CN107254419B - 一种具有头孢抗性且高表达Sir2蛋白的枯草芽孢杆菌及其应用 - Google Patents
一种具有头孢抗性且高表达Sir2蛋白的枯草芽孢杆菌及其应用 Download PDFInfo
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- CN107254419B CN107254419B CN201710437277.4A CN201710437277A CN107254419B CN 107254419 B CN107254419 B CN 107254419B CN 201710437277 A CN201710437277 A CN 201710437277A CN 107254419 B CN107254419 B CN 107254419B
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Abstract
本发明公开了一种具有头孢抗性且高表达Sir2蛋白的枯草芽孢杆菌Bacillus subtilis BS16及其应用。本发明具有头孢抗性且高表达Sir2蛋白的枯草芽孢杆菌Bacillus subtilis BS16保藏于广东省微生物菌种保藏中心,保藏号为GDMCC No.60193,保藏地址为广州市先烈中路100号大院59号楼5楼,保藏日期为2017年6月2日。本发明经实验发现,具有头孢抗性且高表达Sir2蛋白的枯草芽孢杆菌Bacillus subtilis BS16可以增强细胞葡萄糖有氧氧化,提高线粒体呼吸,对ATP能量代谢通路进行调控,因此,枯草芽孢杆菌Bacillus subtilis BS16可实现对细胞衰老、线粒体功能障碍和代谢综合征等多种代谢相关疾病的治疗。
Description
技术领域
本发明属新军中筛选和应用机制领域,更具体地说,本发明涉及一种具有头孢抗性且高表达Sir2蛋白的枯草芽孢杆菌Bacillus subtilis BS16及其应用。
背景技术
Sir2蛋白家族(Sirtuin)是一种保守蛋白,它主要作为NAD+依赖性蛋白去乙酰化酶,其存在于包括古细菌到哺乳动物各种生物中。目前很多的研究已经表明哺乳动物Sirtuin蛋白家族参与调节细胞代谢,并且有助于延缓,甚至阻止代谢相关疾病。另外,越来越多的研究也表示益生菌可以改善代谢疾病等。益生菌也具有Sir2同源蛋白,然而关于sir2蛋白在益生菌中的生物学功能少有报道。
研究表明,sirtuins广泛参与调节机体能量代谢调节,保持机体糖脂代谢稳态,其中以SIRT1、SIRT3、SIRT6研究的最为广泛。SIRT1是该家族研究最多的一个成员,当前的研究已经表明其不但可以调控组蛋白的去乙酰化作用,而且还对非组蛋白也有很好的调节作用。在体内和体外SIRT1可通过PGC-1α转录因子来调节糖异生、糖酵解,导致线粒体数量和功能的增加。另外有研究表明,SIRT1可以改善线粒体功能障碍,当过表达SIRT1时改善高糖诱导的骨骼肌细胞胰岛素抵抗,进一步研究发现这是通过调节SIRT1-SIRT3-线粒体的途径来实现的。在肌肉中,SIRT1能够通过去乙酰化PGC1-α来增强线粒体中的脂肪酸氧化。另外SIRT3、SIRT4和SIRT5属于线粒体Sirtuin蛋白,它们位于线粒体中。目前,SIRT3是最主要的一种线粒体去乙酰化酶,它的很多靶蛋白已被确定,其中有许多参与调节重要的代谢平衡。例如SIRT3去乙酰化而激活异柠檬酸脱氢酶2(IDH2;它参与三羧酸循环(TCA循环))。SIRT3也激活TCA循环谷氨酸脱氢酶(GDH),尽管GDH脱乙酰的生理意义尚不清楚。此外,SIRT3去乙酰化复合体I,复合体II以及复合体III,它们参与氧化磷酸化(OXPHOS),线粒体有氧呼吸的最后阶段。在HEK293T细胞内过表达线粒体sirtuin蛋白改变糖酵解和线粒体功能。SIRT6是仅次于SIRT1、SIRT3相比,研究的比较深入的Sirtuin蛋白。SIRT6参预基因组DNA的稳定和修复、代谢和衰老的作用。已有研究表示SIRT6缺失小鼠会出现低血糖症状,而在SIRT6缺失的细胞中Hif1α活性上升,并且葡萄糖摄取增加,糖酵解增强而线粒体呼吸功能减弱。总结以上研究,不难发现Sirtuin蛋白与细胞能量代谢密切相关,而且在一定程度上它们充当着糖酵解到氧化代谢的开关。
