和Leu选择培养基可以筛选到相应载体的酵母转化子。结果表明选择标记基因功能正常。通过荧光显微镜能够观察到过夜培养的转化子有绿色荧光,表明TPI启动子能够驱动绿色荧光蛋白基因表达。此外,利用Southern blot证明了GFP基因能够整合到酵母染色体中。在非选择性压力下,转化子经过近50代培养后,分别有99%的含有URA3基因的转化子和100%含有Leu-2d基因的转化子仍然能够在各自的选择培养基上生长。表明两种整合载体都能够保证整合于酵母的染色体中的GFP基因的遗传稳定性。在细胞浓度相同条件下,通过荧光显微镜观察到含有pNK1-GFP的转化子中有58%的细胞发出绿色荧光。故选择pNK1来进行人的重组胰高血糖素样肽-1类似物的表达。
以本实验室构建的十拷贝长效口服GLP-1(recombinant oval long-actingGLP-1,rolGLP-1)融合基因作为外源基因,利用多拷贝酿酒酵母整合载体pNK1构建表达载体pNK-GLP。用表达载体转化蛋白酶缺陷型酿酒酵母BJ2407,通过Ura~-营养缺陷培养基筛选转化子。随机挑取三个转化子,经过Southern blot鉴定和生长活力测定,选择转化子SG2用于进一步研究。小量培养的转化子SG2经过玻璃珠破碎,SDS-PAGE后,利用Western blot检测到一条约36kDa的蛋白条带,大小与预期相符,表明rolGLP-1融合蛋白得到表达。经过1:1接种培养的SG2裂解后,用Ni-NTA分离rolGLP-1融合蛋白。根据Bradford定量法和蛋白光密度分析算出rolGLP-1融合蛋白表达量的估计值为0.84mg/g湿细胞。在非选择培养基中的连续培养和Western blot鉴定表明,SG2中的rolGLP-1融合基因保持着良好的稳定性。利用冷冻干燥技术处理培养至对数期末的SG2,用冻干的SG2进行降低高血糖大鼠血糖水平的研究。
Wistar大鼠禁食10小时后,腹腔注射60mg/kg链脲佐菌素构建高血糖模型大鼠。造模成功的大鼠血糖水平为25.1±3.5mM。对高血糖大鼠分别进行5g重组酵母SG2/kg剂量灌胃;0.5 g重组酵母SG2/kg剂量灌胃和0.1 g重组酵母SG2/kg剂量灌胃;阳性对照组进行0.1g二甲双胍/kg剂量灌胃;模型组进行5g酵母BJ2407/kg剂量灌胃,所有处理持续20天。体内活性分析结果表明,模型组大鼠血糖依然维持高水平(26.2±2.9mM)。0.5 g重组酵母SG2/kg和0.1 g重组酵母SG2/kg处理组大鼠血糖水平没有明显降低。0.1g二甲双胍/kg和5g重组酵母SG2/kg服药量能够显著降低高血糖大鼠血糖水平。二甲双胍组大鼠血糖值由灌胃前的25.1±3.9mM下降到19.1±4.3mM;5g重组酵母SG2/kg处理组大鼠血糖水平由24.8±4.0mM下降到21.2±3.8mM。此外,大鼠饮水量、摄食量和体重等异常体征也明显改善。
综上所述,本研究以rDNA序列为整合位点分别构建了含有不同营养缺陷型选择标记和相同组成型启动子的多拷贝整合载体pNK1和pNK2,并以载体pNK1构建了含有十拷贝长效rolGLP-1融合基因的表达载体,通过转化酿酒酵母BJ2407得到稳定表达长效rolGLP-1融合蛋白的重组酵母SG2。生物活性试验结果表明,口服重组酵母SG2能够降低高血糖大鼠血糖水平,为进一步研发糖尿病预防和治疗性酵母饮品、食品或生物药物奠定了基础。
Currently,most peptide and protein drugs are still administered by the parenteral route because of insufficient absorption from the gastrointestinal tract.Therapeutic peptides and proteins are usually used for chronic conditions,and the use of injections during long-term treatment has obvious drawbacks.In contrast to this inconvenient and potentially problematic method of drug administration,the oral route offers the advantages of self-administration with a high degree of patient acceptability and compliance,and avoids the pain,discomfort,and possibility of infections caused by injectable dosage forms.Therefore,developing the effective and successful oral delivery system,in order to maximize the therapeutic potential of this class of agents, has been an ongoing topic in pharmaceutical research.It is an innovative way that recombinant microorganisms expressing therapeutic peptides and proteins are used as drug delivery system.Both bacteria and yeasts could be used as a new drug delivery vehicles to the gastrointestinal tract.
