粗糙脉孢菌漆酶基因的克隆及在毕赤酵母中的初步表达

采用连续延伸PCR方法克隆到粗糙脉孢菌（Neurospora crassa）漆酶基因，并将其克隆到表达载体pPIC9k，重组质粒经线性化、电激转化Pichia pastoris KM71，部分阳性克隆的PCR结果表明：漆酶基因已整合到巴斯德毕赤酵母染色体上，重组菌经甲醇诱导后3～5d产漆酶量最高，为2-3U／mL。     

重组毕赤酵母表达期菌体浓度的软测量模型

建立了用于在线估计高密度重组毕赤酵母培养过程中处于表达阶段的菌体密度软测量模型。分别对比了基于遗传算法（GA）的动力学软测量模型以及基于人工神经网络（ANN）的软测量模型，并对神经网络软测量模型的拓扑结构以及训练参数进行了初步探讨。当采用基于遗传算法（GA）的动力学模型，模型拟舍值的最大误差为7．63％；在采用神经网络软测量技术时，选取合适的模型结构和输入参数，最大误差为3．12％，而且软测量模型可以很好地反映菌体浓度实时变化趋势。该研究结果表明，在酵母细胞的高密度培养过程中采用基于神经网络的软测量模型具有较高的准确度，可以较好地实时反映发酵过程中菌体浓度的变化。     

乙型肝炎病毒S基因密码子最佳化及在毕赤酵母中的高效表达

目的 制备适合酵母表达的乙型肝炎病毒S基因,在毕赤酵母中高效表达重组乙型肝炎病毒S蛋白.方法 选用酵母偏爱的同义密码子替换野生型S基因的酵母稀有密码子,采取基因搭桥法及递归式聚合酶链反应,制备合成基因,捕入酵母表达载体pPICZB.携带有合成S基因的重组质粒转化毕赤酵母菌株KM71 H,经甲醇诱导表达乙型肝炎病毒S蛋白.结果 酶切电泳及DNA测序证实合成的S基因正确克隆到表达载体中;聚丙烯酰胺凝胶电泳及免疫印迹显示优化后的乙型肝炎病毒S基因在毕赤酵母中的重组蛋白表达量远高于野生型基因.结论 密码子优化的S基因能明显提高重组S蛋白在毕赤酵母表达系统中的表达量.     

人源性抗角蛋白单链抗体在巴氏毕赤酵母的分泌表达

目的：用巴氏毕赤酵母表达人源性抗角蛋白单链抗体。方法：从已构建好的质粒p3MH／ScFv中亚克隆目的片段ScFv并插入酵母表达载体p^PIC9K，构建成重组质粒p^PIC9K／ScFv，并测序鉴定。通过电转将重组质粒p^PIC9K／ScFv整合到巴氏毕赤酵母菌GS115的染色体上。经G418筛选得到高拷贝转化子及Mut表型鉴定后，用含0．5％甲醇的培养基诱导其分泌表达。结果：通过6天的诱导，该系统成功表达了抗角蛋白单链抗体，Western blot实验证实表达产物具有特异性。结论：获得了真核表达的抗角蛋白单链抗体，为其应用研究打下了基础。     

Laccase from the white-rot fungus Trametes versicolor: cDNA cloning of lcc1 and expression in Pichia pastoris

A cDNA coding for laccase was isolated from the ligninolytic fungus Trametes versicolor by RNA-PCR. The cDNA corresponds to the gene lcc1, which encodes a laccase isoenzyme of 498 amino-acid residues preceded by a 22-residue signal peptide. The lcc1 cDNA was cloned into the vector pHIL-D2 for expression in Pichia pastoris under the control of the AOX1 promoter. Transformants were found to secrete active recombinant enzyme after induction with methanol. The use of growth medium buffered to pH 6.0 and control of pH during cultivation were found to be important, or even necessary, for obtaining activity in liquid cultures. The effect of exchanging the native secretion signal for the Saccharomyces cerevisiae α-factor pre-pro secretion signal was studied by cloning the portion encoding the mature enzyme into the vector pPIC9. The activity obtained for the construct encoding the native laccase signal sequence was found to be seven-fold higher than for the construct encoding the α-factor secretion signal. Utilisation of the P. pastoris pep4 mutant strain SMD1168 was found to provide a two-fold higher level of activity compared with P. pastoris GS115. Received: 16 May / 8 August 1997     

