WO2022012020A1 - Glp-1类似物多肽的制备方法及在ⅱ型糖尿病中应用 - Google Patents
Glp-1类似物多肽的制备方法及在ⅱ型糖尿病中应用 Download PDFInfo
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- the invention belongs to the technical field of biomedicine, and in particular relates to a preparation method of a GLP-1 analog polypeptide and its application in type II diabetes.
- Diabetes is a series of metabolic disorder syndromes such as protein, fat, water and electrolytes caused by the interaction of genetic and environmental factors, resulting in the absolute or relative insufficient secretion of insulin and the decrease in the sensitivity of target tissue cells to insulin, among which hyperglycemia is the main sign. .
- Typical clinical cases may appear polyuria, polydipsia, polyphagia, weight loss and other manifestations, that is, the symptoms of "three more and one less”. Diabetes is one of the important diseases that seriously threaten human life and health in modern society.
- Diabetes mellitus is an endocrine and metabolic syndrome characterized by chronic hyperglycemia.
- type 2 diabetes mellitus T2DM is the main type of its incidence, accounting for more than 95% of the total population, and it tends to be younger.
- GLP-1RAs glucagon-like peptide-1 receptor agonists
- GLP-1 is easily degraded by dipeptidyl peptidase (DPP-4) in vivo and loses its activity, and has no clinical value. Therefore, modifying the structure of GLP-1 to form GLP-1 analogs with the same pharmacological activity, masking the binding site of DPP-IV, and prolonging the half-life is the main subject of the research and development of this type of drug.
- DPP-4 dipeptidyl peptidase
- modifying the structure of GLP-1 to form GLP-1 analogs with the same pharmacological activity, masking the binding site of DPP-IV, and prolonging the half-life is the main subject of the research and development of this type of drug.
- most of the natural GLP-1 is modified by amino acid substitution, fatty acid side chain connection, coupling of macromolecular fusion proteins, etc., which overcomes the problem of easy access while retaining the efficacy of natural GLP-1.
- the expression method of the key intermediates of GLP-1 prepared by biological method is intracellular soluble expression, and the expression level is low, which is not conducive to industrial amplification, as described in patent CN104745597A; patent CN104592381A discloses that the dissolution and renaturation of inclusion bodies takes too long , the volume required for renaturation is too large, and a large amount of urea is used, which is not conducive to industrial scale-up, has strict requirements on experimental equipment and operators, and is easy to cause environmental pollution.
- the present invention provides a preparation method of a GLP-1 analog polypeptide and its application in type II diabetes.
- the method of the invention realizes the high-efficiency expression of GLP-1 analogs, avoids the procedures of denaturing and renaturing inclusion bodies and connecting side chains used in the prior art, simplifies the process, effectively reduces costs, and reduces the generation of environmental pollutants, It is beneficial to industrial scale-up; nickel column affinity chromatography is used for separation and purification, with high separation degree, good purification effect, few impurities and simple operation.
- the yield of the GLP-1 analog prepared by the method of the present invention is greater than 1 g/L, and the purity of the GLP-1 analog reaches more than 87%.
- a preparation method of a GLP-1 analog polypeptide comprising the following steps:
- step (3) transforming the recombinant expression vector described in step (2) into the Escherichia coli host to construct recombinant engineering bacteria;
- Step (4) The obtained bacterial cells are crushed and centrifuged, and the supernatant is taken for separation and purification, and after enzymatic excision of salts, the GLP-1 analog polypeptide is obtained.
- step (1) the sequence of the encoding gene is any one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3.
- step (2) the expression vector is pET-28(+);
- the recombinant engineered bacteria are single-copy, two-copy or four-copy recombinant engineered bacteria strains.
- the intracellular soluble protein comprises the amino acid sequence shown in SEQ ID NO.4.
- step (4) the specific steps of the recombinant engineering bacteria fermentation induction and expression of intracellular soluble protein are:
- step (S4) the fermentation culture is high-density fermentation culture, and the inducer is lactose.
- the initial fermentation temperature was 30° C.
- the stirring speed was 300 rpm
- the ventilation rate was 4L/min
- the pH was 6.5-7.5
- the stirring rate and ventilation rate were continuously increased to maintain dissolved oxygen. Always at 4.5%-20.5%.
- step (5) adopt nickel column affinity chromatography to carry out described separation and purification, and concrete steps include:
- SS4 The target protein was eluted with 10 column volumes of lysis buffer containing 200 mM imidazole at a flow rate of 15 ml/min, and the separation and purification was completed.
- step (5) the specific operation of the enzyme excising salt is:
- step (b) Draining with a glass rod, pouring the swollen Sephadex medium in step (a) into a 50 ⁇ 300mm medium-pressure chromatography column at one time, the volume of the column is 500ml, and the column height is 12cm. Rinse the column with 5 times the column volume of deionized water, and then equilibrate the column with 5 times the column volume of enzyme digestion buffer;
- step (c) loading the eluted target protein on the equilibrated column in step (b), the loading amount is 20% of the column volume, and each loading is 100 ml, and the flow rate is 8 ml/min;
- the preparation method of the GLP-1 analog polypeptide according to the present invention by first synthesizing the encoding gene, then obtaining a recombinant expression vector with the encoding gene, and constructing a recombinant engineering bacteria to induce and express intracellular soluble protein, so as to realize the similarity of GLP-1
- the high-efficiency expression of the compound avoids the procedures of denaturing and renaturing inclusion bodies and connecting side chains used in the prior art, simplifying the process, effectively reducing costs, reducing the generation of environmental pollutants, and facilitating industrialization amplification; Separation and purification with chromatography has the advantages of high separation degree, good purification effect, few impurities and simple operation.
- the GLP-1 analog prepared by the method of the present invention is more than 1 g/L, and the purity of the GLP-1 analog reaches more than 87%.
- the GLP-1 analog is digested with enterokinase to further prepare the intermediate GLP-1 (7-37) of liraglutide, which is used to prepare liraglutide.
- Fig. 1A-Fig. 1C are fermentation curve diagrams of each copy strain of Example 1-3 of the present invention.
- Figure 2 is a schematic diagram of the expression levels of each copy strain of Examples 1-3 of the present invention.
