CN112831516A - 一种表达glp-1样因子的重组菌及应用 - Google Patents
一种表达glp-1样因子的重组菌及应用 Download PDFInfo
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Abstract
本发明公开了一种表达GLP‑1样因子的重组菌及应用,属于新型生物药物工程技术领域。本发明是通过构建含有GLP‑1样因子基因的重组表达载体并转化到食品级乳酸菌中,获得可发酵产生GLP‑1样因子的重组乳酸菌。该重组乳酸菌促进β细胞增殖,恢复并促进胰岛素的生物合成与分泌,提高外周组织、受体等对胰岛素的敏感性,降低血糖水平。
Description
技术领域
本发明属于新型生物药物工程技术领域,具体涉及一种表达GLP-1样因子的重组菌及应用。
背景技术
Ⅱ型糖尿病又称为非胰岛素依赖型糖尿病,体内的胰岛细胞破坏并不严重,仍然能够产生一定量的胰岛素,但机体对胰岛素产生抵抗,从而导致胰岛素出现相对不足。传统的理论认为Ⅱ型糖尿病发病和进展的一个重要原因是胰岛功能的进行性衰退,包括β细胞胰岛素分泌缺陷和α细胞胰升糖素不适当的分泌增加造成的胰岛素与胰升糖素比例失调,造成血糖控制的不断恶化,并发症的发生。临床主要通过作用于血糖代谢的部分环节来降低血糖,并未针对β细胞功能减退进行治疗。因此,研究新的治疗方法从根本上长期控制血糖稳定十分迫切。
GLP-1是一种在食物营养物质刺激下由肠道细胞合成分泌的肠促胰素,以葡萄糖浓度依赖方式作用于胰腺的β细胞促进胰岛素释放及α细胞抑制不适当的胰升糖素分泌,控制血糖水平。同时GLP-1可抑制胰岛β细胞凋亡和促进其增殖,有助于恢复胰岛细胞的功能,可减缓胃排空和抑制食欲从而控制患者体重,增加外周组织对胰岛素的敏感性等,使其成为最理想的治疗II型糖尿病的药物。
发明内容
针对现有技术中存在的问题,本发明的目的在于提供一种能够表达GLP-1样因子的重组菌,通过口服途径促进β细胞增殖,恢复并促进胰岛素的生物合成与分泌,提高外周组织、受体等对胰岛素的敏感性,从根本上解决胰岛素缺乏从而达到降低血糖水平目的。
为了达到上述目的,本发明采用的技术方案如下:
一种表达GLP-1样因子的重组表达载体,所述重组表达载体是在表达载体中插入有编码GLP-1样因子的核苷酸序列;
所述编码GLP-1样因子的核苷酸序列如SEQ ID No:1所示。
SEQ ID No:1:
在上述方案的基础上,所述表达载体为pNZ8149。
一种表达GLP-1样因子的重组菌,所述重组菌是由上述的重组表达载体转化导入到乳酸菌中所得。
在上述方案的基础上,所述乳酸菌为乳酸乳球菌NZ3900。
在上述方案的基础上,所述重组菌为乳酸乳球菌(Lactococcus lactis)HL19003,保藏编号为CCTCC NO:M 2020968。
上述表达GLP-1样因子的重组菌在制备预防和治疗Ⅱ型糖尿病药物或食品中的应用。
上述表达GLP-1样因子的重组菌在制备具有促进胰岛素分泌和胰岛细胞增殖功效的药物或食品中的应用。
使用上述重组菌表达GLP-1样因子的方法,步骤为:
(1)将保藏编号为CCTCC NO:M 2020968的重组菌株接种到M17培养基,30℃静置培养20h;
(2)取步骤(1)培养后的培养液按3%接种到GM17培养基,30℃恒温静置培养,待OD600nm至0.4,加入Nisin进行诱导表达。
在上述方案的基础上,所述Nisin加入终浓度为30ng/mL;诱导表达2-6h。
一种具有促进胰岛素分泌和胰岛细胞增殖功效的生物制剂,其特征在于,是由上述方法诱导后的菌液,经离心去除菌体后的上清液。
本发明采用的表达载体pNZ8149是由Nisin诱导的食品级的表达载体,并携带乳糖分解基因;NZ3900菌株是一类乳糖缺陷型菌株,当pNZ8149正确转入NZ3900菌株后,使菌株代谢乳糖产生乳酸,pH值的下降使得溴甲酚紫培养基由紫色变为黄色。
本发明有益效果在于,成功构建了用于表达GLP-1样因子基因的重组乳酸乳球菌。实验证明,本发明的重组乳酸菌能够分泌表达GLP-1样因子,促进胰岛素分泌和细胞增殖,可控制血糖水平。
附图说明
