本文選題：促胰島素分泌肽類似物 + PEG修飾�。� 參考：《重慶理工大學》2014年碩士論文

【摘要】：研究目的: 通過化學合成法合成促胰島素分泌肽類似物(Byt-C)，Byt-C是以上市藥物艾塞那肽（Byetta）的39個氨基酸序列為基礎，在其C末端第39位后連接一個半胱氨酸（Cys）而成，采用長效修飾劑馬來酰亞胺活化單甲氧基40kDa分子量的聚乙二醇（mPEG-MAL-40K）對Byt-C的半胱氨酸游離巰基進行單一位點的定點修飾，并且通過優(yōu)化其修飾、純化工藝，達到高修飾率，，得到高純度目標產(chǎn)物。通過質(zhì)譜分子量和質(zhì)量肽圖確認結(jié)構(gòu)中半胱氨酸游離巰基的定點修飾以及修飾劑中馬來酰亞胺為閉環(huán)結(jié)構(gòu)的穩(wěn)定產(chǎn)物。 研究方法: 1)通過化學合成法獲得Byt-C。①Byt-C純度檢測，包括SDS-PAGE，RP-HPLC方法的建立。②Byt-C結(jié)構(gòu)確認，包括氨基酸全序測定，質(zhì)譜分子量測定，質(zhì)量肽圖分析驗證。 2) Byt-C的mPEG-MAL-40K修飾。包括修飾比例，修飾緩沖條件，修飾緩沖體系，修飾濃度，保護劑優(yōu)化。 3) mPEG-MAL-Byt-C分離純化。包括陽離子交換層析、反相層析、凝膠過濾層析。 4) mPEG-MAL-Byt-C非C末端修飾異構(gòu)體研究。包括胰蛋白酶酶切質(zhì)量肽圖，修飾位點，氨基偶聯(lián)分析。 5)馬來酰亞胺為閉環(huán)結(jié)構(gòu)的確證研究。包括水解開環(huán)，氫譜核磁共振分析。 研究結(jié)果: 1. Byt-C多肽的質(zhì)譜分子量與理論值完全一致，氨基酸測序完全正確，RP-HPLC純度99%以上。 2. mPEG-MAL-Byt-C經(jīng)修飾、純化條件優(yōu)化研究，可獲得純度高達98%的單一目標產(chǎn)物。 3. mPEG-MAL-Byt-C非C末端修飾異構(gòu)體研究顯示，證明了mPEG-MAL-Byt-C為修飾位點是單一的Byt-C多肽C末端Cys的游離巰基。 4. mPEG-MAL-Byt-C中馬來酰亞胺為閉環(huán)結(jié)構(gòu)的確認研究表明，目標產(chǎn)物為Byt-C多肽與聚乙二醇修飾劑mPEG-MAL-40k的閉環(huán)馬來酰亞胺偶聯(lián)而成的單一產(chǎn)物，且在修飾、純化以及保存過程中均不會產(chǎn)生PEG的開環(huán)共軛物。
[Abstract]:Objectives of the study: The synthesis of insulin stimulating peptide analogue Byt-C by chemical synthesis is based on the 39 amino acid sequence of the listed drug Eisenapeptide Byettaand is connected to a cysteine CysA at the 39th position of its C-terminal. The polyethyleneglycol mPEG-MAL-40K, activated by Maleimide, was used to modify the cysteine free sulfhydryl group of Byt-C at a single site. By optimizing the modification and purification process, the modification rate was high. High purity target product was obtained. The fixed point modification of cysteine free sulfhydryl group in the structure and the stable product of Maleimide in the modifier were confirmed by mass spectrometry molecular weight and mass peptide map. Research methods: 1) the purity of Byt-C.1Byt-C was determined by chemical synthesis method, including the establishment of SDS-PAGEGE-RP-HPLC method, the confirmation of the structure of 2.2Byt-C, including the determination of total sequence of amino acids, the determination of molecular weight by mass spectrometry, and the verification of mass peptide analysis. 2) mPEG-MAL-40K modification of Byt-C. It includes modification ratio, modification buffer condition, modified buffer system, modification concentration, protection agent optimization. 3) mPEG-MAL-Byt-C separation and purification. It includes cation exchange chromatography, reverse phase chromatography and gel filtration chromatography. 4) study on non-C-terminal modified isomers of mPEG-MAL-Byt-C. It includes trypsin digested mass peptide map, modification site and amino coupling analysis. 5) confirmation of Maleimide as closed loop structure. Including hydrolysis ring opening, hydrogen NMR analysis. Results of the study: 1. The molecular weight of Byt-C polypeptide was completely consistent with the theoretical value, and the purity of RP-HPLC was over 99% by amino acid sequencing. 2. After modification of mPEG-MAL-Byt-C and optimization of purification conditions, a single target product with purity up to 98% was obtained. 3. The non-C-terminal modified isomers of mPEG-MAL-Byt-C showed that mPEG-MAL-Byt-C was the free sulfhydryl group of the C-terminal Cys of a single Byt-C polypeptide. 4. The confirmation of the closed loop structure of Maleimide in mPEG-MAL-Byt-C shows that the target product is a single product of Byt-C polypeptide coupled with the closed loop Maleimide of polyethylene glycol modifier mPEG-MAL-40k. No open loop conjugate of PEG is produced during purification and preservation.
【學位授予單位】：重慶理工大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R914.5

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