- Figure 3 is a mass spectrogram of the GLP-1 analog polypeptide obtained in Example 1 of the present invention.
- Fig. 4 is the HLPC spectrum of eluted target protein in Example 1;
- Fig. 5 is the process of adopting nickel column affinity chromatography to carry out separation and purification in embodiment 2 and product electrophoresis figure;
- Figure 6 is a comparison diagram of pancreatic tissue sections of diabetic model mice and normal mice
- FIG. 7 is a schematic diagram of detecting changes in blood glucose levels in mice at different times.
- the LB medium, fermentation medium, nickel column, lysis buffer, Sephadex medium, enzyme cleavage buffer, etc. in the following examples are all commercially available products known to those skilled in the art.
- the present embodiment provides a method for preparing a GLP-1 analog polypeptide, comprising the following steps:
- step (3) transforming the recombinant expression vector described in step (2) into Escherichia coli host BL21 (DE3) to construct a single-copy recombinant engineering bacterium of the GLP-1 analog;
- the intracellular soluble protein comprises the amino acid sequence described in SEQ ID NO.4, specifically: MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSDYKDDDDKHAEGTFTS DVSSYLEGQAAKEFIAWLVR GRG;
- Step (4) Resuspend 200 g of bacterial cells in 2 L of lysis buffer, homogenize twice with a homogenizer, remove macromolecular impurities in a boiling water bath, and centrifuge at 8000 rpm for 30 min at 4°C to collect the supernatant After separation and purification by nickel-column affinity chromatography, enzymatic cleavage of salts, the GLP-1 analog polypeptide is obtained; it is detected that the yield of GLP-1 analog polypeptide prepared by the method in this example is greater than 1 g/L, and the GLP-1 analog polypeptide is obtained.
- the -1 analog has a purity of more than 75%.
- step (a) Add 100 g of Sephadex G-25 to 500 ml of deionized water after sonication, soak overnight for swelling, and gently stir every 2 hours; (b) drain with a glass rod, Pour the swollen Sephadex medium in step (a) into a 50 ⁇ 300mm medium-pressure chromatography column at one time, the column volume is 500ml, and the column height is 12cm. Rinse with 10 times the column volume of deionized water first.
- the present embodiment provides a method for preparing a GLP-1 analog polypeptide, comprising the following steps:
- step (3) transforming the recombinant expression vector described in step (2) into Escherichia coli host BL21 (DE3) to construct a two-copy recombinant engineering bacterium of the GLP-1 analog;
- (S4) insert the described tertiary seed liquid into the fermentation medium by 3% inoculum, carry out high-density fermentation culture, the initial fermentation temperature is 30 ° C, the stirring speed is 300 rpm, the ventilation volume is 4L/min, and the pH is 6.5 -7.5, and then continuously increase the stirring speed and aeration to maintain the dissolved oxygen at 18% ⁇ 0.5%.
- the carbon source in the initial medium is about to be exhausted, the dissolved oxygen and pH will rise.
- the OD 600 of the fermentation broth reaches 8-10 or more, add lactose to induce two times, put it in a tank, and collect the cells by centrifugation.
- Step (4) Resuspend 200 g of bacterial cells in 2.5 L of lysis buffer, homogenize twice with a homogenizer, remove macromolecular impurities in a boiling water bath, and centrifuge at 8,000 rpm and 4°C for 30 min to collect the supernatant
- the salt is excised by enzyme to obtain the GLP-1 analog polypeptide; the output of the GLP-1 analog polypeptide is greater than 0.5 g/L, and the GLP-1 analog polypeptide is similar to GLP-1.
- the material purity is greater than 85%.
- step (a) Add 100 g of Sephadex G-25 to 500 ml of deionized water after sonication, soak overnight for swelling, and stir gently every 3 hours; (b) drain with a glass rod, Pour the swollen Sephadex medium in step (a) into a 50 ⁇ 300mm medium-pressure chromatography column at one time, the column volume is 500ml, and the column height is 12cm. Rinse with 10 times the column volume of deionized water first.
- step (b) load the eluted target protein on the equilibrated column in step (b), and the loading amount is 20% of the column volume , each loading of 100ml, and the flow rate of 8ml/min;
- step (d) Elution with enzyme digestion buffer, so as to replace the target protein into the digestion buffer for direct digestion; after loading, the recombinant GLP-1 is similar to The substance (protein) is first eluted together with the enzyme digestion buffer, and the salt ion (imidazole, etc.) is then eluted, and the collected protein is the GLP-1 analog polypeptide.
- the present embodiment provides a method for preparing a GLP-1 analog polypeptide, comprising the following steps:
- step (3) transforming the recombinant expression vector described in step (2) into Escherichia coli host BL21 (DE3) to construct a four-copy recombinant engineering bacterium of GLP-1 analog;
- (S3) described secondary seed liquid is inoculated in the LB medium of 250ml by the inoculation amount of 2.0%, under the condition of 37 DEG C, 200rpm shaking culture 12h, promptly obtains tertiary seed liquid;
- (S4) insert the described tertiary seed liquid into the fermentation medium by 5% inoculum, carry out high-density fermentation culture, the initial fermentation temperature is 30 ° C, the stirring speed is 300 rpm, the ventilation volume is 4L/min, and the pH is 6.5 -7.5, and then continuously increase the stirring speed and aeration to maintain the dissolved oxygen at 20% ⁇ 0.5%.
- the carbon source in the initial medium is about to be used up, the dissolved oxygen and pH will rise.
- the OD 600 of the fermentation broth reaches 8-10 or more, add lactose to induce two times, put it in a tank, and collect the cells by centrifugation.
- Step (4) Resuspend 200 g of bacterial cells in 3 L of lysis buffer, use a homogenizer to homogenize twice, remove macromolecular impurities in a boiling water bath, and centrifuge at 8000 rpm and 4°C for 30 min to collect the supernatant After separation and purification by nickel-column affinity chromatography, enzymatic cleavage of salts, the GLP-1 analog polypeptide is obtained; the yield of the GLP-1 analog polypeptide is greater than 1 g/L, and the purity of the GLP-1 analog is greater than 85%.
