【作者】 丁云菲；

【导师】 戴长柏； 袁正宏； 孙茂盛； 马雁冰； 刘红岩；

【作者基本信息】 中国协和医科大学 ， 病原生物学， 2002， 博士

【摘要】 乙肝病毒(HBV)包膜蛋白由3个区域组成：与肝细胞直接作用的含有108或119个氨基酸残基的preS1，通过多聚白蛋白间接与之作用的含有55个氨基酸残基的preS2区域，和含有226个氨基酸残基的S蛋白区。许多研究证明，带有preS1和preS2序列的乙肝表面抗原(HBsAg)有望成为新一代更高效的乙肝疫苗。巴斯德毕赤酵母(Pichia pastoris)是近年兴起的一个重组蛋白生产系统，以其独特的优势在生物工程应用中不断得到推广。本论文就HBV包膜M蛋白在原核大肠杆菌(E．coli)和真核Pichia系统的表达、性质鉴定及免疫原性等进行了系统详细的研究。 在原核系统中表达preS2多肽，为HBV包膜M蛋白的活性检测提供材料。将人工合成preS2的全基因插入E．coli表达载体pThioHisA，以硫氧还蛋白融合蛋白的形式实现高效表达。经渗透压处理初步纯化蛋白，获得了具有preS2抗原性的thioredoxin-preS2融合蛋白。 为了在Pichia系统中高效表达HBV包膜M蛋白，用限制性内切酶SacI+SalI消化质粒pPIC3.5k和pAO815，替换对等部分，组成新的毕赤酵母高拷贝整合型表达载体pAO818。用它构建表达HBV包膜S蛋白的重组质粒，获得稳定的高表达。证实新载体改建成功后，将M蛋白编码基因插入酵母整合型表达质粒pAO818的醇氧化酶(AOX1)启动子下游，构建携带8拷贝M表达盒的重组载体，经电转化SMD1168菌株和G418筛选，得到了高效分泌表达M蛋白的毕赤酵母菌株，表达量达到50mg／L。通过CsC1等密度梯度离心的初步纯化，分泌型重组M蛋白具有preS2和S抗原性，可以形成大小在22nm左右的颗粒，并具有一定程度的糖基化。 E．coli系统表达的thioredoxin-preS2融合蛋白和Pichia系统分泌表达的HBV包膜M蛋白颗粒的免疫原性研究表明，实验组小鼠均出现抗-preS2阳性，其抗体水平明显高于对照组；与细胞免疫相关的γ干扰素水更多还原

【Abstract】 The hepatitis B virus (HBV) envelope (env) protein is composed of three regions: the 108- or 119-residue preS1 region involved in the direct interaction with hepatocytes, the 55-residue preS2 region associated with the polymerized albumin-mediated interaction, and the major 226-residue S protein region. Many studies have provided evidence that hepatitis B surface antigen (HBsAg) including preSl and preS2 sequences could be an ideal candidate for a new HBV vaccine with higher efficacy. The methylotrophic yeast Pichia pastoris has been developed as production systems for recombinant proteins recently. The favourable and most advantageous characteristics of the species have resulted in an increasing number of biotechnological applications.To determine the activity of HBV env protein M, polypeptide preS2 was expressed in prokaryotic system. A gene coding for preS2 was artificially synthesized. The gene was subcloned into Escherichia coli expression vector pThioHisA and expressed at high level as a fusion protein of thioredoxin. The thioredoxin-preS2 fusion protein was initial purified by the treatment of osmotic pressure, displayed preS2 antigenicity.To express HBV env protein M at a high level in P. pastoris, the same segments of pPIC3.5k and pAO815 digested with restriction enzyme SacI and SalI were exchanged to obtain the new vector pAO818. Genes of HBV env protein S was placed in this vector and expressed at sable and high level. After success in reconstruction of this high level expression vector, gene of HBV env protein M was placed under the control of AOX1 promoter in this integrative Pichia expression vector carrying eight copies of the expression cassette. Through electroporation transformation of SMD1168 and G418 selection, a strain of Pichia pastoris capable of secreting protein M into the更多还原