利用IMPACT-TWIN系統(tǒng)制備降鈣素和胰島素類似物的研究
發(fā)布時(shí)間：2019-01-03 13:53
【摘要】：生物活性肽是一類活性好、利用率較高的化合物。目前,已有多種生物活性多肽作為藥物用于臨床治療。本文研究了兩種活性肽的生物制備方法。論文包括兩個(gè)部分,第一部分是人源降鈣素類似物的生物制備。第二部分是重組B27賴氨酸去三肽胰島素前體(MIP, monomeric B27Lys destripeptide insulin precursor)融合蛋白包含體復(fù)性方法的研究。 在第一部分,實(shí)驗(yàn)制備了環(huán)狀和線性的人源降鈣素類似物。骨質(zhì)疏松現(xiàn)已成為世界范圍的健康問題,目前全球發(fā)病率已超過25%,躍居常見病、多發(fā)病的第7位。降鈣素是臨床應(yīng)用中治療骨質(zhì)疏松最有效的藥物之一。因?yàn)槿嗽唇碘}素來源有限,目前臨床常用的降鈣素為鮭魚降鈣素。但是鮭魚降鈣素存在一定的免疫源性,所以改進(jìn)人源降鈣素的制備方法是亟待解決的一個(gè)課題。降鈣素生理活性與其C末端酰胺化結(jié)構(gòu)密切相關(guān)。因此,通過基因工程方法制備人源降鈣素,必須進(jìn)行體外的C末端酰胺化,這導(dǎo)致制備過程復(fù)雜且成本較高。目前的研究表明對于末端酰胺化的活性肽可以通過頭尾環(huán)化的方式得到與線性肽活性相近的環(huán)狀類似物,這不僅解決了線性肽C末端酰胺化的問題,而且環(huán)肽具有更穩(wěn)定的骨架結(jié)構(gòu)和功能活性。本實(shí)驗(yàn)采用了大腸桿菌IMPACT-TWIN (Intein Mediated Purification with an Affinity Chitin-binding Tag-Two Intein)表達(dá)系統(tǒng)。該系統(tǒng)通過不同的克隆方法可以獲得頭尾相連的環(huán)肽或線性肽。在制備過程中經(jīng)幾丁質(zhì)親和純化和內(nèi)含肽剪接即可獲得無冗余氨基酸殘基的活性肽。整個(gè)制備過程具有步驟少、時(shí)間短等優(yōu)點(diǎn)。論文制備了兩種人源降鈣素類似物。其中,人源降鈣素類似物chCalcitonin (△AP)(cyclic human calcitonin)為C末端缺失兩個(gè)氨基酸殘基的環(huán)狀人源降鈣素類似物。另一種人源降鈣素類似物hCalcitonin (human calcitonin)為全長線性的人源降鈣素類似物。采用IMPACT-TWIN表達(dá)系統(tǒng)獲得18.5mg/L環(huán)狀人源降鈣素chCalcitonin (△AP)初純品,經(jīng)質(zhì)譜鑒定其分子量與理論值一致,表明這一生物制備方法是可行的。在人源降鈣素類似物hCalcitonin的研究中,對誘導(dǎo)時(shí)間、溫度、IPTG濃度進(jìn)行了優(yōu)化,確定最佳誘導(dǎo)表達(dá)條件為16℃,16h,0.05mM IPTG。 在論文第二部分,主要探討重組B27賴氨酸去三肽胰島素前體MIP融合蛋白包含體的復(fù)性方法。速效胰島素是一類通過基因工程方式獲得,與餐后正常人體內(nèi)胰島素分泌代謝時(shí)相最為接近的胰島素類似物。B27賴氨酸去三肽胰島素(B27K-DTrI)是由本實(shí)驗(yàn)室制備的一類速效胰島素類似物。其活性為野生型人胰島素的80%,具有潛在的臨床應(yīng)用價(jià)值。在大腸桿菌中重組表達(dá)的B27賴氨酸去三肽胰島素前體(MIP)融合蛋白為包含體,需要經(jīng)過體外變復(fù)性后才能恢復(fù)其生物活性。因此,提高其復(fù)性率是獲得大量有活性的B27K-DTrI的關(guān)鍵步驟。本實(shí)驗(yàn)比較了三種復(fù)性方法(透析法、稀釋法及凝膠過濾層析法)對MIP復(fù)性率的影響。實(shí)驗(yàn)結(jié)果表明復(fù)性效果較好為43.6%。在此基礎(chǔ)上,對復(fù)性緩沖液中各種因子進(jìn)行優(yōu)化。以GSSG:GSH、Arg、PEG:Pro為三因素,設(shè)計(jì)正交實(shí)驗(yàn)。經(jīng)極差和析因分析結(jié)果表明最佳的復(fù)性條件為2mM GSH、0.4mM GSSG、0.8M PEG:Pro=2:1。在此最優(yōu)條件下,進(jìn)行驗(yàn)證實(shí)驗(yàn)。實(shí)驗(yàn)結(jié)果表明,MIP復(fù)性效率為72%,產(chǎn)率為1.56mg/L。復(fù)性效率及產(chǎn)率均為優(yōu)化前兩倍,基本達(dá)到優(yōu)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，
本文編號(hào)：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，
本文編號(hào)：2399467
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