- step (a) Add 100 g of Sephadex G-25 to 500 ml of deionized water after ultrasonication, soak overnight for swelling, and stir gently every 4 hours; (b) drain with a glass rod, Pour the swollen Sephadex medium in step (a) into a 50 ⁇ 300mm medium-pressure chromatography column at one time, the column volume is 500ml, and the column height is 12cm. Rinse with 10 times the column volume of deionized water first.
- step (b) load the eluted target protein on the equilibrated column in step (b), and the loading amount is 20% of the column volume , each loading of 100ml, and the flow rate of 8ml/min;
- step (d) Elution with enzyme digestion buffer, so as to replace the target protein into the digestion buffer for direct digestion; after loading, the recombinant GLP-1 is similar to The substance (protein) is first eluted together with the enzyme digestion buffer, and the salt ion (imidazole, etc.) is then eluted, and the collected protein is the GLP-1 analog polypeptide.
- mice 50 6-week-old (18-20g) BALB/C mice (male), the mice were reared in separate cages, the temperature of the animal room was controlled at 25 ⁇ 2°C, the humidity was 50 ⁇ 10%, the light cycle was 12h and the dark was 12h, and the mice were adapted to the environment for one week. .
- Mice were randomly divided into cages in groups of 8. All mice were fasted for 12 h and their body weight and fasting blood-glucose (FBG) were measured. The control group was fed with standard feed for 4 weeks, and the model group was fed with high-fat feed for 4 weeks. After 4 weeks, the body weight and FBG of the mice in each group were measured. After fasting for 12 hours, STZ was injected intraperitoneally at a dose of 60 mg/kg (body weight) for 3 consecutive days.
- FBG blood-glucose
- mice with FBG ⁇ 11.1 mmol/L and stable for one week were selected as the type II diabetes model mice, with 8 mice/group. Cage randomly.
- the comparison of pancreatic tissue sections of diabetic model mice and normal mice is shown in Figure 6, confirming that the modeling was successful.
- mice were randomly divided into three groups: diabetes negative control group (DCN), diabetes positive control group (DCP), and diabetes treatment group (DT), with 8 mice in each group.
- DCN diabetes negative control group
- DCP diabetes positive control group
- DT diabetes treatment group
- mice After overnight fasting for 12 h, all mice were given D-glucose by gavage at 1.5 g/kg (body weight).
- GLP-1 analogs (12 ⁇ g/50 g body weight) were then injected subcutaneously (s.c.), and the DCP and DCN groups were injected with the same volume of normal saline (0.9% NaCl) and commercial liraglutide injection, respectively.
- the invention provides a preparation method of GLP-1 analog polypeptide and its application in type II diabetes.
