修饰的含前S1、前S2免疫决定簇乙肝表面抗原融合多肽在毕赤酵母中的共表达

Simultaneous Expression of Modified Hepatitis B Surface Antigen Fusion Polypeptides Containing PreS1, PreS2 Epitopes in Pichia pastoris

用BamHⅠ和BglⅡ双酶切合SS2嵌合基因片段的重组质粒pAO815-SS2,分离含AOX1启动子区、SS2嵌合基因和AOX1转录终止序列的表达盒.将分离的BamHⅠ-BglⅡ表达盒与经BamHⅠ线性化的pAO815-SS1质粒连接,转化大肠杆菌Top10F＇,提取质粒,用BamHⅠ和BglⅡ双酶切分析重组质粒并筛选正向插入SS2表达盒的重组子.将该重组质粒用电转化法导入Pichia pastoris GS115细胞,经表型筛选、小试表达和产物鉴定,构建了重组表达菌株GS115-SS1S2.重组菌株经甲醇诱导后制备抗原粗提液,进一步进行ELISA和Western blot鉴定.ELISA结果显示,产物同时具有S、前S1和前S2抗原性.Western blot分析进一步表明,表达产物既能与S抗体结合,也能与前S1和前S2抗体结合.工程菌经高密度发酵,表达量达到300～600mg/L.抗原经纯化后进行电镜观察,形成直径20～35nm的球形颗粒.纯化抗原经SDS-PAGE分析,SS1和SS2多肽仍然保持完整,基本无降解.

At present time, the widely used hepatitis B virus（HBV） vaccines consist of only the small hepatitis B surface antigen expressed in yeast or CHO cells. The frequency of non-responders to these vaccines has increased the demand for a more immunogenie vaccine. Some studies have suggested that the addition of preS region to the vaccine will improve its efficacy. However, the large protein（L） containing the whole preS region can not be effectively expressed in vitro. To overcome this problem, two ehimerle eontruets, SS1, surface gene containing preS1 region at C-terminus and SS2, surface gene containing preS2 region at C-terminus, were constructed and effectively expressed in our previous studies. Here we further constructed an expression vector containing both SS1 and SS2 expression cassettes by separation and ligation the SS2 cassette to a linearized SS1 expression vector pAOS15-SS1. The recombinant vector was transformed into Pichia pastoris GS115 by eleetroporation. A highlevel expression strain （GS115-SS1S2） was established by primary screening for His^＋ transformants and further analysis for induction products. ELISA results demonstrated that the expressed protein had S, preS1 and preS2 antigenicities simultaneously. Western blotting showed that the product can bind to all of the three antibodies, anti-S, anti-preS1 and anti-preS2. The expression protein was present in the form of particles of 20 - 35nm diameter and the yield of recombinant particles reached 300 - 600mg/L by fermentation. The SS1 and SS2 polypeptides kept intact in purified particles, suggesting that the stability of preS region has been significantly improved.