新型融合序列在长效GLP-1药物开发中的应用

发布时间：2018-01-31 15:02

本文关键词： GLP-1 半衰期分离纯化生物活性　出处：《上海医药工业研究院》2017年硕士论文　论文类型：学位论文

【摘要】：众所周知,多肽类治疗药物易被体内蛋白酶降解导致半衰期较短,临床上往往通过增加给药频率或给药剂量来实现治疗效果,这无疑给患者造成了巨大的痛苦和经济负担,因此,药企和研发机构不断探索新型长效策略来延长多肽类药物的半衰期。血清白蛋白结合序列(SABA序列),XTEN序列和PAS序列是国外最新报道可显著延长多肽药物半衰期的长效序列,但在国内医药领域并没有相关的文献报道。本研究以胰高血糖素样肽-1(GLP-1)为研究对象,采用大肠杆菌重组表达的方式,将上述三种长效序列与GLP-1序列融合,考察融合蛋白的表达情况和生物活性,验证上述三种长效序列实施方法的可行性,为开发其他长效多肽药物提供参考。文中就三个长效序列-SABA序列,XTEN序列和PAS序列分别与GLP-1序列进行融合,设计了18个不同连接肽和不同组合方式的SABA-GLP-1融合序列、4个不同长度的XTEN-GLP-1融合序列、2个不同长度的PAS-GLP-1融合序列。上述构建的24个新型长效融合序列均可成功在大肠杆菌中进行表达。本研究通过优化发酵诱导条件增加了融合蛋白的表达量,并对不同的融合蛋白建立了相应的分离纯化方法,得到了纯度相对较高的融合蛋白产物。本研究还建立了GLP-1类化合物体外活性检测方法,应用该方法对长效药物阿必鲁肽和GLP-1(7-37)进行了活性验证,同时对上述得到的所有长效GLP-1融合蛋白进行了活性检测和对比分析。结果表明:SABA-GLP-1融合蛋白均以包涵体形式表达,后期需要对其进行变性和复性,但复性后纯化工艺相对简单。不同连接肽连接的SABA-GLP-1融合蛋白在表达量和生物学活性方面也有较大差异,且该系列融合蛋白的体外生物学活性普遍不高,其中生物活性最高的S12融合蛋白的半数有效浓度为9.296 nmol/L;而XTEN-GLP-1和PAS-GLP-1融合蛋白均以可溶性形式表达,且纯化工艺复杂,但体外生物活性普遍较SABA-GLP-1融合蛋白高,其中活性较高的AE2和PAS1的半数有效浓度分别为5.068nmol/L和2.820 nmol/L。通过本研究表明,验证了利用大肠杆菌重组表达的方式制备上述三种长效融合蛋白方法的可行性。SABA序列,XTEN序列和PAS序列3个长效序列与多肽药物融合后可在一定程度上保持生物活性,且不同长效序列的长度、Linker和组合方式的变化均对融合蛋白的生物活性产生显著影响,后续可以通过优化序列、Linker和不同组合方式进一步提高其生物活性。
[Abstract]:It is well known that polypeptide treatment drugs are easy to be degraded by protease in vivo resulting in a shorter half-life, clinical treatment often by increasing the frequency or dosage of drugs to achieve therapeutic effect. This undoubtedly caused great suffering and economic burden to patients, therefore. Pharmaceutical companies and R & D institutions have been exploring new long-term strategies to prolong the half-life of polypeptide drugs. Serum albumin binding sequence (SABA). XTEN sequence and PAS sequence are the new long acting sequences which can prolong the half life of polypeptide drugs. However, there is no related literature in the field of medicine in China. In this study, glucagon like peptide-1 (GLP-1) was used as the research object, and Escherichia coli recombinant expression was used. The fusion of the three long-acting sequences with GLP-1 sequence was performed to investigate the expression and biological activity of the fusion protein and to verify the feasibility of the implementation of the three long-acting sequences. For the development of other long-acting polypeptide drugs, three long-acting sequences-SABA sequence XTEN sequence and PAS sequence were fused with GLP-1 sequence respectively. Eighteen SABA-GLP-1 fusion sequences with different binding peptides and different combinations were designed, and four XTEN-GLP-1 fusion sequences with different lengths were designed. Two PAS-GLP-1 fusion sequences of different lengths were constructed. The 24 novel fusion sequences were successfully expressed in Escherichia coli. In this study, the fusion was increased by optimizing the fermentation induction conditions. Protein expression. The different fusion proteins were separated and purified, and the relative high purity fusion protein products were obtained. In this study, the in vitro activity detection method of GLP-1 compounds was also established. This method was used to verify the activity of the long-acting drugs Abilutine and GLP-1 (7-37). At the same time, the activity of all the long-acting GLP-1 fusion proteins were detected and compared. The results showed that all of the fusion proteins were expressed in the form of inclusion body. The denaturation and renaturation were needed in the later stage, but the purification process after renaturation was relatively simple. The expression and biological activity of SABA-GLP-1 fusion protein with different ligation peptides were also different. The biological activity of S12 fusion protein was not high in vitro, and the median effective concentration of S12 fusion protein with the highest bioactivity was 9.296 nmol / L; However, both XTEN-GLP-1 and PAS-GLP-1 fusion proteins were expressed in soluble form, and the purification process was complicated, but the bioactivity in vitro was generally higher than that of SABA-GLP-1 fusion protein. The median effective concentrations of AE2 and PAS1 with high activity were 5.068 nmol / L and 2.820 nmol / L, respectively. The feasibility of preparing the three kinds of long-term fusion protein by E. coli recombinant expression was verified. The fusion of XTEN sequence and PAS sequence with polypeptide drugs can maintain biological activity to a certain extent and the length of different long-acting sequences. The changes of Linker and combination mode had a significant effect on the bioactivity of the fusion protein, which could be further improved by optimizing the sequence of linker and different combinations.
【学位授予单位】：上海医药工业研究院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R915

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