Glucagon-like peptide-1(GLP-1),so-called incretin,is a peptide hormone secreted from gut endocrine cells.It has lots of physiological actions,including glucose-dependent insulinotropic effect,stimulation ofβ-cell proliferation and so on. Therefore,GLP-1 has great therapeutic potential to treat diabetes type 2.However, the clinical application of native GLP-1 is limited by its very short half-life in vivo due principally to its rapid degradation by dipeptidyl peptidaseⅣ(DPPⅣ) and renal clearance.Accordingly,developing long-acting therapeutic GLP-1 analogs is of considerable interest.
S.cerevisiae(baker yeast) is rich in vitamins,digestive enzymes and so on.As we know,it is a long history that S.cerevisiae is used as tea or beverage in Taiwan, Hong Kong and other places.If S.cerevisiae cells could be utilized as a drug vehicle, or named sustained release "capsules",to intracellularly stably express therapeutic polypeptide drugs for diabetes type 2,we might develop its therapeutic use as functional drinks and food,and even biodrug.Thus,we can prevent and treat diabetes or other chronic diseases as we enjoy these delicious drinks or food.This strategy would be very attractive.
This study aims to construct recombinant S.cerevisiae intracellularly stably expressing polypeptide for the treatment of diabetes type 2 in order to lay the foundation for the development of the therapeutic use of yeast as drinks,food or biodrug.We start with the construction of yeast integrating vector containing the backbone of the multiple plasmid pUC18.For targeted integration of foreign gene into reiterated multi-copy rDNA sequence on yeast chromosome,we firstly designed two pairs of primers to amply two rDNA fragment,AB and CD.These two fragment located in a 2.2kb recombination hot region between 25SrDNA gene and 5SrDNA gene.Two fragments of AB and CD were successively cloned into EcoRⅠ/SacⅠand PstⅠsites of pUC18.The plasmid named pUC18-Sr was obtained.Using vector pYX212 as a template,two pairs of primers,U1-U2 and T1-T2,were designed to amply auxotropic selective marker URA3 gene and expression cassette containing TPI promoter,respectively.Afterwards,amplied URA3 gene and expression cassette fragment,were ligated into SalI/XbaI and XbaI/KpnI sites of pUC18-Sr.The vector pNK1 was obtained.In addition,expression cassette fragment and selective marker Leu2-d gene fragment from pYEX were successively ligated into XbaI/KpnI and blunted XbaI sites of pUC18-Sr.Vector pNK2 was generated.
To test the function of vectors pNK1 and pNK2,BamHI/HpaI fragment from plasmid pEGFP-N1 containing green fluorescent protein gene(GFP) was ligated into BamHI/XmaI site in expression cassette to construct expression vectors pNK1-GFP and pNK2-GFP.The linearized pNK1-GFP and pNK2-GFP were transformed into Saccharomycess cerevisiae.The transformants containing URA3 gene were selected on Ura selective medium.The transformants containing Leu-2d gene were selected on Leu selective medium.The results showed that the function of two selective genes is normal.After transformants were cultured overnight,green fluorescence from transforms was observed by fluorescence microscope,indicating promoter TPI might drive the expression of GFP gene.Furthermore,the results of Southern blot demonstrated that GFP gene was integrated into yeast genome.Under the non-selective condition,Above 99%transformant containing URA3 gene SG2 and 100%transforms containing Leu-2d gene still grew on Ura~- selective medium and on Leu~- selective medium,respectively,throughout sequential passage of 50 generations. The above results indicated that both pNK1 and pNK2 guarantee the mitotic stability of GFP gene integrated into yeast chromosome.Because 58%cells from transformants containing pNK1-GFP were of green fluorescence by fluorescence microscope observation in the same cell concentration,pNK1 was selected to express recombinant oral long-acting glucagons-like peptide-1(rolGLP-1).