益生菌,作为肠道微生物显示了良好的改善宿主代谢疾病的作用,但是其具体的分子机制并不清楚。Sir2基因家族是一种保守的从古细菌到哺乳动物都存在的NAD+依赖的组蛋白/非组蛋白去乙酰化酶。目前很多的研究已经表明哺乳动物Sirtuin蛋白家族可以调节细胞代谢,包括细胞呼吸,糖酵解,糖异生,脂肪酸代谢等。sirtuins正常活性的维持有助于延缓,甚至阻止代谢相关疾病的发生。目前,有关益生菌sir2蛋白生物学功能少有报道。是否益生菌Sir2蛋白参与调节宿主代谢,并作为益生菌调节代谢疾病的一个靶点值得研究。
目前对于益生菌Sir2同源蛋白的研究还处于起始阶段,只有枯草芽孢杆菌Sir2同源蛋白被确定,是由yhdz基因编码。而其他益生菌的sir2同源蛋白并没有相关的研究。以往对于哺乳动物Sirtuin蛋白家族的研究,表明其广泛参与糖脂代谢的调控及维持能量代谢平衡,并且有望成为代谢疾病的靶点。那么是否益生菌Sir2蛋白参与细胞代谢,并且充当益生菌调节宿主代谢的分子靶点目前并没有相关研究,这些是值得深入研究的。
发明内容
本发明筛选出具有头孢抗性且高表达Sir2蛋白的枯草芽孢杆菌Bacillussubtilis BS16并研究其作用机制,使其在疾病治疗中发挥作用。
本发明提供了一种具有头孢抗性且高表达Sir2蛋白的枯草芽孢杆菌Bacillussubtilis BS16,保藏于广东省微生物菌种保藏中心,保藏号为GDMCC No.60193,保藏地址为广州市先烈中路100号大院59号楼5楼,保藏日期为2017年6月2日。
本发明具有头孢抗性且高表达Sir2蛋白的枯草芽孢杆菌Bacillus subtilisBS16可用于制备治疗抗衰老、线粒体功能障碍或代谢综合征的药物。
本发明还提供了一种抗衰老食品,包括有效剂量的作为活性成分的具有头孢抗性且高表达Sir2蛋白的枯草芽孢杆菌Bacillus subtilis BS16和/或其菌体蛋白成分。
本发明还提供了一种抗衰老药品,包括有效剂量的作为活性成分的具有头孢抗性且高表达Sir2蛋白的枯草芽孢杆菌Bacillus subtilis BS16和/或其菌体蛋白成分,以及药学上可接受的载体。
本发明还提供了一种预防和治疗细胞线粒体功能障碍的食品,包括有效剂量的作为活性成分的具有头孢抗性且高表达Sir2蛋白的枯草芽孢杆菌Bacillus subtilis BS16和/或其菌体蛋白成分。
本发明还提供了一种预防和治疗细胞线粒体功能障碍的药物,包括有效剂量的作为活性成分的具有头孢抗性且高表达Sir2蛋白的枯草芽孢杆菌Bacillus subtilis BS16和/或其菌体蛋白成分,以及药学上可接受的载体。
本发明还提供了一种预防和治疗代谢综合征的食品,包括有效剂量的作为活性成分的具有头孢抗性且高表达Sir2蛋白的枯草芽孢杆菌Bacillus subtilis BS16和/或其菌体蛋白成分。
本发明还提供了一种预防和治疗代谢综合征的药物,包括有效剂量的作为活性成分的具有头孢抗性且高表达Sir2蛋白的枯草芽孢杆菌Bacillus subtilis BS16和/或其菌体蛋白成分,以及药学上可接受的载体。
相对于现有技术,本发明经实验发现,具有头孢抗性且高表达Sir2蛋白的枯草芽孢杆菌Bacillus subtilis BS16可以增强细胞葡萄糖有氧氧化,提高线粒体呼吸,对ATP能量代谢通路进行调控,因此,枯草芽孢杆菌Bacillus subtilis BS16可实现对细胞衰老、线粒体功能障碍和代谢综合征等多种代谢相关疾病的治疗和预防。
附图说明
图1为实施例1枯草芽孢杆菌sir2基因鉴定结果。
图2为实施例1枯草芽孢杆菌蛋白电泳图。
