利用IMPACT-TWIN系统制备降钙素和胰岛素类似物的研究
发布时间：2019-01-03 13:53
【摘要】：生物活性肽是一类活性好、利用率较高的化合物。目前,已有多种生物活性多肽作为药物用于临床治疗。本文研究了两种活性肽的生物制备方法。论文包括两个部分,第一部分是人源降钙素类似物的生物制备。第二部分是重组B27赖氨酸去三肽胰岛素前体(MIP, monomeric B27Lys destripeptide insulin precursor)融合蛋白包含体复性方法的研究。 在第一部分,实验制备了环状和线性的人源降钙素类似物。骨质疏松现已成为世界范围的健康问题,目前全球发病率已超过25%,跃居常见病、多发病的第7位。降钙素是临床应用中治疗骨质疏松最有效的药物之一。因为人源降钙素来源有限,目前临床常用的降钙素为鲑鱼降钙素。但是鲑鱼降钙素存在一定的免疫源性,所以改进人源降钙素的制备方法是亟待解决的一个课题。降钙素生理活性与其C末端酰胺化结构密切相关。因此,通过基因工程方法制备人源降钙素,必须进行体外的C末端酰胺化,这导致制备过程复杂且成本较高。目前的研究表明对于末端酰胺化的活性肽可以通过头尾环化的方式得到与线性肽活性相近的环状类似物,这不仅解决了线性肽C末端酰胺化的问题,而且环肽具有更稳定的骨架结构和功能活性。本实验采用了大肠杆菌IMPACT-TWIN (Intein Mediated Purification with an Affinity Chitin-binding Tag-Two Intein)表达系统。该系统通过不同的克隆方法可以获得头尾相连的环肽或线性肽。在制备过程中经几丁质亲和纯化和内含肽剪接即可获得无冗余氨基酸残基的活性肽。整个制备过程具有步骤少、时间短等优点。论文制备了两种人源降钙素类似物。其中,人源降钙素类似物chCalcitonin (△AP)(cyclic human calcitonin)为C末端缺失两个氨基酸残基的环状人源降钙素类似物。另一种人源降钙素类似物hCalcitonin (human calcitonin)为全长线性的人源降钙素类似物。采用IMPACT-TWIN表达系统获得18.5mg/L环状人源降钙素chCalcitonin (△AP)初纯品,经质谱鉴定其分子量与理论值一致,表明这一生物制备方法是可行的。在人源降钙素类似物hCalcitonin的研究中,对诱导时间、温度、IPTG浓度进行了优化,确定最佳诱导表达条件为16℃,16h,0.05mM IPTG。 在论文第二部分,主要探讨重组B27赖氨酸去三肽胰岛素前体MIP融合蛋白包含体的复性方法。速效胰岛素是一类通过基因工程方式获得,与餐后正常人体内胰岛素分泌代谢时相最为接近的胰岛素类似物。B27赖氨酸去三肽胰岛素(B27K-DTrI)是由本实验室制备的一类速效胰岛素类似物。其活性为野生型人胰岛素的80%,具有潜在的临床应用价值。在大肠杆菌中重组表达的B27赖氨酸去三肽胰岛素前体(MIP)融合蛋白为包含体,需要经过体外变复性后才能恢复其生物活性。因此,提高其复性率是获得大量有活性的B27K-DTrI的关键步骤。本实验比较了三种复性方法(透析法、稀释法及凝胶过滤层析法)对MIP复性率的影响。实验结果表明复性效果较好为43.6%。在此基础上,对复性缓冲液中各种因子进行优化。以GSSG:GSH、Arg、PEG:Pro为三因素,设计正交实验。经极差和析因分析结果表明最佳的复性条件为2mM GSH、0.4mM GSSG、0.8M PEG:Pro=2:1。在此最优条件下,进行验证实验。实验结果表明,MIP复性效率为72%,产率为1.56mg/L。复性效率及产率均为优化前两倍,基本达到优化目的。
[Abstract]:The bioactive peptide is a compound with good activity and high utilization rate. Currently, a plurality of biologically active polypeptides have been used as medicaments for clinical treatment. The biological preparation of two active peptides is studied in this paper. The thesis consists of two parts, the first part is the biological preparation of the human procalcitonin analog. The second part is a study of the renaturation method of the fusion protein of the recombinant B27 lysine detripeptide insulin precursor (MIP). In the first part, the experiment prepared a ring-like and linear human procalcitonin Osteoporosis has now become a world-wide health problem, with a global incidence of more than 25%, a common and frequently-occurring disease 7 Bit. Calcitonin is the most effective drug for the treatment of osteoporosis in clinical use. 1. Because of the limited source of procalcitonin, the commonly used calcitonin is the calcium-reducing calcium of the salmon. but salmon calcitonin has a certain immune source, so the method of improving the human source calcitonin is a class to be solved urgently. in that study, the physiological activity of the calcitonin is closely related to the C-terminal and the amine-like structure of the C-terminal. Off. Therefore, human source calcitonin is prepared by a genetic engineering method, which necessitates an in vitro C-terminal deamination, which results in a complex preparation process and a lower cost High. The present study shows that the active peptides with the end-to-end amination can be obtained by the end-to-end cyclization to obtain a cyclic analogue close to the activity of the linear peptide, which not only solves the problem that the terminal of the linear peptide C is amide, but also has a more stable framework structure and function activity. The expression of IMPACT-TWIN (Intein Mediated Purification with an Affinity Chitin-binding Tag-Two Ingenin) was used in this experiment. Series. The system can obtain the end-to-end linked cyclic peptide or linear through different cloning methods peptide. The activity of the non-redundant amino acid residue can be obtained by the chitin affinity purification and the inclusion of peptide splicing in the preparation process. The whole preparation process has the advantages of less steps, short time and the like. Point. The paper prepared two kinds of human procalcitonin. The human source calcitonin analogue, chCalcitonin, is similar to the cyclic human source calcitonin in which the C-terminal is missing two amino acid residues another human source calcitonin analogue, hCalcitonin, is a full-length linear human source calcitonin An IMPACT-TWIN expression system was used to obtain the initial pure product of 18. 