Utilizing a ten-tandem-copy rolGLP-1 fusion gene constructed in our lab as a foreign gene,the expression vector pNK-GLP was constructed by introducing rolGLP-1 fusion gene into multi-copy Yip(pNK1).The linearized pNK-GLP was transformed into protease-deficient S.cerevisiae BJ2407,and the transformants were selected on auxotrophic(Ura~-) selective plate.Three transformants were randomly selected.Then,the positive transformants were identified by Southern blot.After growth determination in YPD medium,transformant SG2 was selected and used for further research.The small-scale cultures of transformant SG2,using glass beads,was broken.The cell lysate separated on the SDS-PAGE were analyzed by West blot,an approx.36kD protein band corresponding to the predictive molecular weight was detected.The analysis result indicated that the rolGLP-1 fusion protein was intracellularly expressed in transformant SG2.After growth in YPD medium with the inoculum and medium ratio of 1:1,SG2 was disrupted.The cell lysate was applied to Ni-NTA resin column,and rolGLP-1 fusion protein was purified.The expression level of rhGLP-1 analogue fusion protein was assessed by densitometric scaning,and the amount of rolGLP-1 fusion protein evaluated by Bradford method reached 0.84mg/g wet cells.After SG2 continuously growing in non-selective medium, rolGLP-1 fusion gene was of mitotically stable,and still was expressed in SG2 by analysis of Western blot.The SG2 cells in late exponential phase were dried by freeze-drying technique,and then lyophilized SG2 cells were used for the assessment of glucose-lowering effects in hyperglycemic rats.
After 10h fasting,Wistar rats were intravenously infected with 60mg streptozotocin/kg to induce the hyperglycemia.The serum glucose level of hyperglycemic rat was 25.1±3.5mM.The hyperglycemic rats were administered various doses(5g/kg,0.5g/kg,0.1g/kg) of recombinant yeast SG2,positive control group were given 0.1g/kg Metformin Hydrochloride and model group were given 5g/kg yeast BJ2407 by gastric gavages for 20 days.The analysis of in vivo activity indicated that serum glucose value of rats in model group still retained high level (26.2±2.9mM).Oral administration of recombinant yeast SG2 at 0.5g/kg/d and 0.1g/kg/d could not obviously lower serum glucose level.Taking 0.1g/kg/d Metformin Hydrochloride or 5g/kg/d SG2 significantly lowered serum glucose level. Oral administration of 0.1g/kg/d Metformin Hydrochloride decreased serum glucose level of hyperglycemic rats from 25.1±3.9 to 19.1±4.3mM(P＜0.01).Oral administration of SG2 at 5g/kg/d for 20 days also lowered serum glucose level of hyperglycemic rats from 24.8±4.0 to 21.2±3.8mM(P＜0.05).In addition,the abnormal changes of drinking amount,food intake and body weight of hyperglycemic rats also were significantly improved.
In summary,two multi-copy integration vectors of pNK1 and pNK2,using strategy based on integration into the ribosomal DNA cluster,were successfully constructed in this study.They contain,except for constitutive promoter TPI, auxotrophic selective marker URA3 gene and Leu-2d gene,respectively.Whereafter, the expression vector containing ten-tandem-copy rolGLP-1 fusion gene,pNK-GLP, was constructed and transformed into S.cerevisiae BJ2407.recombinant yeast SG2 stably expressing rolGLP-1 fusion protein was obtained.Meanwhile,the result of bioactivity test indicated that oral administration of recombinant yeast SG2 could lower serum glucose level of hyperglycemic rats.It laid the well foundation for further development of the use of recombinant S.cerevisiae as beverage,food or biodrug for the prevention and treatment of diabetes type 2.