图3为实施例1枯草芽孢杆菌Sir2蛋白Western blot结果。
图4为实施例2枯草芽孢杆菌sir2基因的PCR片段大小鉴定。
图5为实施例2pEGFP-N1-BS-sir2双酶切鉴定结果。
图6为实施例2pEGFP-N1-BS-sir2转染HEK293T细胞的荧光显微镜结果,其中,从左至右依次为对照组、空质粒转染组vector,重组质粒pEGFP-N1-BS-sir2转染组Sir2。
图7为实施例2重组质粒转染HEK293T细胞所表达融合蛋白的Western Blot蛋白检测结果,其中,1为空白对照组,2为pEGFP-N1空质粒组,3为pEGFP-N1-BS-sir2组。
图8为实施例2重组质粒转染HEK293T细胞后的ATP水平。
图9为实施例2免疫共沉淀结果BS-sir2基因对葡萄糖代谢酶活力的影响。
图10为实施例2BS-sir2基因乳酸水平的影响。
图11为实施例2BS-sir2影响线粒体呼吸结果。
具体实施方式
以下结合实施例,对本发明进行进一步详细说明。
实施例1
1.实验方法材料
1.1益生菌初筛
收集南阳市人民医院的3个青年病人粪便样品各1.0g,用LB培养基扩增培养,用溴化乙锭法去除质粒,蒸馏水稀释至10-8共9个稀释度,取1mL,涂布到LB固体培养基上,置于培养箱中,37培养48h。培养结束后,取出平板挑选出表面粗糙不透明,呈污白色或微黄色菌落,并转接至LB液体培养基中在37培养48h,并放入4冰箱保存备用。
1.2菌株的形态学观察。
将分离纯化的菌株纯培养物接种到增菌液体培养基中,37培养24h复活后,经革兰氏染色操作后,在光学显微镜下观察其细胞形态特征,并用带有数字成像系统的显微镜选取合适视野进行照相记录。
1.3生理生化特征鉴定
根据《伯杰氏细菌鉴定手册》和《常见细菌系统鉴定手册》鉴定分离菌株。
1.4枯草芽孢杆菌分子生物学-16S rDNA鉴定。
16S rDNA全长通用引物:
引物1:27F--SEQ ID NO:1;
引物2:1492R--SEQ ID NO:2;
PCR反应体系(25μL):引物1/1.0μL;引物2/1.0μL;10×PCR Buffer/2.5μL;dNTPmix/2.0μL;Taq酶/0.3μL;DNA模板/1.0μL;超纯水/25μL。
PCR反应条件为:先94——4min;再94——30s,55——40s,72——90s下循环30次,最后72——10min。
以无菌去离子水替代模板作为阴性对照。扩增结束后取3.0μL扩增后的产物进行琼脂糖凝胶电泳。电泳结束后,切割待测胶带送上海生工进行序列测定。将测定菌株的序列结果与NCBI中已知的16S rDNA序列进行Blast对比分析得到最相近的菌株,以确定实验所分离细菌的种属。
1.5抑菌活性测定
待测菌为上述获得的Bacillus subtilis BS12,Bacillus subtilis BS14,Bacillus subtilis BS16,分别取各菌100μL于100mL的LB液体培养基中进行活化复苏。指示菌为Escherichia coli ATCC11105、Clostridium difficile NY-5、Staphylococcusaureus NT-12。
1.6药敏试验
选择10种抗菌药物药敏纸片:头孢吡肟(FEP,20μg),头孢噻肟(CTX,20μg),利福平(Rf,10μg),氨苄西林(AM,10μg),四环素(TE,20μg),氯霉素(CHL,20μg),环丙沙星(CIP,10μg),阿米卡星(AK,20μg),庆大霉素(GM,10μg),甲氧苄氨嘧啶(TMP,10μg),均购自广州市宜康生物科技有限公司(Oxoid)。
根据美国临床实验室标准化协会颁布的KB法进行药敏试验。首先用无菌镊子分别夹取含有不同抗生素的药敏纸片,分别贴在已接种待测菌的各平板(200μL待测菌菌悬液含菌数为3.0×108CFU/mL,分别加入到温度为50左右的LB琼脂培养基)中,迅速混合均匀,倒平板。每个平板贴大约5张药敏纸片,各纸片间距离大致相等,并做好标记。在37过夜培养,观察平板抑菌情况,游标卡尺测定抑菌圈的大小并记录