5 mg/ L ring-type human procalcitonin (BAP), and the molecular weight was determined by mass-spectrum to be consistent with the theoretical value, indicating that this method is feasible. The induction time, temperature and IPTG concentration were optimized in the study of human source calcitronin. The optimal induction conditions were 16 鈩，
本文编号：2399467
[Abstract]:The bioactive peptide is a compound with good activity and high utilization rate. Currently, a plurality of biologically active polypeptides have been used as medicaments for clinical treatment. The biological preparation of two active peptides is studied in this paper. The thesis consists of two parts, the first part is the biological preparation of the human procalcitonin analog. The second part is a study of the renaturation method of the fusion protein of the recombinant B27 lysine detripeptide insulin precursor (MIP). In the first part, the experiment prepared a ring-like and linear human procalcitonin Osteoporosis has now become a world-wide health problem, with a global incidence of more than 25%, a common and frequently-occurring disease 7 Bit. Calcitonin is the most effective drug for the treatment of osteoporosis in clinical use. 1. Because of the limited source of procalcitonin, the commonly used calcitonin is the calcium-reducing calcium of the salmon. but salmon calcitonin has a certain immune source, so the method of improving the human source calcitonin is a class to be solved urgently. in that study, the physiological activity of the calcitonin is closely related to the C-terminal and the amine-like structure of the C-terminal. Off. Therefore, human source calcitonin is prepared by a genetic engineering method, which necessitates an in vitro C-terminal deamination, which results in a complex preparation process and a lower cost High. The present study shows that the active peptides with the end-to-end amination can be obtained by the end-to-end cyclization to obtain a cyclic analogue close to the activity of the linear peptide, which not only solves the problem that the terminal of the linear peptide C is amide, but also has a more stable framework structure and function activity. The expression of IMPACT-TWIN (Intein Mediated Purification with an Affinity Chitin-binding Tag-Two Ingenin) was used in this experiment. Series. The system can obtain the end-to-end linked cyclic peptide or linear through different cloning methods peptide. The activity of the non-redundant amino acid residue can be obtained by the chitin affinity purification and the inclusion of peptide splicing in the preparation process. The whole preparation process has the advantages of less steps, short time and the like. Point. The paper prepared two kinds of human procalcitonin. The human source calcitonin analogue, chCalcitonin, is similar to the cyclic human source calcitonin in which the C-terminal is missing two amino acid residues another human source calcitonin analogue, hCalcitonin, is a full-length linear human source calcitonin An IMPACT-TWIN expression system was used to obtain the initial pure product of 18. 5 mg/ L ring-type human procalcitonin (BAP), and the molecular weight was determined by mass-spectrum to be consistent with the theoretical value, indicating that this method is feasible. The induction time, temperature and IPTG concentration were optimized in the study of human source calcitronin. The optimal induction conditions were 16 鈩，
本文编号：2399467
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