- the high-efficiency expression of GLP-1 analogs is realized, and the inclusion body of the prior art is avoided.
- the process of denaturation and renaturation and connection of side chains simplifies the process, effectively reduces the cost, reduces the generation of environmental pollutants, and is beneficial to industrialized amplification; finally, the present invention adopts nickel column affinity chromatography for separation and purification, with high separation degree and good purification effect. Less impurities, simple operation.
- the yield of the GLP-1 analog prepared by the method of the present invention is greater than 1 g/L, and the purity of the GLP-1 analog reaches more than 87%.
- the GLP-1 analog is digested with enterokinase to further prepare the intermediate GLP-1 (7-37) of liraglutide, which is used for preparing liraglutide, and has good economic value and application prospect.
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Abstract
提供了一种GLP-1类似物多肽的制备方法及其在Ⅱ型糖尿病中的应用,通过先合成编码基因,再获得带有该编码基因的重组表达载体,构建重组工程菌发酵诱导表达胞内可溶性蛋白,该蛋白可采用镍柱亲和层析进行分离纯化。该GLP-1类似物经肠激酶酶切可进一步制备利拉鲁肽中间体GLP-1(7-37),用于制备利拉鲁肽。
Description
交叉引用
本申请要求2020年7月17日提交的专利名称为“GLP-1类似物多肽的制备方法及在Ⅱ型糖尿病中的应用”的第202010690185.9号中国专利申请的优先权,其全部公开内容通过引用整体并入本文。
本发明属于生物医药技术领域,具体涉及一种GLP-1类似物多肽的制备方法及其在Ⅱ型糖尿病中的应用。
糖尿病是由于遗传和环境因素相互作用,引起胰岛素绝对或相对分泌不足以及靶组织细胞对胰岛素敏感性降低,引起蛋白质、脂肪、水和电解质等一系列代谢紊乱综合征,其中以高血糖为主要标志。临床典型病例可出现多尿、多饮、多食、消瘦等表现,即“三多一少”症状。糖尿病是现代社会严重威胁全球人类生命健康的重要疾病之一。国际糖尿病联盟于2019年更新的数据显示:全球约有4.63亿成人患有糖尿病,预计2045年全世界糖尿病患者将达到7.002亿;2019年,中国糖尿病患者约1.164亿,成为全球糖尿病患者最多的国家。糖尿病是一种以慢性高血糖为特征的内分泌代谢综合征,其中,2型糖尿病(T2DM)是其发病的主要类型,占发病总人群的95%以上,而且呈年轻化趋势。
近年来胰高血糖素样肽-1(glucagon-likepeptide-1,GLP-1)受体激动剂(GLP-1RAs)治疗T2DM成为研究热点,GLP-1通过葡萄糖依赖的方式促进胰岛素的合成和分泌,发挥降糖作用。其不仅具有优异的降糖效果,还有控制体重、调节血脂、改善胰岛β细胞功能等特点,同时低血糖的不良反应发生率较低。GLP-1及其类似物在治疗2型糖尿病的优良效果,使其近年来在糖尿病治疗药物市场中逐渐占据重要地位。
然而,天然GLP-1在体内易被二肽基肽酶(DPP-4)降解而失去活性, 不具有临床使用价值。因此对GLP-1结构修饰,形成具有同样药理活性的GLP-1类似物,并掩盖DPP-Ⅳ的结合位点,延长半衰期是该类药物研发的主要课题。为了延长GLP-1类似物半衰期,大多通过对天然GLP-1进行氨基酸替换、连接脂肪酸侧链、偶联大分子融合蛋白等方式对其进行修饰,在保留天然GLP-1功效的同时克服了易被DPP-4降解的缺点,达到延长半衰期的目的。截至目前为止,已经有7个GLP-1RAs经美国食品药品监督管理局批准上市,即艾塞那肽、利拉鲁肽、阿必鲁肽、度拉糖肽、利西拉肽和索马鲁肽等,该类药物在未来10年里将会是抗糖尿病药物的重要增长点。
目前,GLP-1类似物的关键中间体31肽制备较为困难,多是通过化学法制备,需要通过氨基酸一个一个链接,中国专利也公开了一系列化学合成方法,如CN105732798、CN102875665、CN102286092等。对于GLP-1这样长度的多肽来说,虽然可以通过固相自动化合成,但生产周期长,产率低。另外,生物法制备GLP-1关键中间体的表达方式为胞内可溶性表达,表达量较低,不利于工业化放大,如专利CN104745597A所述;专利CN104592381A中公开了包涵体溶解复性耗时过长,复性所需体积过大,使用大量的尿素,不利于工业放大,对实验设备和操作者的要求较为严格,并且易造成环境的污染。
发明内容
为了解决现有技术存在的上述问题,本发明提供了一种GLP-1类似物多肽的制备方法及其在Ⅱ型糖尿病中的应用。本发明所述方法,实现GLP-1类似物的高效表达,避免了现有技术采用的对包涵体变性复性和连接侧链的工序,简化工艺,有效降低成本,减少环境污染物的产生,利于工业化放大;采用镍柱亲和层析进行分离纯化,分离度高,纯化效果好,杂质少,操作简单。本发明方法制备得到GLP-1类似物的产量大于1g/L,GLP-1类似物纯度达到87%以上。
本发明所采用的技术方案为:
一种GLP-1类似物多肽的制备方法,包括如下步骤:
(1)合成编码基因;
(2)将所述编码基因连接到表达载体中,得到带有编码基因的重组表达载体;
(3)将步骤(2)所述重组表达载体转化到大肠杆菌宿主中,构建重组工程菌;
(4)利用所述重组工程菌发酵诱导表达胞内可溶性蛋白;