1.7Sir2基因的鉴定。
以待测菌的基因组DNA为模板;
枯草芽孢杆菌sir2引物:
BS-sir2-F:SEQ ID NO:3;
BS-sir2-R:SEQ ID NO:4。
PCR反应体系:模板——1μL;引物BS-sir2-F——0.5μL;引物BS-sir2-R——0.5μL;dNTP(10mM)——0.5μL;LA Taq(5U/μL)——0.5μL;10×PCR buffer——2.5μL;灭菌水——19.5μL。
1.8(1)配置蛋白胶。分离胶和浓缩胶配方如下:
表1
(2)灌胶:混匀后用移液枪将凝胶溶液沿玻棒小心注入到长、短玻璃板间的狭缝内(胶高度距样品模板梳齿下缘约1cm);
(3)液封:在凝胶表面沿短玻板边缘轻轻加一层水以隔绝空气,使胶面平整。静置约30min观察胶面变化,当看到水与凝固的胶面有折射率不同的界限时,表明胶已完全凝固,倒掉上层水,并用滤纸吸干残留的水液;
(4)插入制胶梳:混匀后用移液枪将凝胶溶液注入到长、短玻璃板间的狭缝内(分离胶上方),轻轻加入样品模板梳,小心避免气泡的出现。约30分钟,聚合完全;
(5)安装电泳槽:将制备好的凝胶板取下,小心拔下梳子。两块10%的凝胶板分别插到U形橡胶框的两边凹形槽中,可往上提起使凝胶板紧贴橡胶。将装好玻璃板的胶模框平放在仰放的贮槽框上,其下缘与贮槽框下缘对齐,放入电泳槽内。倒入1X tris-gly电泳缓冲液;
(6)样品处理:对于蛋白样品直接取80μl的样品,依次加上20μl 5x buffer(加了B-巯基乙醇),混匀。对于菌体或组织等固体样品,取少量样品加100ul 2x buffer(加了B-巯基乙醇)煮沸10分钟;
(7)加样:用移液枪取处理过的样品溶液10μl,小心地依次加入到各凝胶凹形样品槽内,marker加入到其中一个槽内,为区别两块板,marker可加在不同的孔槽中;
(8)剥离胶:把电泳槽取出,两块板拿下来,用刮片从长短玻片中间翘起,再把浓缩胶刮掉,取下;
(9)染色:放于加有R250染色液的染色皿中,染液漫过胶即可,置于摇床上,转速约为45r/min,时间约1小时,完成后染液倒掉并用水洗掉染液;
(10)凝胶成像系统对蛋白电泳胶拍照,并保存图片。
1.9Western blotting
(1)制胶:配置12%的分离胶并注入玻璃板间隙至离上边缘1.5cm处,上层加适量75%乙醇;待凝固,倒掉上层液体,注入5%浓缩胶,插入梳子,自然晾干。
(2)上样:将蛋白质样品100水浴3min,在电泳槽中倒入新配置的电泳液,分别在两边泳道加入8μl和5μl蛋白maker,用1×蛋白上样缓冲液补足体积到10μl,其余泳道各加10μl样品,50V恒压跑电泳。
(3)转膜:电泳结束后,剪取11cm×8cm滤纸和合适大小的0.22μm的PVDF膜,用甲醇活化5min,按照“海绵-6层滤纸-凝胶-PVDF膜-6层滤纸-海绵”组装转印夹层,夹板组装后转移至转膜槽,200mA恒流转移60min。
(4)孵育抗体:转膜结束后,TBS液洗膜5min,5%的脱脂奶粉封闭1h,TBST液洗膜3次,每次5min,加一抗兔抗人SIRT3多克隆抗体稀释液4过夜孵育;TBST液洗膜3次,每次5min,加二抗兔抗稀释液室温孵育1h。
(5)发光检测:TBST液洗膜,加发光液孵育3min,用滤纸吸干发光液,将胶片和PVDF膜放入压片盒中压片1min,取出胶片定影30s,用水清洗,烘干,扫描。
2.实验结果
2.1枯草芽孢杆菌生理生化结果如表2。
表2生理生化试验
2.2 16S rDNA鉴定结果。
进入NCBI核酸数据库http://blast.ncbi.nlm.nih.gov/Blast.cgi,输入待测菌株的16S rDNA序列,点击Blast进入比对。
比对结果显示,有三种菌株的16S rDNA序列与其它多株枯草芽孢杆菌的16S rDNA序列有99%的相似性,初步判断为枯草芽孢杆菌(Bacillus subtilis),将其命名为Bacillus subtilis BS12,Bacillus subtilis BS14,Bacillus subtilis BS16。