乙型肝炎病毒C基因在毕赤酵母中的表达

目的:研究乙肝病毒核心(C)基因在毕赤(Pichia pastoris)酵母中的表达,以期获得高效表达的具有良好免疫反应性和特异性的重组乙肝病毒核心蛋白(HBcAg)。方法:采用PCR法从含HBV全基因序列的质粒pHBV1中扩增C基因,亚克隆到pGEM-T载体中,经DNA序列分析后将目的基因定向克隆到酵母表达载体pPIC9中,构建重组质粒pPIC9-cAg。然后用电转法将重组质粒转化入酵母菌GS115,0.5%甲醇诱导表达。采用SDS-PAGE、Western blot和ELISA法对表达产物进行分析。结果:限制性内切酶酶切和DNA序列分析证实HBV C基因已正确克隆到酵母表达载体pPIC9中;SDS-PAGE结果显示重组HBcAg在毕赤酵母细胞中表达;ELISA及Western blot分析表明,表达产物具有良好的免疫反应性和特异性,重组HBcAg的滴度可达1∶12 800。结论:成功构建了pPIC9-cAg重组质粒,并在毕赤酵母中高效表达了具有良好免疫反应性和特异性的重组HBcAg,为进一步研制抗-HBc诊断试剂盒奠定了基础。     

巴斯德毕赤酵母表达系统表达重组蛋白的影响因素及优化

巴斯德毕赤酵母表达系统是一种优秀的重组蛋白表达系统，近年来被广泛应用于重组蛋白的表达。本文介绍了巴斯德毕赤酵母表达系统的特点及其用于外源蛋白表达的基本策略。随后重点讨论了发酵过程中影响外源蛋白表达的主要环境因素、影响机制及控制策略，这些影响因素包括诱导温度、甲醇浓度、溶解氧、氮源浓度和类型及诱导pH。它们影响重组蛋白的表达量和品质，并且这几种因素的作用是相互影响和制约的，因此需要系统优化。     

Killer toxin production in Pichia acaciae is associated with linear DNA plasmids

Summary We have identified a strain of the yeast Pichia acaciae which produces a killer toxin active against the yeast Debaryomyces tamarii. The killer phenotype was associated with the presence of two DNA plasmids, pPacl-1 (13.6 kilobase pairs) and pPacl-2 (7.3 kilobase pairs). P. acaciae strains, cured of these plasmids by irradiation with ultraviolet light, lacked killer activity and were sensitive to toxin produced by the parental strain. A partially cured strain, GS-1215, missing only the smaller plasmid, pPacl-2, also exhibited loss of both toxin activity and immunity. Exonuclease studies revealed that both plasmids were linear double-stranded DNA molecules with 5 protected ends. The P. acaciae system differs from that of the well-studied Kluyveromyces lactis killer system both in the range of susceptible strains and in the sizes of the plasmids involved. Our studies contradict previous reports that Pichia killer systems are invariably chromosomal.     

人内皮抑素在毕赤酵母中的组成型表达及其活性检测

为获得大量具有生物学活性的重组人内皮抑素(Endostatin),在毕赤酵母中进行组成型表达的研究。将由毕赤酵母偏爱密码子组成的Endostatin cDNA插入组成型载体pGAPZαA中,构建表达载体pGAPZαA-ENDO,并转化到毕赤酵母X-33中。重组菌株在甘油醛-3-磷酸脱氢酶(GAP)启动子的调控作用下,进行组成型表达Endostatin蛋白。重组人Endostatin产量达到102mg/L。Western blot显示:表达蛋白能与兔抗Endostatin的多克隆抗体特异性结合。纯化后的重组Endostatin具有抑制人血管内皮细胞系ECV-304细胞增殖的活性。利用毕赤酵母组成型表达系统能获得大量具有生物活性的Endostatin蛋白。