(5)步骤(4)得到菌体进行破碎后离心,取上清液进行分离纯化、酶切除盐后,即得所述GLP-1类似物多肽。
步骤(1)中,所述编码基因的序列为SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3中的任一种。
步骤(2)中,所述表达载体为pET-28(+);
步骤(3)中,所述重组工程菌为单拷贝、二拷贝或四拷贝的重组工程菌菌株。
步骤(4)中,所述胞内可溶性蛋白包含SEQ ID NO.4所示的氨基酸序列。
步骤(4)中,所述重组工程菌发酵诱导表达胞内可溶性蛋白的具体步骤为:
(S1)将所述重组工程菌的单菌落接种于含有卡那霉素的LB培养基中,在37℃、200rpm条件下振荡培养12h,即得一级种子液;
(S2)将所述一级种子液按0.5-2.0%的接种量接种于LB培养基中,在37℃、200rpm条件下振荡培养12h,即得二级种子液;
(S3)将所述二级种子液按0.5-2.0%的接种量接种于LB培养基中,在37℃、200rpm条件下振荡培养6-12h,即得三级种子液;
(S4)将所述三级种子液按1-5%接种量接入发酵培养基中,进行发酵培养,当发酵液OD
600达到8-10以上时加入诱导剂进行两次诱导,诱导完成后放罐,离心收集菌体。
步骤(S4)中,所述发酵培养为高密度发酵培养,所述诱导剂为乳糖。
步骤(S4)中,进行所述发酵培养时,初始发酵温度为30℃,搅拌速度为300rpm,通气量为4L/min,pH为6.5-7.5,之后不断提高搅拌速度和通气量以维持溶解氧始终在4.5%-20.5%。
步骤(5)中,采用镍柱亲和层析法进行所述分离纯化,具体步骤包括:
(SS1)先采用10倍柱体积的去离子水和5倍柱体积的裂解缓冲液平衡柱子;
(SS2)将所述上清液上样于平衡处理后的镍柱,上样流速为5ml/min;
(SS3)分别用20倍柱体积的裂解缓冲液、15倍柱体积含20mM咪唑的裂解缓冲液、10倍柱体积含50mM咪唑的裂解缓冲液清洗未结合的杂蛋白,清洗流速均为15ml/min;
(SS4)采用10倍柱体积含200mM咪唑的裂解缓冲液以15ml/min流速洗脱目的蛋白,即完成所述分离纯化。
步骤(5)中,所述酶切除盐的具体操作为:
(a)取葡聚糖凝胶介质加入500ml超声后的去离子水中,浸泡过夜进行溶胀,期间进行数次搅拌;
(b)用玻璃棒引流,将步骤(a)溶胀后葡聚糖凝胶介质一次性倒入50×300mm的中压层析柱中,装完柱体积为500ml,柱高12cm,先用10倍柱体积的去离子水冲洗柱子,再用5倍柱体积的酶切缓冲液平衡柱子;
(c)将所述洗脱下来的目的蛋白在步骤(b)平衡后的柱子上进行上样,上样量为20%柱体积,每次上样100ml,流速为8ml/min;
(d)采用酶切缓冲液进行洗脱,收集蛋白即为所述GLP-1类似物多肽。
所述方法制得的GLP-1类似物多肽在制备治疗Ⅱ型糖尿病药物中的 应用。
本发明的有益效果为:
本发明所述的GLP-1类似物多肽的制备方法,通过先合成编码基因,再获得带有该编码基因的重组表达载体,构建重组工程菌发酵诱导表达胞内可溶性蛋白,实现GLP-1类似物的高效表达,避免了现有技术采用的对包涵体变性复性和连接侧链的工序,简化工艺,有效降低成本,减少环境污染物的产生,利于工业化放大;最后本发明采用镍柱亲和层析进行分离纯化,分离度高,纯化效果好,杂质少,操作简单。本发明方法制备得到GLP-1类似物的大于1g/L,GLP-1类似物纯度达到87%以上。所述GLP-1类似物经肠激酶酶切可进一步制备利拉鲁肽中间体GLP-1(7-37),用于制备利拉鲁肽。
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1A-图1C为本发明实施例1-3各拷贝菌株的发酵曲线图;
图2为本发明实施例1-3各拷贝菌株的表达量示意图;
图3为本发明实施例1所得GLP-1类似物多肽的质谱图;
图4为实施例1中洗脱目的蛋白的HLPC谱图;
图5为实施例2中采用镍柱亲和层析法进行分离纯化的过程及产物电泳图;
图6为糖尿病模型小鼠和正常小鼠的胰腺组织切片对比图;
图7为不同时间检测小鼠血糖水平变化示意图。
为使本发明的目的、技术方案和优点更加清楚,下面将对本发明的技 术方案进行详细的描述。显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施方式,都属于本发明所保护的范围。
下述实施例中的LB培养基、发酵培养基、镍柱、裂解缓冲液、葡聚糖凝胶介质、酶切缓冲液等均为本领域技术人员所知晓的市售产品。
实施例1
本实施例提供一种GLP-1类似物多肽的制备方法,包括如下步骤:
(1)合成编码基因,所述编码基因的序列如SEQ ID NO.1所示,具体为:ATGG GTTCTTCTCA CCACCACCAC CACCACTCTT CTGGTCTGGT TCCGCGTGGT TCTCACATGG CTTCTATGAC CGGTGGTCAG CAGATGGGTC GTGGTTCTGA CTACAAAGAC GACGACGACA AACACGCTGA AGGTACCTTC ACCTCTGACG TTTCTTCTTA CCTGGAAGGT CAGGCTGCTA AAGAATTCAT CGCTTGGCTG GTTCGTGGTC GTGGTTAA;
(2)将所述编码基因连接到表达载体pET-28(+)中,得到带有编码基因的重组表达载体pET-28a-GLP-1;
(3)将步骤(2)所述重组表达载体转化到大肠杆菌宿主BL21(DE3)中,构建GLP-1类似物的单拷贝重组工程菌;
(4)利用所述重组工程菌发酵诱导表达胞内可溶性蛋白,所述胞内可溶性蛋白包含SEQ ID NO.4所述的氨基酸序列,具体为:MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSDYKDDDDKHAEGTFTS DVSSYLEGQAAKEFIAWLVR GRG;
所述重组工程菌发酵诱导表达胞内可溶性蛋白的具体步骤为:
(S1)将所述重组工程菌的单菌落接种于含有卡那霉素的5ml的LB培养基中,在37℃、200rpm条件下振荡培养12h,即得一级种子液;
(S2)将所述一级种子液按0.5%的接种量接种于50ml的LB培养基 中,在37℃、200rpm条件下振荡培养12h,即得二级种子液;
(S3)将所述二级种子液按0.5%的接种量接种于250ml的LB培养基中,在37℃、200rpm条件下振荡培养6h,即得三级种子液;