2.3抑菌活性测定
抑菌活性的试验结果,见表3。其中Bacillus subtilis BS12和Bacillussubtilis BS16都有一定的抗菌能力,但Bacillus subtilis BS14的抗菌能力较弱。
表3 Bacillus subtilis抑菌活性的试验结果
2.4药敏试验,结果见表4所示。我们筛选到的两株Bacillus subtilis BS12和Bacillus subtilis BS16都具有很强的头孢抗性。
表4 Bacillus subtilis的药敏试验结果
注:S-敏感;R-耐药;I-中度耐药。
2.5 Sir2基因PCR鉴定结果
如图1所示,在744bp左右分别扩增出二条枯草芽孢杆菌sir2基因条带。
2.6 SDS-PAGE结果
如图2所示,两株枯草芽孢杆菌Sir2蛋白分别在27KD处有一条明亮的条带。
2.7 Western blotting
Western blot结果显示,Bacillus subtilis BS16的Sir2蛋白表达量最高,因此,成功筛选到一株带有头孢抗性且高表达Sir2蛋白的枯草芽孢杆菌Bacillus subtilisBS16。
实施例2枯草芽孢杆菌Bacillus subtilis BS16sir2(BS-sir2)基因转染细胞及胞内功能研究
1.实验材料
1.1细菌、质粒和细胞
1.2主要培养基及试剂
(1)缓冲液A:由150mM氯化钠,500mM Tris-HCl,0.1%TritoX-100,pH8.0。
(2)缓冲液B:由50mM Tris-HCl,0.1%TritonX-100,pH8.0构成。
(3)抗体稀释液:称取BSA粉末10g溶解于200mL TBST缓冲液中,即为5%BSA溶液,4℃保存备用。
(4)细胞生长液:胎牛血清(终浓度为10%)和青霉素链霉素溶液(终浓度为100单位/毫升)被添加到培养液中,4℃保存备用。
(5)细胞维持液:胎牛血清和青霉素链霉素溶液(终浓度为100单位/毫升)(终浓度为2%)被添加到培养液中,4℃保存备用。
(6)胰蛋白酶溶液:称取氯化钠8.0g,氯化钾0.2g,磷酸氢二钠0.2g,磷酸二氢钾0.2g,胰酶2.5g,加超纯水定容至1L,再过滤除菌,分装并封口,-20℃保存备用。
(7)10%SDS(十二烷基硫酸钠):取10g十二烷基硫酸钠溶解在90mL去离子水中,然后调节溶液pH值至7即可。4%SDS:取SDS 4g溶解于96mL去离子水中,调节溶液pH到7.0。
(8)PBS磷酸盐缓冲液:称取氯化钠1.6g,氯化钾0.04g,NaHPO4-12H2O0.698g,磷酸二氢钾0.04g,加超纯水至200mL,均匀混合,调PH至7.2,将配制好的PBS溶液高压灭菌30min,之后分装并封住瓶口,-20℃保存备用。
(9)主要试剂
1.4实验方法
1.4.1真核表达载体构建及鉴定
1.4.1.1 BS-sir2基因扩增
(1)利用Primier 5.0软件设计特异性引物,BS-sir2基因两端分别加入EcoR I、BamH I酶切位点。
BS-sir2-FF:SEQ ID NO:5,其中,GAATTC为EcoR I酶切位点
BS-sir2-RR:SEQ ID NO:6,其中,GGATCC为BamH I酶切位点)
(2)PCR反应体系:
(3)PCR反应条件
1.4.1.2 BS-sir2基因连接进入质粒pEGFP-N1
(1)插入片段的双酶切
(2)pEGFP-N1的双酶切
(3)内切酶灭活:酶切结束后,在60℃水浴放置25min
(4)连接反应
连接后,37℃下,PCR仪恒温反应20h,用1%琼脂糖凝胶检测并回收目的质粒pEGFP-N1-BS-sir2。
1.4.1.3重组质粒pEGFP-N1-BS-sir2的鉴定
