【作者】 张莹宽；

【导师】 汪天虹；

【作者基本信息】 山东大学 ， 微生物学， 2008， 硕士

【摘要】 药用糖蛋白是生物工程产品中最重要的一类。随着基因工程技术和现代医学的不断发展,药用人源或动物来源的重组蛋白质的生产对各种宿主表达系统提出了更多的糖基化修饰的要求,以确保药物产品的安全性、药效和均一性。研究人员已开始对各种基因工程宿主表达系统,包括细菌、酵母、丝状真菌、昆虫细胞、CHO细胞及转基因植物等表达系统进行糖基化修饰途径的改造,以期望得到能够产生与人或哺乳动物糖基化形式相近的工程菌株,从而应用于人源糖蛋白的生产,提高药用糖蛋白的活性和稳定性。在目前常用的真核来源药用蛋白表达系统中,由于原核生物表达系统缺少必要的蛋白质糖基化修饰,而哺乳动物细胞表达系统虽然能够生产人源化糖蛋白,但由于重组蛋白产量低、培养时间长及成本昂贵等原因,其应用规模与范围受到较大的限制。酵母和丝状真菌是最常用的工业发酵用真核微生物,具有易操作、发酵周期短及成本低廉等优点。与酵母相比,丝状真菌糖蛋白N-聚糖糖基化的程度低,其寡聚糖加工系统和N-糖基化类型更接近于人的加工系统和人源糖蛋白的寡聚糖类型。因此,丝状真菌作为一种新的极有希望的真核表达系统已引起广泛关注。丝状真菌瑞氏木霉（Trichoderma reesei）具有极强的蛋白分泌能力和真核生物的翻译后修饰、加工过程,是非常有应用前景的真核表达系统。但瑞氏木霉与哺乳动物问的糖基化修饰形式仍存在一定的差异。哺乳动物N-聚糖的经典加工途径产生复合型N-聚糖,而瑞氏木霉的N-聚糖一般为寡甘露糖型,使利用瑞氏木霉为宿主生产的人源重组糖蛋白临床理化性质和药理作用受到一定的影响。因此,有必要开展瑞氏木霉N-聚糖工程,使瑞氏木霉所产糖蛋白N-聚糖类型与哺乳动物典型的复合型N-聚糖尽可能接近。瑞氏木霉N-聚糖加工途径的早期阶段与哺乳动物相同,产生Glc₃Man₉GlcNAc₂聚糖前体结构。此后,由于缺乏必要的甘露糖苷酶而不能完成甘露糖残基的修剪,又缺乏必要的糖基转移酶而不能完成多种单糖组分在特异前体的添加,最终只能形成寡甘露糖型N-聚糖。而哺乳动物复合型N-聚糖是在多种甘露糖苷酶和N-乙酰氨基葡萄糖转移酶、半乳糖基转移酶、唾液酸转移酶等糖基转移酶的共同作用下形成。本研究欲改造瑞氏木霉N-聚糖途径,首先敲除瑞氏木霉固有的甘露糖基转移酶基因,以去除冗余的末端甘露糖残基,产生蛋白糖链的核心五糖结构。然后转入并表达外源N-乙酰氨基葡萄糖转移酶、半乳糖基转移酶和唾液酸转移酶,从而向核心五糖上依次添加N-乙酰氨基葡萄糖、半乳糖和唾液酸,产生末端唾液酸化结构的复合型N-聚糖,最终实现瑞氏木霉N-糖基化修饰途径的人源化系统改造,为人源糖蛋白的生产做前期准备。为了敲除瑞氏木霉自身的α-1,3-甘露糖基转移酶基因（α-1,3-mannosyltransferase gene,alg3）,同时在瑞氏木霉中表达外源α-2,6-唾液酸转移酶基因（α-2,6-sialyltransferase gene,ST6GalI）,本文构建了含同源敲除表达盒Palg3-ST6-Talg3的载体pSΔM。用质粒pSΔM与含有来自A.niger的pyrG基因的丝状真菌表达质粒pAB4-1共转化瑞氏木霉pyrG营养缺陷型菌株M23,pyrG基因的转入和成功表达可以使M23恢复野生型性状。用PCR进行初筛从约三千株转化子中获得5株可能的同源整合转化子,但其中4株转化子不能正常产生孢子和传代培养。对这4株原始转化子染色体DNA样品进行PCR和荧光定量PCR分析,显示该DNA样品为目标突变株和出发菌株M23染色体DNA的混合体系,即存在出发菌株M23染色体DNA的干扰。其中M23染色体DNA含量分别占样品总量的82%、69%、77%和79%,表明这4株突变株中alg3基因确实被敲除,并推测由于alg3基因通过同源双交换被敲除使突变株不能存活,显示alg3基因编码产物是瑞氏木霉生存所必需的。另外一株转化子T.reesei b-8-1能够存活并多次传代,PCR分析结果显示b-8-1中Palg3-ST6-Talg3同源敲除表达盒是插入在alg3基因序列下游约1800 bp处的终止子编码区,而alg3基因编码区未被破坏;RT-PCR结果表明alg3和ST6GalI基因均有表达活性,但alg3基因的表达活性降低;荧光定量RT-PCR结果显示b-8-1的alg3基因表达量为出发菌株M23的80%。分析原因认为表达盒Palg3-ST6-Talg3的同源整合和外源ST6GalI基因的表达在一定程度上影响了alg3基因的表达活性。为了在T.reesei中表达外源β-1,4-半乳糖基转移酶（β-1,4-galactosyltransferase,GalT）基因和N-乙酰氨基葡萄糖转移酶（N-acetylglucosaminyl transferase I,GnTI）基因,构建了含Pcbh1-GnTI-Tcbh1和PpgdA-GalT-TtrpC双表达盒的真核表达载体pGG。用质粒pGG与携带有E.coli潮霉素抗性基因的丝状真菌表达质粒pAN7-1共转化T.reesei b-8-1,获得2株双表达盒稳定整合的潮霉素抗性转化子T.reesei GF4和GF52。RT-PCR结果显示GF4和GF52中外源ST6GalI、GalT和GnTI基因均在转录水平成功表达,alg3基因仍然有转录活性;荧光定量RT-PCR结果显示GF4和GF52中alg3基因转录水平降低,仅为M23中转录水平的62.5%。分离纯化T.reesei M23和GF4的胞外分泌蛋白CBHI,对此蛋白糖链的结构变化分析正在进行中。本文成功的进行了对瑞氏木霉N-糖基化途径的初步改造,表达了外源N-乙酰氨基葡萄糖转移酶、半乳糖基转移酶和唾液酸转移酶,敲低了瑞氏木霉α-1,3-甘露糖基转移酶基因的表达水平。研究结果有利于推动工业重要丝状真菌糖工程的发展,具有一定的理论价值和潜在的工业应用前景。更多还原