图1为重组表达载体pNZ8149-glp-1的构建流程。
具体实施方式
在本发明中所使用的术语,除非有另外说明,一般具有本领域普通技术人员通常理解的含义。
下面结合具体实施例,并参照数据进一步详细的描述本发明。以下实施例只是为了举例说明本发明,而非以任何方式限制本发明的范围。
实施例1
构建含有GLP-1样因子基因的pNZ8149载体
GLP-1样因子基因的DNA序列如SEQ ID NO:1所示,两端带上NcoI和SphI酶切位点,同时引入Usp45的信号肽碱基序列,把设计好的碱基序列(SEQ ID No:2)送至上海生工生物公司进行序列合成,随后将合成片段插入到pNZ8149中,得到质粒pNZ8149-glp-1,其构建过程如图1所示。
SEQ ID No:1:
SEQ ID No:2:
实施例2
乳酸乳球菌感受态细胞的制备
挑取NZ3900单菌落接种于5mLGM17培养基(M17培养基、5g/L葡萄糖)中,30℃厌氧培育12h,进行活化;待12h后,在10mLSGM17培养基(M17培养基、0.5mol/L蔗糖、25g/L甘氨酸、5g/L葡萄糖)中转接入500μL已活化12h的菌液,30℃培养12h。将10mL培养液整体加至100mL SGM17培养液中,30℃培育4h后冰浴20min,5000r/min离心10min后弃上清液。用灭菌预冷的40mLOSPS缓冲液(0.5M蔗糖+10%甘油)重悬菌体,冰上静置20min离心弃上清液,25mL预冷溶液II(0.5mol/L蔗糖+100mL/L甘油+0.05mol/L EDTA)重悬菌体冰上静置20min后离心取菌体。再用15mL OSPS缓冲液重悬菌体,冰上静置20min后离心弃上清液。最后加入1mLOSPS缓冲液重悬菌体,80μL分装,置于-80℃保存或直接使用。
实施例3
乳酸乳球菌的电转化
将质粒pNZ8149-glp-1和实施例2制备的NZ3900感受态细胞在电转化杯中混合,在电阻200Ω、电容25μF、电场强度10kV/cm条件下进行电转化。将转化后的菌体,加入预冷的GM17MC恢复培养基(M17培养基、5g/L葡萄糖、20mmol/L MgCl2、2mmol/L CaCl2),30℃静置孵育2h。洗涤细胞,涂布于筛选培养基(M17培养基、0.04g/L溴甲酚紫、琼脂)上,静置培养24h。进一步挑取平板上黄色生长的菌株活化并进行菌落PCR和双酶切验证,并进行测序分析。
测序分析为pNZ8149-glp-1乳酸乳球菌的阳性转化子即为表达GLP-1样因子的重组菌株NZ3900/pNZ8149-glp-1。
对NZ3900/pNZ8149-glp-1重组乳酸乳球菌进行生物保藏,保藏信息如下:
保藏时间:2020年12月24日
保藏单位名称:中国典型培养物保藏中心
保藏编号:CCTCC NO:M 2020968
保藏单位地址:湖北省武汉市武昌区八一路299号武汉大学校内(武汉大学第一附小对面),武汉大学保藏中心
分类命名:乳酸乳球菌HL19003 Lactococcus lactis HL19003
实施例4
glp-1样因子基因在重组乳酸菌中的表达和含量测定
将构建好的重组菌株NZ3900/pNZ8149-glp-1以1%的接种量接种于M17培养基,30℃静置培养20h,第二天取培养液按3%接种量再次接种于新鲜的GM17培养基中,30℃恒温静置培养约4h,待OD 600nm至0.4左右,加入Nisin至终浓度30ng/mL进行诱导表达,以不添加Nisin的重组菌株NZ3900/pNZ8149-glp-1培养液作为对照;取不同诱导时间的菌液于4℃、4000rpm离心5min,取上清进行glp-1含量的检测。
通过检测,在Nisin诱导2.5h时,glp-1的分泌量达到最大,为256nmol/L,此时菌体的浓度约为6×108CFU/mL,在Nisin诱导3-6h,glp-1的分泌量基本保持稳定,而未加入Nisin诱导的对照组没有表达。
实施例5
重组乳酸菌NZ3900/pNZ8149-glp-1对控糖作用的研究