(S4)将所述三级种子液按1%接种量接入发酵培养基中,进行高密度发酵培养,初始发酵温度为30℃,搅拌速度为300rpm,通气量为4L/min,pH为6.5-7.5,之后不断提高搅拌速度和通气量以维持溶解氧始终在5%±0.5%,等到初始培养基中碳源即将用尽,溶氧和pH会有所上升,开始流加补料,当发酵液OD
600达到8-10以上时加入乳糖进行两次诱导后,放罐,离心收集菌体。在发酵培养的过程中,每隔两小时取样测菌体OD
600值,并且进行SDS-PAGE电泳检测蛋白表达量,分别如图1A和图2中A所示(箭头处),图中可以看出:实现了目标蛋白表达。
(5)步骤(4)得到菌体200g重悬于2L裂解缓冲液,采用均质机进行均质两次,沸水浴除去大分子杂蛋白,经8000rpm、4℃离心30min后,收集上清液采用镍柱亲和层析法进行分离纯化后,酶切除盐,即得所述GLP-1类似物多肽;检测本实施例方法制得GLP-1类似物多肽的产量大于1g/L,GLP-1类似物纯度达到75%以上。
采用镍柱亲和层析法进行所述分离纯化,具体步骤包括:
(SS1)先采用10倍柱体积的去离子水和5倍柱体积的裂解缓冲液平衡柱子;(SS2)将所述上清液上样于平衡处理后的镍柱,上样流速为5ml/min;(SS3)分别用20倍柱体积的裂解缓冲液、15倍柱体积含20mM咪唑的裂解缓冲液、10倍柱体积含50mM咪唑的裂解缓冲液清洗未结合的杂蛋白,清洗流速均为15ml/min;(SS4)采用10倍柱体积含200mM咪唑的裂解缓冲液以15ml/min流速洗脱目的蛋白,即完成所述分离纯化。
所述酶切除盐的具体操作为:
(a)取100g葡聚糖凝胶介质(Sephadex G-25)加入500ml超声后的去离子水中,浸泡过夜进行溶胀,期间每隔2小时进行一次轻轻搅拌;(b)用玻璃棒引流,将步骤(a)溶胀后葡聚糖凝胶介质一次性倒入 50×300mm的中压层析柱中,装完柱体积为500ml,柱高12cm,先用10倍柱体积的去离子水冲洗柱子,再用5倍柱体积的酶切缓冲液平衡柱子;(c)将所述洗脱下来的目的蛋白在步骤(b)平衡后的柱子上进行上样,上样量为20%柱体积,每次上样100ml,流速为8ml/min;(d)采用酶切缓冲液进行洗脱,以便将目的蛋白置换到酶切缓冲液中直接进行酶切;上样后,重组GLP-1类似物(蛋白)随酶切缓冲液一起先被洗脱下来,盐离子(咪唑等)随后被洗脱下来,收集蛋白即为所述GLP-1类似物多肽。经质谱鉴定产物正确(鉴定结果如图3所示),经HPLC鉴定目的蛋白的纯度75%以上(如图4箭头所指)。
实施例2
本实施例提供一种GLP-1类似物多肽的制备方法,包括如下步骤:
(1)合成编码基因,所述编码基因的序列如SEQ ID NO.2所示,具体为:
ATGG GTTCTTCTCA CCACCACCAC CACCACTCTT CTGGTCTGGT TCCGCGTGGT TCTCACATGG CTTCTATGAC CGGTGGTCAG CAGATGGGTC GTGGTTCTGA CTACAAAGAC GACGACGACA AACACGCTGA AGGTACCTTC ACCTCTGACG TTTCTTCTTA CCTGGAAGGT CAGGCTGCTA AAGAATTCAT CGCTTGGCTG GTTCGTGGTC GTGGTTAAATGG GTTCTTCTCA CCACCACCAC CACCACTCTT CTGGTCTGGT TCCGCGTGGT TCTCACATGG CTTCTATGAC CGGTGGTCAG CAGATGGGTC GTGGTTCTGA CTACAAAGAC GACGACGACA AACACGCTGA AGGTACCTTC ACCTCTGACG TTTCTTCTTA CCTGGAAGGT CAGGCTGCTA AAGAATTCAT CGCTTGGCTG GTTCGTGGTC GTGGTTAA;
(2)将所述编码基因连接到表达载体pET-28(+)中,得到带有编码基因的重组表达载体pET-28a-GLP-1;
(3)将步骤(2)所述重组表达载体转化到大肠杆菌宿主BL21(DE3)中,构建GLP-1类似物的二拷贝重组工程菌;
(4)利用所述重组工程菌发酵诱导表达胞内可溶性蛋白,所述胞内可溶性蛋白包含SEQ ID NO.4所述的氨基酸序列;
所述重组工程菌发酵诱导表达胞内可溶性蛋白的具体步骤为:
(S1)将所述重组工程菌的单菌落接种于含有卡那霉素的5ml的LB培养基中,在37℃、200rpm条件下振荡培养12h,即得一级种子液;
(S2)将所述一级种子液按1.2%的接种量接种于50ml的LB培养基中,在37℃、200rpm条件下振荡培养12h,即得二级种子液;
(S3)将所述二级种子液按1.2%的接种量接种于250ml的LB培养基中,在37℃、200rpm条件下振荡培养9h,即得三级种子液;
(S4)将所述三级种子液按3%接种量接入发酵培养基中,进行高密度发酵培养,初始发酵温度为30℃,搅拌速度为300rpm,通气量为4L/min,pH为6.5-7.5,之后不断提高搅拌速度和通气量以维持溶解氧始终在18%±0.5%,等到初始培养基中碳源即将用尽,溶氧和pH会有所上升,开始流加补料,当发酵液OD
600达到8-10以上时加入乳糖进行两次诱导后,放罐,离心收集菌体。在发酵培养的过程中,每隔两小时取样测菌体OD
600值,并且进行SDS-PAGE电泳检测蛋白表达量,分别如图1B和图2中B所示(箭头所指),图中可以看出:目标蛋白表达显著提高。
(5)步骤(4)得到菌体200g重悬于2.5L裂解缓冲液,采用均质机进行均质两次,沸水浴除去大分子杂蛋白,经8000rpm、4℃离心30min后,收集上清液采用镍柱亲和层析法进行分离纯化后,酶切除盐,即得所述GLP-1类似物多肽;所述GLP-1类似物多肽的产量大于0.5g/L,GLP-1类似物纯度大于85%。
采用镍柱亲和层析法进行所述分离纯化,具体步骤包括:
(SS1)先采用10倍柱体积的去离子水和5倍柱体积的裂解缓冲液平衡柱子;(SS2)将所述上清液上样于平衡处理后的镍柱,上样流速为 5ml/min;(SS3)分别用20倍柱体积的裂解缓冲液、15倍柱体积含20mM咪唑的裂解缓冲液、10倍柱体积含50mM咪唑的裂解缓冲液清洗未结合的杂蛋白,清洗流速均为15ml/min;(SS4)采用10倍柱体积含200mM咪唑的裂解缓冲液以15ml/min流速洗脱目的蛋白,即完成所述分离纯化。
所述酶切除盐的具体操作为:
(a)取100g葡聚糖凝胶介质(Sephadex G-25)加入500ml超声后的去离子水中,浸泡过夜进行溶胀,期间每隔3小时进行一次轻轻搅拌;(b)用玻璃棒引流,将步骤(a)溶胀后葡聚糖凝胶介质一次性倒入50×300mm的中压层析柱中,装完柱体积为500ml,柱高12cm,先用10倍柱体积的去离子水冲洗柱子,再用5倍柱体积的酶切缓冲液平衡柱子;(c)将所述洗脱下来的目的蛋白在步骤(b)平衡后的柱子上进行上样,上样量为20%柱体积,每次上样100ml,流速为8ml/min;(d)采用酶切缓冲液进行洗脱,以便将目的蛋白置换到酶切缓冲液中直接进行酶切;上样后,重组GLP-1类似物(蛋白)随酶切缓冲液一起先被洗脱下来,盐离子(咪唑等)随后被洗脱下来,收集蛋白即为所述GLP-1类似物多肽。
实施例3
本实施例提供一种GLP-1类似物多肽的制备方法,包括如下步骤:
(1)合成编码基因,所述编码基因的序列如SEQ ID NO.3所示,具体为:
ATGG GTTCTTCTCA CCACCACCAC CACCACTCTT CTGGTCTGGT TCCGCGTGGT TCTCACATGG CTTCTATGAC CGGTGGTCAG CAGATGGGTC GTGGTTCTGA CTACAAAGAC GACGACGACA AACACGCTGA AGGTACCTTC ACCTCTGACG TTTCTTCTTA CCTGGAAGGT CAGGCTGCTA AAGAATTCAT CGCTTGGCTG GTTCGTGGTC GTGGTTAAATGG GTTCTTCTCA CCACCACCAC CACCACTCTT CTGGTCTGGT TCCGCGTGGT TCTCACATGG CTTCTATGAC CGGTGGTCAG CAGATGGGTC GTGGTTCTGA CTACAAAGAC GACGACGACA AACACGCTGA AGGTACCTTC ACCTCTGACG TTTCTTCTTA CCTGGAAGGT CAGGCTGCTA AAGAATTCAT CGCTTGGCTG GTTCGTGGTC GTGGTTAA ATGG GTTCTTCTCA CCACCACCAC CACCACTCTT CTGGTCTGGT TCCGCGTGGT TCTCACATGG CTTCTATGAC CGGTGGTCAG CAGATGGGTC GTGGTTCTGA CTACAAAGAC GACGACGACA AACACGCTGA AGGTACCTTC ACCTCTGACG TTTCTTCTTA CCTGGAAGGT CAGGCTGCTA AAGAATTCAT CGCTTGGCTG GTTCGTGGTC GTGGTTAAATGG GTTCTTCTCA CCACCACCAC CACCACTCTT CTGGTCTGGT TCCGCGTGGT TCTCACATGG CTTCTATGAC CGGTGGTCAG CAGATGGGTC GTGGTTCTGA CTACAAAGAC GACGACGACA AACACGCTGA AGGTACCTTC ACCTCTGACG TTTCTTCTTA CCTGGAAGGT CAGGCTGCTA AAGAATTCAT CGCTTGGCTG GTTCGTGGTC GTGGTTAA;
(2)将所述编码基因连接到表达载体pET-28(+)中,得到带有编码基因的重组表达载体pET-28a-GLP-1;
(3)将步骤(2)所述重组表达载体转化到大肠杆菌宿主BL21(DE3)中,构建GLP-1类似物的四拷贝重组工程菌;
(4)利用所述重组工程菌发酵诱导表达胞内可溶性蛋白,所述胞内可溶性蛋白包含SEQ ID NO.4所述的氨基酸序列;
所述重组工程菌发酵诱导表达胞内可溶性蛋白的具体步骤为:
(S1)将所述重组工程菌的单菌落接种于含有卡那霉素的5ml的LB培养基中,在37℃、200rpm条件下振荡培养12h,即得一级种子液;
(S2)将所述一级种子液按2.0%的接种量接种于50ml的LB培养基中,在37℃、200rpm条件下振荡培养12h,即得二级种子液;
(S3)将所述二级种子液按2.0%的接种量接种于250ml的LB培养 基中,在37℃、200rpm条件下振荡培养12h,即得三级种子液;
(S4)将所述三级种子液按5%接种量接入发酵培养基中,进行高密度发酵培养,初始发酵温度为30℃,搅拌速度为300rpm,通气量为4L/min,pH为6.5-7.5,之后不断提高搅拌速度和通气量以维持溶解氧始终在20%±0.5%,等到初始培养基中碳源即将用尽,溶氧和pH会有所上升,开始流加补料,当发酵液OD
600达到8-10以上时加入乳糖进行两次诱导后,放罐,离心收集菌体。在发酵培养的过程中,每隔两小时取样测菌体OD
600值,并且进行SDS-PAGE电泳检测蛋白表达量,分别如图1C和图2中C所示(箭头所指),图中可以看出:目标蛋白表达大幅度提高。
(5)步骤(4)得到菌体200g重悬于3L裂解缓冲液,采用均质机进行均质两次,沸水浴除去大分子杂蛋白,经8000rpm、4℃离心30min后,收集上清液采用镍柱亲和层析法进行分离纯化后,酶切除盐,即得所述GLP-1类似物多肽;所述GLP-1类似物多肽的产量大于1g/L,GLP-1类似物纯度大于85%。
采用镍柱亲和层析法进行所述分离纯化,具体步骤包括:
(SS1)先采用10倍柱体积的去离子水和5倍柱体积的裂解缓冲液平衡柱子;(SS2)将所述上清液上样于平衡处理后的镍柱,上样流速为5ml/min;(SS3)分别用20倍柱体积的裂解缓冲液、15倍柱体积含20mM咪唑的裂解缓冲液、10倍柱体积含50mM咪唑的裂解缓冲液清洗未结合的杂蛋白,清洗流速均为15ml/min;(SS4)采用10倍柱体积含200mM咪唑的裂解缓冲液以15ml/min流速洗脱目的蛋白,即完成所述分离纯化。收集各洗脱组分进行Tris-Tricine-SDS-PAGE电泳,纯化过程及产物电泳如图5所示。
所述酶切除盐的具体操作为:
(a)取100g葡聚糖凝胶介质(Sephadex G-25)加入500ml超声后的去离子水中,浸泡过夜进行溶胀,期间每隔4小时进行一次轻轻搅拌;(b)用玻璃棒引流,将步骤(a)溶胀后葡聚糖凝胶介质一次性倒入 50×300mm的中压层析柱中,装完柱体积为500ml,柱高12cm,先用10倍柱体积的去离子水冲洗柱子,再用5倍柱体积的酶切缓冲液平衡柱子;(c)将所述洗脱下来的目的蛋白在步骤(b)平衡后的柱子上进行上样,上样量为20%柱体积,每次上样100ml,流速为8ml/min;(d)采用酶切缓冲液进行洗脱,以便将目的蛋白置换到酶切缓冲液中直接进行酶切;上样后,重组GLP-1类似物(蛋白)随酶切缓冲液一起先被洗脱下来,盐离子(咪唑等)随后被洗脱下来,收集蛋白即为所述GLP-1类似物多肽。
实验例
对实施例1所得GLP-1类似物多肽(GLP-1类似物)在Ⅱ型糖尿病中的降糖效果进行评定,具体操作如下:
