【作者】 陈伟；

【导师】 熊盛；

【作者基本信息】 暨南大学 ， 微生物与生化药学， 2010， 硕士

【摘要】 蓝藻抗病毒蛋白N (cyanovirin-N, CVN)是一种备受关注的HIV特异性microbicide候选物,作为一种异源多肽,存在半衰期短、易引起免疫应答等缺点；PEG修饰是解决蛋白质类药物在药用过程中存在的稳定性差,半衰期短等问题的有效途径,但CVN蛋白N-末端氨基酸、半胱氨酸和赖氨酸残基都位于其活性中心或活性中心附近,直接的PEG修饰会影响CVN与病毒表面糖蛋白gpl20的结合,从而影响其抗病毒活性。本实验中,在CVN的N-末端接入了(GGGGS)3的亲水柔性Linker序列(即LCVN),通过His6-SUMO的融合表达系统,SUMO-LCVN蛋白在大肠杆菌中以可溶的形式表达,表达量占菌体总蛋白的30%。SUMO-LCVN融合蛋白比SUMO-CVN蛋白对SUMO蛋白酶更敏感,能被SUMO蛋白酶充分酶切,因此LCVN蛋白的制备更加高效,更适合大规模生产。分别以分子量为10KDa和20KDa的mPEG-ALD对LCVN的N-末端进行定点修饰,对反应pH、LCVN与mPEG-ALD的摩尔比进行筛选；得到了最佳的PEG修饰条件。反应时间对PEG修饰结果影响较小,室温2h即可反应完全。SP sepHarose阳离子交换亲和层析成功分离单PEG修饰产物(monoPEGyalted-LCVN),其纯度>95%。在纳摩尔浓度范围,LCVN、monoPEGyalted-LCVN与gp120蛋白有较高的亲和力,并呈现一定的浓度依赖性。体外抗HSV-1和HIV-1活性实验表明：LCVN是一种细胞毒性更低、活性更强的CVN的衍生物；monoPEGyalted-LCVN抗病毒活性得到一定程度的保持,细胞毒性显著降低,能够有效抑制HIV感染细胞与未感染细胞的融合。本研究成功制备了纯度>95%的重组LCVN蛋白及monoPEGyalted-LCVN, LCVN细胞毒性更低、活性更强；monoPEGyalted-LCVN细胞毒性显著降低,纳摩尔浓度范围能有效抑制HIV感染细胞与未感染的融合,这位开发基于CVN的新型、高效抗病毒药物打下了基础。更多还原

【Abstract】 Cyanovirin-N is an 11-KD anti-HIV protein originally isolated from extracts of a cyanobacterium, Nostoc ellipsosporum. The protein binds with high affinity to the viral envelope glycoprotein gp120 and aborts cell-to cell fusion and transmission of HIV-1 infection.Due to its cyanobacterial origin it is likely to show the typical drawbacks associated with pHarmaceutical use of foreign proteins such as short plasma half-life, proteolysis and immunogenicity. Poly (ethylene glycol)(PEG) is a widely investigated polymer used for the covalent modification of biological macromolecules for many pHarmaceutical and biotechnical applications, especially of peptides and proteins. PEGylation reagents could conjugate N-terminal residues (a-amine groups), lyscine (ε-amine) and cystine residues, but random PEGyaltion usually result in inactive molecules as multi-PEGyaltion production or PEGyaltion sites located proximal to the active site.Here, the (GGGGS)3 flexible Linker was linked to the N-terminus of CVN and constructed the vector PET3c-SUMO-LCVN. This SUMO-fused LCVN was expressed in the cytoplasm of E. coli in a folded and soluble form, up to 30% of the total soluble protein. The preparation of LCVN was very efficinent and high yield with lower cost due to SUMO-LCVN was very susceptible to SUMO protease and fusion protein could be cleaved completely without DTT, so LCVN protein was more propitious for large-scale production. Poly-ethylene glycol (PEG) with average molecular weight of 20,000 and 30,000 daltons was covalently attached to the N-terminus of LCVN. MonoPEGylated-LCVN was separated by SP sepHarose chromatograpHy. The bioactivity assay demonstrated that LCVN and monoPEGyalted-LCVN bound to gp120 with nanomolar concentration. In addition, LCVN possessed enhanced in vitro anti-HSV-land anti-HIV/ⅢB activity along with decreased cytotoxicity compared to native CVN. MonoPEGyalted-LCVN remarkerablely reduce the cell-to-cell fusion and polykaryoctyes formation, and retain significantly anti-HIV and anti-HSV-1 activity with pronounced decreased cytotoxicity.更多还原