CaCl2法将连接体系pEGFP-N1-BS-sir2转化大肠杆菌感受态细胞后涂布于Kan抗性的LB固体平板上进行筛选,置于37℃生化恒温培养箱中倒置培养过夜。随机挑选转化子进行菌落PCR验证,筛选阳性菌落。提质粒,用EcoR I、BamH I进行双酶切鉴定,挑选阳性克隆菌液进行测序分析。
1.4.2脂质体转染
根据Lipofectamine 2000脂质体转染试剂盒说明,具体操作如下:
(1)在24孔板中(每孔1-2×105细胞)接种500μl不含抗生素和血清的DMEM培养基,至转染时细胞可长至90-95%融合;
(2)质粒DNA和Lipofectamine 2000分别单独用50μl Opti-ME I无血清培养基稀释,充分混匀,室温孵育5分钟;
(3)将上述所稀释的DNA和Lipofectamine 2000混匀(总体积为100μl),室温静置20分钟;
(4)加入100μl转染液在每孔细胞中,轻轻摇匀;
(5)37℃,5%CO2培养4-6h后可更换培养基,24h后裂解细胞,以备检测基因表达。
1.4.3.检测绿色荧光蛋白(GFP)的表达
质粒pEGFP-N1-BS-sir2经Lipofectamine 2000介导转染293T细胞,37℃CO2培养箱中培养4h后,将无抗无血清培养基换成正常培养基继续培养。48h左右荧光显微镜观察绿色荧光,拍照。
1.4.4通过Western blot检测BS-sir2的表达情况
(1)收集经筛选的稳定表达细胞株于1.5ml EP管,加入100ul预冷的细胞裂解液和1ul蛋白酶抑制剂,涡旋两次,每次间隔5min,每次5s。
(2)冰上静置20min后4、12000g离心7min,收集上清液即为细胞总蛋白。采用BCAProtein Assay试剂盒定量。采用BCA Protein Assay试剂盒定量。
(3)取20ug蛋白加入上样缓冲液,沸水中煮5min后进行SDS-PAGE分离(分离胶浓度为10%),100V恒压转移1h50min至PVDF膜上,于含5%脱脂奶粉的TBST缓冲液封闭1h,以封闭液稀释小鼠抗Anti-EGFP单抗(1∶500)4过夜。
(4)次日用0.1%PBST洗三次,每次10min,加入用PBS稀释的羊抗鼠IgG-HRP二抗(1∶1000)室温避光摇床孵育1h,用0.1%PBST洗三次,洗涤同上,再用0.1M PBS洗5min后扫膜。以β-actin作为内参。
1.4.5 ATP含量测定
根据ATP含量测定试剂盒说明书操作:
(1)在冰上溶解待用的试剂,利用ATP测定裂解液将ATP标准溶液稀释成0.1、1、10μM/L的浓度,制备标准曲线。
(2)细胞培养皿中加入200μl裂解液,反复吹打至充分裂解,4下12000rmp离心10min,取上清。
(3)稀释ATP检测工作液,每个样品重复3次,每个监测孔中加100μl ATP检测工作液,室温放置5min,消耗掉本地ATP,在检测孔中加上100μl待测样品或标准样品,充分混匀,间隔2s,立即用生物发光仪检测CMP值。根据计算公式利用测出的标准曲线计算待测样品中ATP浓度。
1.4.6丙酮酸激酶活力检测
根据丙酮酸激酶试剂盒说明书操作:
(1)取400μl细胞提取液,超声波破碎细胞(功率20%,超声3s,间隔10s,重复30次),8000g,4离心10min,保存上清待用。
(2)将试剂盒中试剂四与试剂五混匀,37水浴5min,加入30μl样本,开始计时,在340nm波长下记录20s时初始吸光度A1。
(3)之后快速将比色皿水浴37水浴中准确反应2min,快速取出比色皿后用擦镜纸擦干,340nm下比色,记录2min20s时的吸光度A2,计算A=A1-A2。
(4)PK酶活力计算公式:PK(U/104cell)=反应总体积÷样本体积÷反应时间÷NADPH消光系数×A÷活细胞密度。
1.4.7果糖-6-磷酸激酶活力检测
试剂盒的说明书操作如下:
(1)细胞处理方法同3.3.7中步骤一。
(2)PFK工作液800μl、样本30μl、试剂六5μl、试剂七5μl依次加入到1mL比色皿中,340nm波长纪录20s时的吸光度为A1。