胰高血糖素样肽-1(glucagon-like peptide-1,GLP-1)称为肠促胰岛素,是一种分泌自肠道细胞的肽激素,具有血糖依赖性促胰岛素分泌、刺激β-细胞增殖等多种生理功能。在2型糖尿病的临床治疗中具有较好的应用前景。但由于体内二肽基肽酶-Ⅳ(DPP-Ⅳ)的快速降解和肾脏代谢作用,导致其半衰期很短,使其临床应用受到了很大限制。因而,开发长效药用GLP-1类似物已成为受关注的一个热点。
酿酒酵母(面包酵母)本身富含维生素、消化酶等,我国台湾、香港等地素有饮用酵母茶或酵母饮料的悠久历史。若使其胞内再能稳定表达多肽类糖尿病治疗药物,以酿酒酵母细胞作为药物载体——缓释“胶囊”,研发治疗性功能酵母饮品、食品或生物药物,使人们在品尝美味饮品或食品的同时,起到预防和治疗糖尿病等慢性疾病的作用,前景十分诱人。
本研究旨在构建一种胞内稳定表达多肽类糖尿病治疗药物的酿酒酵母,为研发治疗性功能酵母饮品、食品或生物药物奠定基础。首先从构建以质粒pUC18作为骨架的整合型载体入手,以酿酒酵母染色体中多拷贝串联排列的rDNA序列作为外源基因多拷贝整合的位点,设计两对引物扩增rDNA序列中位于25S和5S rDNA之间的一段2.2kb的同源重组热点区域,得到AB和CD两条rDNA片段。将AB、CD分别连接到pUC18的SacI/EcoRI和PstI位点,得到pUC18-Sr。以质粒pYX212作为模板,设计两对引物U1/U2和T1/T1,分别扩增得到营养标记基因URA3和包括组成型磷酸丙糖异构酶(triose-phosphate isomerase,TPI)启动子在内的表达盒(TPI cassette)片段,将URA3基因和表达盒依次连接到pUC18-Sr的SalI/XbaI和XbaI/KpnI位点,得到载体pNK1。此外,将表达盒和来自于质粒pYEX的选择标记Leu2-d基因片段依次连入载体pUC18-Sr的XbaI/KpnI和平端化的XbaI位点,得到载体pNK2。
为检验载体pNK1和pNK2的功能,将来自质粒pEGFP-N1的绿色荧光蛋白基因(GFP)的BamHI/HpaI片段连接到pNK1和pNK2的BamHI/XmaI位点,构建表达载体pNK1-GFP和pNK2-GFP。将两个载体线性化后转化酿酒酵母,在Ura和Leu选择培养基可以筛选到相应载体的酵母转化子。结果表明选择标记基因功能正常。通过荧光显微镜能够观察到过夜培养的转化子有绿色荧光,表明TPI启动子能够驱动绿色荧光蛋白基因表达。此外,利用Southern blot证明了GFP基因能够整合到酵母染色体中。在非选择性压力下,转化子经过近50代培养后,分别有99%的含有URA3基因的转化子和100%含有Leu-2d基因的转化子仍然能够在各自的选择培养基上生长。表明两种整合载体都能够保证整合于酵母的染色体中的GFP基因的遗传稳定性。在细胞浓度相同条件下,通过荧光显微镜观察到含有pNK1-GFP的转化子中有58%的细胞发出绿色荧光。故选择pNK1来进行人的重组胰高血糖素样肽-1类似物的表达。
以本实验室构建的十拷贝长效口服GLP-1(recombinant oval long-actingGLP-1,rolGLP-1)融合基因作为外源基因,利用多拷贝酿酒酵母整合载体pNK1构建表达载体pNK-GLP。用表达载体转化蛋白酶缺陷型酿酒酵母BJ2407,通过Ura~-营养缺陷培养基筛选转化子。随机挑取三个转化子,经过Southern blot鉴定和生长活力测定,选择转化子SG2用于进一步研究。小量培养的转化子SG2经过玻璃珠破碎,SDS-PAGE后,利用Western blot检测到一条约36kDa的蛋白条带,大小与预期相符,表明rolGLP-1融合蛋白得到表达。经过1:1接种培养的SG2裂解后,用Ni-NTA分离rolGLP-1融合蛋白。根据Bradford定量法和蛋白光密度分析算出rolGLP-1融合蛋白表达量的估计值为0.84mg/g湿细胞。在非选择培养基中的连续培养和Western blot鉴定表明,SG2中的rolGLP-1融合基因保持着良好的稳定性。利用冷冻干燥技术处理培养至对数期末的SG2,用冻干的SG2进行降低高血糖大鼠血糖水平的研究。
Wistar大鼠禁食10小时后,腹腔注射60mg/kg链脲佐菌素构建高血糖模型大鼠。造模成功的大鼠血糖水平为25.1±3.5mM。对高血糖大鼠分别进行5g重组酵母SG2/kg剂量灌胃;0.5 g重组酵母SG2/kg剂量灌胃和0.1 g重组酵母SG2/kg剂量灌胃;阳性对照组进行0.1g二甲双胍/kg剂量灌胃;模型组进行5g酵母BJ2407/kg剂量灌胃,所有处理持续20天。体内活性分析结果表明,模型组大鼠血糖依然维持高水平(26.2±2.9mM)。0.5 g重组酵母SG2/kg和0.1 g重组酵母SG2/kg处理组大鼠血糖水平没有明显降低。0.1g二甲双胍/kg和5g重组酵母SG2/kg服药量能够显著降低高血糖大鼠血糖水平。二甲双胍组大鼠血糖值由灌胃前的25.1±3.9mM下降到19.1±4.3mM;5g重组酵母SG2/kg处理组大鼠血糖水平由24.8±4.0mM下降到21.2±3.8mM。此外,大鼠饮水量、摄食量和体重等异常体征也明显改善。
综上所述,本研究以rDNA序列为整合位点分别构建了含有不同营养缺陷型选择标记和相同组成型启动子的多拷贝整合载体pNK1和pNK2,并以载体pNK1构建了含有十拷贝长效rolGLP-1融合基因的表达载体,通过转化酿酒酵母BJ2407得到稳定表达长效rolGLP-1融合蛋白的重组酵母SG2。生物活性试验结果表明,口服重组酵母SG2能够降低高血糖大鼠血糖水平,为进一步研发糖尿病预防和治疗性酵母饮品、食品或生物药物奠定了基础。
Currently,most peptide and protein drugs are still administered by the parenteral route because of insufficient absorption from the gastrointestinal tract.Therapeutic peptides and proteins are usually used for chronic conditions,and the use of injections during long-term treatment has obvious drawbacks.In contrast to this inconvenient and potentially problematic method of drug administration,the oral route offers the advantages of self-administration with a high degree of patient acceptability and compliance,and avoids the pain,discomfort,and possibility