【Abstract】 Filamentous fungi have long been used for the production of metabolites and enzymes,expecially the proteins that cannot be actively expressed in Escherichia coli or require glycosylation for proper folding and biological activity.The benefits of the filamentous fungus Trichoderma reesei include its simplicity,rapidity,and ability to glycosylate recombinant proteins.However,for the production of therapeutic glycoproteins intended for use in humans,fungi glycosylation is of the high-mannose type,which confers a short in vivo half-life to the protein and may render it less efficacious or even immunogenic.An enzymatically modulation of host glycosylation pathways in vivo is needed to change their ability to modify proteins with human glycosylation structures.Thinking about the cost of manufacturing and purification,the conversion of the fungi-type high-mannose glycans to mammalian N-type complex glycans is showing a significant promise and challenging the current dominance of therapeutic protein production based on mammalian cell culture.In this perspective,we have constructed an expression plasmid vector pS△M containing Palg3-ST6-Talg3 expressive box-a ratα-2,6-sialyltransferase(ST6GalI) cDNA was subcloned between the promoter and terminator of T.reeseiα-1,3-mannosyltransferase gene(alg3).The resulting construct was used to knockout T.reesei alg3 and express rat ST6GalI gene at the same time at the case of homointergration.Co-transformation of the auxotroph strains T.reesei M23 with plasmid vectors pS△M and pAB4-1 containing the A.niger pyrG selectable gene has been performed to isolate a stably-transformed T.reesei variant.Five homointergrants were identified from about 3000 heterointergrants,while four of them can not survive no matter what effort we have attempted.It supposed that a lethal knockout happened. The only survival designated T.reesei b-8-1 was identitied partial homointergrant at the downstream side of alg3 gene.The expression activity of alg3 gene was suppessed at a certain extent,while ST6GalI gene was found an expression activity in T.reesei b-8-1. Then we constructed the second expression plasmid vector pGG containing Pebh1-GnTI-Tcbh1-PpgdA-GalT-TtrpC expressive box-a N-acetylglucosaminyl transferaseⅠ(GnTI)gene and a bovineβ-1,4-galactosyltransferase(GalTI)eDNA were subcloned into the fungous promoter and terminator.The resulting construct was used to express GnTI gene and GalTI gene at the same time.Co-transformation of T.reesei b-8-1 with plasmid vectors pGG and pAN7-1 carrying the Escherichia coliβ-glucuronidase gusA gene has been performed to isolate a stably-transformed T. reesei variant.Two transformants were identified and designated T.reesei GF4 and T. reesei GF52.The expression activity of GnTI and GalTI gene was found through RT-PCR experiment and a declined expression activity of alg3 gene was found through Real Time RT-PCR in T.reesei GF4 and GF52.Analysis to N-glycan of secret protein CBHI in T.reesei GF4 is ongoing.更多还原