重组乳酸菌NZ3900/pNZ8149-glp-1按3%接种量接种于新鲜的GM17培养基中,30℃恒温静置培养约4h,无菌加入终浓度为30ng/mL的Nisin诱导2.5h后,4000rpm无菌离心10min,取上清并与RPMI 1640培养基等比例混合备用。同时,将大鼠胰岛素瘤细胞系INS-1细胞按1×105/孔的细胞数接种在96孔培养板中培养36h,弃上清,试验组加入重组乳酸菌培养上清与RPMI 1640培养基的等比混合液,对照组只加入RPMI 1640培养基,加样量均为100uL/孔,继续孵育12h后测定胰岛素含量和活细胞数。
结果表明,空白对照组胰岛素浓度为58ng/mL,其活细胞数计为100%,试验组胰岛素浓度为72.2ng/mL,其活细胞数为108.9%。由此可见,重组乳酸菌NZ3900/pNZ8149-glp-1可显著促进胰岛素的分泌和胰岛细胞增殖,可控血糖。
以上所述,仅是本发明的较佳实施例而已,并非是对本发明作其它形式的限制,任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例。但是凡是未脱离本发明技术方案内容,依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与改型,仍属于本发明技术方案的保护范围。
序列表
<110> 青岛海华莱康生物医药技术有限公司
青岛海华生物医药技术有限公司
<120> 一种表达GLP-1样因子的重组菌及应用
<160> 2
<170> SIPOSequenceListing 1.0
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<212> DNA
<213> 人类(Homo sapiens)
<400> 1
cataaggaag ggacctttac cagtgatgta agttcttatt tggaaggcca agctgccaag 60
gaattcattg cttggctggt gaaaggccga gga 93
<210> 2
<211> 1560
<212> DNA
<213> 人工序列(Artificial Sequence)
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ccatggattc aaccgaactt aatgggagga aaaattaaaa aagaacagtt atgaaaaaaa 60
agattatctc agctatttta atgtctacag tgatactttc tgctgcagcc ccgttgtcag 120
gtgtttacgc tgacacaaac tcagatattg ctaaacaaga tgcgacaatt tcaagcgcgc 180
aatctgctaa agcacaagca caagcacaag ttgatagctt gcaatcaaaa gttgacagct 240
tacaacaaaa gcaaacaagt actaaagcac aaatcgctaa aatcgaaagc gaactgaaag 300
cacttaatgc tcaaattgct actttgaacg aaagtatcaa agaacgtaca aagacattgg 360
aagctcaagc acgtagtgct caagttaaca gctcagcaac aaattatatg gatgctgttg 420
ttaattcaaa atctttgaca gatgttattc aaaaagtaac agctattgct actgtttcta 480
gtgccaacaa acaaatcttg gaacaacaag aaaaagagca aaaagagctt agccaaaagt 540
cagaaactgt taaaaagaac tacaaccagt tcgtttctct ttcacaaagt ttggattctc 600
aagctcaaga attgacttca caacaagctg aactcaaagt tgcgactttg aactatcaag 660
caacaattgc aactgcgcaa gataaaaaac aagctttatt agatgaaaaa gcagctgcag 720