of infections caused by injectable dosage forms.Therefore,developing the effective and successful oral delivery system,in order to maximize the therapeutic potential of this class of agents, has been an ongoing topic in pharmaceutical research.It is an innovative way that recombinant microorganisms expressing therapeutic peptides and proteins are used as drug delivery system.Both bacteria and yeasts could be used as a new drug delivery vehicles to the gastrointestinal tract.
Glucagon-like peptide-1(GLP-1),so-called incretin,is a peptide hormone secreted from gut endocrine cells.It has lots of physiological actions,including glucose-dependent insulinotropic effect,stimulation ofβ-cell proliferation and so on. Therefore,GLP-1 has great therapeutic potential to treat diabetes type 2.However, the clinical application of native GLP-1 is limited by its very short half-life in vivo due principally to its rapid degradation by dipeptidyl peptidaseⅣ(DPPⅣ) and renal clearance.Accordingly,developing long-acting therapeutic GLP-1 analogs is of considerable interest.
S.cerevisiae(baker yeast) is rich in vitamins,digestive enzymes and so on.As we know,it is a long history that S.cerevisiae is used as tea or beverage in Taiwan, Hong Kong and other places.If S.cerevisiae cells could be utilized as a drug vehicle, or named sustained release "capsules",to intracellularly stably express therapeutic polypeptide drugs for diabetes type 2,we might develop its therapeutic use as functional drinks and food,and even biodrug.Thus,we can prevent and treat diabetes or other chronic diseases as we enjoy these delicious drinks or food.This strategy would be very attractive.
This study aims to construct recombinant S.cerevisiae intracellularly stably expressing polypeptide for the treatment of diabetes type 2 in order to lay the foundation for the development of the therapeutic use of yeast as drinks,food or biodrug.We start with the construction of yeast integrating vector containing the backbone of the multiple plasmid pUC18.For targeted integration of foreign gene into reiterated multi-copy rDNA sequence on yeast chromosome,we firstly designed two pairs of primers to amply two rDNA fragment,AB and CD.These two fragment located in a 2.2kb recombination hot region between 25SrDNA gene and 5SrDNA gene.Two fragments of AB and CD were successively cloned into EcoRⅠ/SacⅠand PstⅠsites of pUC18.The plasmid named pUC18-Sr was obtained.Using vector pYX212 as a template,two pairs of primers,U1-U2 and T1-T2,were designed to amply auxotropic selective marker URA3 gene and expression cassette containing TPI promoter,respectively.Afterwards,amplied URA3 gene and expression cassette fragment,were ligated into SalI/XbaI and XbaI/KpnI sites of pUC18-Sr.The vector pNK1 was obtained.In addition,expression cassette fragment and selective marker Leu2-d gene fragment from pYEX were successively ligated into XbaI/KpnI and blunted XbaI sites of pUC18-Sr.Vector pNK2 was generated.