1)高脂饲料联合STZ诱导Ⅱ型糖尿病小鼠模型
50只6周龄(18-20g)BALB/C小鼠(雄),将小鼠分笼饲养,控制动物房温度25±2℃,湿度50±10%,光照12h黑暗12h循环,适应环境一周。将小鼠以8只/组随机分笼。所有小鼠禁食12h后测定体重及空腹血糖(Fasting blood-glucose,FBG),对照组以标准饲料继续饲喂4周,建模组以高脂饲料持续饲喂4周。4周后,测定各组小鼠体重及FBG。禁食12h后,按60mg/kg(体重)的剂量腹腔注射STZ,连续注射3天。
分别于建模后第3、7、10、14天测定建模组小鼠体重及FBG,选取FBG≥11.1mmol/L且稳定一周的小鼠作为Ⅱ型糖尿病模型小鼠,以8只/组随机分笼。糖尿病模型小鼠和正常小鼠的胰腺组织切片对比如图6所示,证实建模成功。
2)GLP-1类似物在Ⅱ型糖尿病小鼠中的降糖效果
将小鼠随机分三组:糖尿病负对照组(DCN),糖尿病正对照组(DCP),糖尿病治疗组(DT),每组8只小鼠。
所有小鼠过夜禁食12h后,按1.5g/kg(体重)灌胃D-葡萄糖。
然后皮下注射(s.c.)GLP-1类似物(12μg/50g体重),DCP和DCN组分别注射相同体积的生理盐水(0.9%NaCl)和市售利拉鲁肽注射液。
注射后0、15、30、60、120、180min、300min后断尾取血,用罗氏血糖试纸测定血糖水平,结果如图7所示,从图中可以看出:本发明制得的所述GLP-1类似物(His-31p)与市售利拉鲁肽(Lir)具有同等的降血糖效果。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到变化或替换,都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应以所述权利要求的保护范围为准。
本发明提供一种GLP-1类似物多肽的制备方法及其在Ⅱ型糖尿病中的应用。通过先合成编码基因,再获得带有该编码基因的重组表达载体,构建重组工程菌发酵诱导表达胞内可溶性蛋白,实现GLP-1类似物的高效表达,避免了现有技术采用的对包涵体变性复性和连接侧链的工序,简化工艺,有效降低成本,减少环境污染物的产生,利于工业化放大;最后本发明采用镍柱亲和层析进行分离纯化,分离度高,纯化效果好,杂质少,操作简单。本发明方法制备得到GLP-1类似物的产量大于1g/L,GLP-1类似物纯度达到87%以上。所述GLP-1类似物经肠激酶酶切可进一步制备利拉鲁肽中间体GLP-1(7-37),用于制备利拉鲁肽,具有较好的经济价值和应用前景。
Claims (10)
- 一种GLP-1类似物多肽的制备方法,其特征在于,包括如下步骤:(1)合成编码基因;(2)将所述编码基因连接到表达载体中,得到带有编码基因的重组表达载体;(3)将步骤(2)所述重组表达载体转化到大肠杆菌宿主中,构建重组工程菌;(4)利用所述重组工程菌发酵诱导表达胞内可溶性蛋白;(5)步骤(4)得到菌体进行破碎后离心,取上清液进行分离纯化、酶切除盐后,即得所述GLP-1类似物多肽。
- 根据权利要求1所述的GLP-1类似物多肽的制备方法,其特征在于,步骤(1)中,所述编码基因的序列为SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3中的任一种。
- 根据权利要求1所述的GLP-1类似物多肽的制备方法,其特征在于,步骤(2)中,所述表达载体为pET-28(+);步骤(3)中,所述重组工程菌为单拷贝、二拷贝或四拷贝的重组工程菌菌株。
- 根据权利要求1所述的GLP-1类似物多肽的制备方法,其特征在于,步骤(4)中,所述胞内可溶性蛋白包含SEQ ID NO.4所示的氨基酸序列。
- 根据权利要求1所述的GLP-1类似物多肽的制备方法,其特征在于,步骤(4)中,所述重组工程菌发酵诱导表达胞内可溶性蛋白的具体步骤为:(S1)将所述重组工程菌的单菌落接种于含有卡那霉素的LB培养基中,在37℃、200rpm条件下振荡培养12h,即得一级种子液;(S2)将所述一级种子液按0.5-2.0%的接种量接种于LB培养基中,在37℃、200rpm条件下振荡培养12h,即得二级种子液;(S3)将所述二级种子液按0.5-2.0%的接种量接种于LB培养基中,在37℃、200rpm条件下振荡培养6-12h,即得三级种子液;(S4)将所述三级种子液按1-5%接种量接入发酵培养基中,进行发酵培养,当发酵液OD 600达到8-10以上时加入诱导剂进行两次诱导,诱导完成后放罐,离心收集菌体。
- 根据权利要求5所述的GLP-1类似物多肽的制备方法,其特征在于,步骤(S4)中,所述发酵培养为高密度发酵培养,所述诱导剂为乳糖。
- 根据权利要求5所述的GLP-1类似物多肽的制备方法,步骤(S4)中,进行所述发酵培养时,初始发酵温度为30℃,搅拌速度为300rpm,通气量为4L/min,pH为6.5-7.5,之后不断提高搅拌速度和通气量以维持溶解氧在4.5%-20.5%。
- 根据权利要求1所述的GLP-1类似物多肽的制备方法,其特征在于,步骤(5)中,采用镍柱亲和层析法进行所述分离纯化,具体步骤包括:(SS1)先采用10倍柱体积的去离子水和5倍柱体积的裂解缓冲液平衡柱子;(SS2)将所述上清液上样于平衡处理后的镍柱,上样流速为5ml/min;(SS3)分别用20倍柱体积的裂解缓冲液、15倍柱体积含20mM咪唑的裂解缓冲液、10倍柱体积含50mM咪唑的裂解缓冲液清洗未结合的杂蛋白,清洗流速均为15ml/min;(SS4)采用10倍柱体积含200mM咪唑的裂解缓冲液以15ml/min流速洗脱目的蛋白,即完成所述分离纯化。
- 根据权利要求1所述的GLP-1类似物多肽的制备方法,其特征在于,步骤(5)中,所述酶切除盐的具体操作为:(a)取葡聚糖凝胶介质加入500ml超声后的去离子水中,浸泡过夜 进行溶胀,期间进行数次搅拌;(b)用玻璃棒引流,将步骤(a)溶胀后葡聚糖凝胶介质一次性倒入50×300mm的中压层析柱中,装完柱体积为500ml,柱高12cm,先用10倍柱体积的去离子水冲洗柱子,再用5倍柱体积的酶切缓冲液平衡柱子;(c)将所述洗脱下来的目的蛋白在步骤(b)平衡后的柱子上进行上样,上样量为20%柱体积,每次上样100ml,流速为8ml/min;(d)采用酶切缓冲液进行洗脱,收集蛋白即为所述GLP-1类似物多肽。
- 权利要求1-9任一项所述方法制得的GLP-1类似物多肽在制备治疗Ⅱ型糖尿病药物中的应用。
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