(3)然后37水浴10min,取出用擦镜纸擦干后,再在340nm下比色,测定10min20s时的吸光度为A2,计算A=A1-A2。
(4)PFK酶活力计算公式:同4.3.3中计算公式。
1.4.8乳酸脱氢酶活力检测
根据乳酸脱氢酶试剂盒的说明书操作如下:
(1)细胞处理同3.3.3中步骤一;
(2)样品为50升,试剂为L为250,试剂为50L(仅为实验组),蒸馏水为50升(仅对照组加),除充分搅拌,然后37水浴15min。最后再加试剂三,继续水浴15min。然后再加试剂四,充分混匀,室温静置3min,450nm下测吸光度并记录;
(3)LDH酶活力计算公式:
LDH(U/104cell)=1379×(测定管吸光度-对照管吸光度)/细胞密度(104cell/mL)。
1.4.9细胞耗氧量检测
(1)把细胞接种至海马测试24孔微孔板中,其中每孔都加入了200μl普通高糖DMEM,然后置于5%CO2的恒温细胞培养箱中,37过夜培养;
(2)培养24h后,用高糖的海马XF测试培养液(25mmol/L葡萄糖浓)清洗细胞2次,再补足培养体系,最终每孔的海马XF测试培养液体积为525μl,最后将24孔微孔板置于37、无CO2的恒温培养箱中培养1h。
(3)然后测试板添加到海马生物能源计XF24完成校准程序,然后放入细胞培养板测试。
(4)在实验前24小时需要对海马生物能量测定仪XF24进行启动预热,海马检测板提前24h加入海马活化液并置于37、无CO2的恒温培养箱中进行检测探头的活化。
(5)在生物能量分析仪XF24通过测量耗氧率每孔的压力(耗氧、率、OCR)反映在细胞氧化磷酸化的程度。
1.4.10乳酸含量检测
根据乳酸含量测定试剂盒说明书操作:
(1)细胞培养皿中加入200μl裂解液,反复吹打至尽量充分裂解,4下12000rmp离心10min,取上清。
(2)取5mL待测样品加入40μl LD显色液,混匀,37水浴5min,然后立即加入终止液240μl终止反应。
(3)用酶标仪检测340nm下的吸光度。
1.4.11统计学处理
采用SPSS18.0软件分析数据,两组比较作t检验,多组均数间比较采用One-wayANOVA分析,显著性差异用p<0.05或者p<0.01表示。
2.实验结果
2.1重组真核表达质粒的构建及鉴定
PCR扩增BS-sir2基因(在其5端引入EcoR I酶切位点,在3’段引入BamH I酶切位点),经琼脂糖凝胶电泳分析,可见约750bp水平处的特异性条带,大小与目的基因理论值744bp相符,见图4。
BS-sir2基因克隆后,经过EcoR I和BamH I酶切,连接到pEGFP-N1质粒,即得到表达BS-sir2的重组质粒质粒pEGFP-N1-BS-sir2,CaCl2法将pEGFP-N1-sir2重组质粒转化大肠杆菌感受态细胞后涂布于Kan抗性的LB固体平板上进行筛选,置于37生化恒温培养箱中倒置培养过夜。随机挑选转化子进行菌落PCR验证,筛选阳性菌落。提质粒,用EcoR I、BamHI进行双酶切鉴定,挑选阳性克隆菌液进行测序分析。EcoR I、BamH I酶切产物琼脂糖凝胶电泳鉴定见图5所示,在4700、750bp左右有目的条带,与预期一致。阳性质粒进行测序和序列比较NCBI。结果表明,该序列与模板基因相同,氨基酸序列100%正确,pEGFP-N1-BS-sir2构建成功。
2.2 BS-sir2基因转染HEK293T细胞
2.2.1荧光显微镜检测
通过Lipofectamine2000脂质体介导,将重组质粒pEGFP-N1-BS-sir2和空质粒pEGFP-N1分别转染到HEK293细胞,48h后倒置荧光显微镜观察GFP的表达,发现:重组质粒pEGFP-N1-BS-sir2组可见GFP表达;空质粒pEGFP-N1组可见GFP表达;未转染处理的HEK293细胞组未见GFP表达。见图6,从左至右分别代表48小时三组荧光显微镜观察结果。
2.2.2 Western blot验证融合蛋白表达