To test the function of vectors pNK1 and pNK2,BamHI/HpaI fragment from plasmid pEGFP-N1 containing green fluorescent protein gene(GFP) was ligated into BamHI/XmaI site in expression cassette to construct expression vectors pNK1-GFP and pNK2-GFP.The linearized pNK1-GFP and pNK2-GFP were transformed into Saccharomycess cerevisiae.The transformants containing URA3 gene were selected on Ura selective medium.The transformants containing Leu-2d gene were selected on Leu selective medium.The results showed that the function of two selective genes is normal.After transformants were cultured overnight,green fluorescence from transforms was observed by fluorescence microscope,indicating promoter TPI might drive the expression of GFP gene.Furthermore,the results of Southern blot demonstrated that GFP gene was integrated into yeast genome.Under the non-selective condition,Above 99%transformant containing URA3 gene SG2 and 100%transforms containing Leu-2d gene still grew on Ura~- selective medium and on Leu~- selective medium,respectively,throughout sequential passage of 50 generations. The above results indicated that both pNK1 and pNK2 guarantee the mitotic stability of GFP gene integrated into yeast chromosome.Because 58%cells from transformants containing pNK1-GFP were of green fluorescence by fluorescence microscope observation in the same cell concentration,pNK1 was selected to express recombinant oral long-acting glucagons-like peptide-1(rolGLP-1).
Utilizing a ten-tandem-copy rolGLP-1 fusion gene constructed in our lab as a foreign gene,the expression vector pNK-GLP was constructed by introducing rolGLP-1 fusion gene into multi-copy Yip(pNK1).The linearized pNK-GLP was transformed into protease-deficient S.cerevisiae BJ2407,and the transformants were selected on auxotrophic(Ura~-) selective plate.Three transformants were randomly selected.Then,the positive transformants were identified by Southern blot.After growth determination in YPD medium,transformant SG2 was selected and used for further research.The small-scale cultures of transformant SG2,using glass beads,was broken.The cell lysate separated on the SDS-PAGE were analyzed by West blot,an approx.36kD protein band corresponding to the predictive molecular weight was detected.The analysis result indicated that the rolGLP-1 fusion protein was intracellularly expressed in transformant SG2.After growth in YPD medium with the inoculum and medium ratio of 1:1,SG2 was disrupted.The cell lysate was applied to Ni-NTA resin column,and rolGLP-1 fusion protein was purified.The expression level of rhGLP-1 analogue fusion protein was assessed by densitometric scaning,and the amount of rolGLP-1 fusion protein evaluated by Bradford method reached 0.84mg/g wet cells.After SG2 continuously growing in non-selective medium, rolGLP-1 fusion gene was of mitotically stable,and still was expressed in SG2 by analysis of Western blot.The SG2 cells in late exponential phase were dried by freeze-drying technique,and then lyophilized SG2 cells were used for the assessment of glucose-lowering effects in hyperglycemic rats.
After 10h fasting,Wistar rats were intravenously infected with 60mg streptozotocin/kg to induce the hyperglycemia.The serum glucose level of hyperglycemic rat was 25.1±3.5mM.The hyperglycemic rats were administered various doses(5g/kg,0.5g/kg,0.1g/kg) of recombinant yeast SG2,positive control group were given 0.1g/kg Metformin Hydrochloride and model group were given 5g/kg yeast BJ2407 by gastric gavages for 20 days.The analysis of in vivo activity indicated that serum glucose value of rats in model group still retained high level (26.2±2.9mM).Oral administration of recombinant yeast SG2 at 0.5g/kg/d and 0.1g/kg/d could not obviously lower serum glucose level.Taking 0.1g/kg/d Metformin Hydrochloride or 5g/kg/d SG2 significantly lowered serum glucose level. Oral administration of 0.1g/kg/d Metformin Hydrochloride decreased serum glucose level of hyperglycemic rats from 25.1±3.9 to 19.1±4.3mM(P＜0.01).Oral administration of SG2 at 5g/kg/d for 20 days also lowered serum glucose level of hyperglycemic rats from 24.8±4.0 to 21.2±3.8mM(P＜0.05).In addition,the abnormal changes of drinking amount,food intake and body weight of hyperglycemic rats also were significantly improved.
In summary,two multi-copy integration vectors of pNK1 and pNK2,using strategy based on integration into the ribosomal DNA cluster,were successfully constructed in this study.They contain,except for constitutive promoter TPI, auxotrophic selective marker URA3 gene and Leu-2d gene,respectively.Whereafter, the expression vector containing ten-tandem-copy rolGLP-1 fusion gene,pNK-GLP, was constructed and transformed into S.cerevisiae BJ2407.recombinant yeast SG2 stably expressing rolGLP-1 fusion protein was obtained.Meanwhile,the result of bioactivity test indicated that oral administration of recombinant yeast SG2 could lower serum glucose level of hyperglycemic rats.It laid the well foundation for further development of the use of recombinant S.cerevisiae as beverage,food or biodrug for the prevention and treatment of diabetes type 2.
引文
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