aaaaagcagc tcaagaagca gctaaaaaac aagcggctta tgaagctcaa caaaaagaag 780
cagcacaagc acaagcagct tcaacagcag caactgctaa agctgtagaa gcagcaactt 840
catcagcttc tgcttcatct agtcaagctc cacaagtaag tacaagcact gataatacaa 900
catcaaatgc tagtgcctca aacagttcta atagttcatc aaactcaagt tcaagttcta 960
gcagttcatc aagctcaagc tcaagctcaa gtaattctaa tgctggtggg aatacaaatt 1020
caggcactag tactggaaat actggaggaa caactactgg tggtagcggt ataaatagtt 1080
caccaattgg aaatccttat gctggtggtg gatgtactga ctatgtatgg caatactttg 1140
ctgcacaagg aatttatatc agaaatatca tgcctggtaa tggtggacaa tgggcttcta 1200
atggacctgc ccaaggcgtg ctccatgttg taggagctgc tcctggtgtt atcgcatcaa 1260
gcttctcagc tgattttgtt ggatatgcaa actcacctta cggtcacgta gctattgtaa 1320
aatcagttaa ttcagatggt acaattacta tcaaagaagg cggatatggt acaacttggt 1380
ggggacatga acgtactgta agtgcgtctg gtgttacttt cttgatgcca aactagaaaa 1440
aagtcttaat aaataaaaaa tagcataagg aagggacctt taccagtgat gtaagttctt 1500
atttggaagg ccaagctgcc aaggaattca ttgcttggct ggtgaaaggc cgaggagcat 1560
Claims (10)
1.一种表达GLP-1样因子的重组表达载体,其特征在于,所述重组表达载体是在表达载体中插入有编码GLP-1样因子的核苷酸序列;
所述编码GLP-1样因子的核苷酸序列如SEQ ID No:1所示。
2.根据权利要求1所述的表达GLP-1样因子的重组表达载体,其特征在于,所述表达载体为pNZ8149。
3.一种表达GLP-1样因子的重组菌,其特征在于,所述重组菌是由权利要求1或2所述的重组表达载体转化导入到乳酸菌中所得。
4.根据权利要求3所述表达GLP-1样因子的重组菌,其特征在于,所述乳酸菌为乳酸乳球菌NZ3900。
5.根据权利要求3或4所述表达GLP-1样因子的重组菌,其特征在于,所述重组菌为乳酸乳球菌(Lactococcus lactis)HL19003,保藏编号为CCTCC NO:M 2020968。
6.权利要求5所述表达GLP-1样因子的重组菌在制备预防和治疗Ⅱ型糖尿病药物或食品中的应用。
7.权利要求5所述表达GLP-1样因子的重组菌在制备具有促进胰岛素分泌和胰岛细胞增殖功效的药物或食品中的应用。
8.使用权利要求5所述重组菌表达GLP-1样因子的方法,其特征在于,步骤为:
(1)将保藏编号为CCTCC NO:M 2020968的重组菌株接种到M17培养基,30℃静置培养20h;
(2)取步骤(1)培养后的培养液按3%接种到GM17培养基,30℃恒温静置培养,待OD600nm至0.4,加入Nisin进行诱导表达。
9.根据权利要求8所述重组菌表达GLP-1样因子的方法,其特征在于,所述Nisin加入终浓度为30ng/mL;诱导表达2-6h。
10.一种具有促进胰岛素分泌和胰岛细胞增殖功效的生物制剂,其特征在于,是由权利要求9所述方法诱导后的菌液,经离心去除菌体后的上清液。
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