【作者】 李大力；

【导师】 王关林；

【作者基本信息】 大连理工大学 ， 生物化学工程， 2000， 博士

【摘要】 蜂毒溶血肽（Melittin）又称蜂毒肽，是蜜蜂毒中的主要多肽成分，具有重要的医学价值。蜂毒溶血肽不仅是重要的抗炎物质，而且对肿瘤细胞也显示高度的毒理作用，还对辐射有预防和治疗作用，可使受辐射动物的生存率显著提高。另外，蜂毒溶血肽具有很强的表面活性，其透过卵磷脂膜和混合脂膜的速度为任何表面活性剂所不及，这一性质使其成为生物膜研究的重要工具。 目前，人们主要通过从蜜蜂毒液中分离提取或化学合成来获得蜂毒溶血肽，但因为磷脂酶A₂的存在而造成的纯化困难和蛋白质化学合成的高成本，使蜂毒溶血肽的广泛应用受到了限制。而生物工程技术的发展为生产蜂毒溶血肽提供了一条新的途径。为此本研究克隆了蜂毒溶血肽和其前体蛋白的cDNA，并用分子生物学技术对克隆得到的cDNA进行了改造，建立了在大肠杆菌中表达生产蜂毒溶血肽的基本系统。 本研究在实验中发现1～18日龄蜜蜂工蜂产生较多的蜂毒溶血肽，因此选择此阶段的工蜂为实验材料。首先在异硫氰酸胍-酚-氯仿一步法的基础上加以改进，建立了简便而有效的工蜂毒腺RNA提取方法。然后克隆了蜂毒溶血肽的cDNA，并在与蜂毒溶血肽对应的序列前加了蛋白质开始翻译所必须的起始密码子ATG，使此cDNA可在合适启动子的调控下表达产生蜂毒溶血肽。 由于蜂毒溶血肽可破坏生物膜引起细胞内容物泄露而导致细胞死亡。蜂毒溶血肽直接在原核或真核表达系统中表达对表达细胞是致死的，此困难一直阻滞基因工程生产蜂毒溶血肽的实施。蜂毒溶血肽前体蛋白是蜂毒溶血肽的天然融合蛋白，从理论上推测蜂毒溶血肽前体蛋白对表达细胞是无毒的，但这个推测必须用实验来证实。本研究克隆得到蜂毒溶血肽前体蛋白的cDNA，并构建了其大肠杆菌表达载体，证实了与β-半乳糖苷酶的部分序列相融合的蜂毒溶血肽前体蛋白在大肠杆菌中表达对表达细胞无致死性。 在证实了与β-半乳糖苷酶的部分序列相融合的蜂毒溶血肽前体蛋白在大肠杆菌中表达对表达细胞无致死性后，就可利用融合蛋白的形式在工程菌中表达生产蜂毒溶血肽，但在工程菌中表达的融合蛋白必须切除其担体蛋白部分才能得到目的蛋白。为此，本研究对克隆得到的蜂毒溶血肽融合蛋白cDNA进行了定点突变，在蜂毒溶血肽序列前引入了天冬酰胺密码子AAT，并实现了突变蛋白在大肠杆菌中的表达，定点突变后的cDNA编码合成的蛋白质中，引入的天冬酰胺 丰大力：蜂毒MM｝ic人其前体蛋白 CDNA的克隆、定点突变和在大肠杆菌中的表达残基和蜂毒溶血肽的第一个氨基酸残基构成了羟胺的化学裂解位点，融合蛋自可经羟胺的化学裂解修饰加工成有活性的蜂毒溶血肽。更多还原

【Abstract】 Melitttn, a main polypeptide component in the bee poison, is of great importance to medical science. It not only possesses the parmacalogical activity of anti- inflammation, anti-arthritis, anti-bacterial, hut also the toxicologic effect to tumor cells. It also can pies ent and treat radiation sickness, therefore enhance the survival rate of the radiated animals. Moreover, melittin has strong surface activity, it can pass through the lecithin’s and the mixed lipid membranes much faster than any other surface-active agents This character has made it an effective tool for bio-membrane research So far, melittin has been mainly obtained by separating and extracting from bee venom or by chemical synthesis. The spreading of melittin was limited because of the following two reasons first, the purification difficulty caused by the phospholipase A2, second, the higher cost of protein chemical synthesis The development of biotechnology provides a new way for melittin production. So in this study the cDNA encoding melittin and its pre-protein were cloned and modified by molecular bio- technique, the basic expression system was established in E. coli. In the work, it was found that 1-18 days of work bees can produce more melittin, so bees at this age were selected as test materials. First, a simple and effective modit5ed uanidine isothiocyanate method was established to extract RNA from poison gland. Then cDNA encoding melittin was cloned, and the initiation codon ATG which is necessary to the initiation of protein translation was added to the cloned sequence, and the eDNA could be expressed to produce melittin under the control of a suitable prornotor The melittin directly expressed in procaryotic or eucaryotic expression systems is deadly to the cells, because it can destroy the bio-membrane and cause the leak of endocvte This has been hindering the melittin production by genetic engineering. Promelittin is the natural fusion protein of melittin. It was supposed theoretically that promelittin was innoxious to the expression cells. This presumption has been proved by experiment In this study, the cDNA encoding promelittin was cloned, and its vector expressed in F ccli was constructed. This proved that promelittin with partial sequence ol P-gdIactoiddse was ilOfl-tetIldl w tile expression cells. After proving the fact, it was possible to express and produce melittin in the tbrrn of fusion protein in the energetic bacteria. In order to obtain the objective protein, the supporter protein has to be excised from the fusion protein For this purpose, the melittin cDNA obtained by cloning was site-directed mutagenized, AAT codon encoding asparamide was arranged before the mehttin sequence, and the mutational protein was expressed in E coli. The induced asparamide residue and the first residueof melittin constitUted the hydroxylamine cleavage site of the ndational prednencoded by the cDN^ and the fiJsion protein could be proceed intO actiVe melittin bythe hydroxylamine cleavne modification.更多还原