BS-sir2蛋白的分子量约为27KD,增强型绿色荧光蛋白(EGFP)的分子量约为27KD,EGFP-BS-sir2融合蛋白分子量约为54KD。1-3泳道分别为空白对照组(blank)、空载体转染组(Vetor)、pEGFP-N1-BS-sir2转染组(Sir2)。第3泳道可在GFP检测水平见到54kDa大小条带,为重组质粒转染HEK293T细胞所表达融合蛋白(图7)。
2.3 BS-Sir2对细胞葡萄糖有氧氧化途径的影响
糖酵解和TCA循环是哺乳动物细胞中心碳代谢的中枢。通过这两种途径,葡萄糖被氧化,以NADH和ATP的形式产生能量,或者转化为氨基酸、脂类和核苷酸的前体。
2.3.1 BS-sir2转染对细胞产生ATP的影响
BS-sir2转染HEK293T细胞后,胞内ATP水平明显增加。BS-sir2转染细胞约有2倍的更高水平的ATP比WT和空载体转染的细胞在转染48h后,24h后略有增加(图8)。
2.3.2 BS-sir2转染对细胞葡萄糖代谢酶活性的影响
糖酵解途径调控的关键限速酶是果糖-6-磷酸激酶(PFK-1)和丙酮酸激酶(PK)。BS-sir2显著增加PFK-1和PK活性在转染48h后(图9)。同时还分析了乳酸脱氢酶(LDH)活性,相反BS-sir2显著降低LDH活性在转染48h后相比转染空载体组和正常细胞组。
2.3.3 BS-sir2转染对细胞乳酸生成的影响
上面的结果证明了BS-sir2增强葡萄糖代谢,为了进一步证明葡萄糖有氧氧化增加,通过试剂盒对乳酸水平进行检测。结果表明,BS-sir2转染细胞显示乳酸水平显著低于空载体转染和野生型细胞(图10)。
2.3.4 BS-sir2转染对细胞线粒体呼吸的影响
利用细胞能量仪,对细胞呼吸能力进行检测。其中寡霉素(Oligomycin)、解偶联剂(FCCP)、鱼藤酮(Rot.)。结果显示BS-sir2转染也导致细胞氧耗量增加,线粒体呼吸增强。以上的这些结果表明BS-sir2转染增强葡萄糖有氧氧化,表现在线粒体呼吸增强,而糖酵解的抑制(无氧呼吸)(图11)。
成功构建了pEGFP-N1-sir2真核表达载体,并转染HEK293T细胞;荧光显微镜和Western blot结果显示,枯草芽孢杆菌Sir2蛋白在HEK293T细胞中稳定表达。接着通过利用相关的试剂盒分别检测了葡萄糖有氧氧化过程中细胞中ATP含量、PFK-1、PK、LDH的酶活力,事实表明BS-sir2增加细胞葡萄糖代谢及ATP含量;另外对BS-sir2转染细胞耗氧量及乳酸水平进行检测,发现细胞耗氧量增加,乳酸水平降低。以上的这些结果表明,BS-sir2转染HEK293T细胞增加细胞葡萄糖有氧氧化,减弱细胞无氧呼吸。
序列表
SEQ ID NO:1 27F
5'-AGAGTTTGATCCTGGCTCAG-3'
SEQ ID NO:2 1492R
5'-GGTTACCTTGTTACGACTT-3'
SEQ ID NO:3 BS-sir2-F
5'-CATGGAAACATTCAAAAGTAT-3'
SEQ ID NO:4 BS-sir2-R
5'-CGCTTTTTCATGCCCAAAC-3'
SEQ ID NO:5 BS-sir2-FF
5'-GGAATTCCATGGAAACATTCAAAAGTAT-3'
SEQ ID NO:6 BS-sir2-RR
CGGGATCCCGCTTTTTCATGCCCAAAC
Claims (2)
1.一种具有头孢抗性且高表达Sir2蛋白的枯草芽孢杆菌( Bacillus subtilis)BS16,其特征在于,保藏于广东省微生物菌种保藏中心,保藏号为GDMCC No.60193,保藏地址为广州市先烈中路100号大院59号楼5楼,保藏日期为2017年6月2日。
2.权利要求1所述具有头孢抗性且高表达Sir2蛋白的枯草芽孢杆菌( Bacillussubtilis) BS16在制备增加细胞葡萄糖代谢和ATP含量、增加细胞葡萄糖有氧氧化、减弱细胞无氧呼吸的药物中的应用。
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