论文标题:hGM-CSF在鱼腥藻7120中的表达及基因修饰对其表达效率影响的研究
Studies on the Expression of hGM-CSF in Anabaena sp.PCC 7120 and the Effects on Its Efficiency by the Gene Modification
论文作者 魏兰珍
论文导师 王全喜；施定基,论文学位 硕士,论文专业 水生生物学
论文单位 上海师范大学,点击次数 70,论文页数 88页File Size5368k
2005-05-01论文网 http://www.lw23.com/lunwen_829233117/ 人粒-巨噬细胞集落刺激因子;鱼腥藻7120;三亲接合转移;转基因蓝藻;生长曲线;光合放氧;基因修饰
Human granulocyte-macrophage colony stimulating factor; Anabaena sp.PCC 7120; triparental conjugative transfer; transgenic cyanobacteria; Western Blot; growth curve; photosynthetic oxygen evolution; gene modification
自从1982 年全世界第一个基因重组药物—“人胰岛素”在美国上市以来,已有近百种生物技术药品上市。我国批准上市的生物工程药品也有15 种之多,而且生物药品种类在大幅度增加,销售额也以每年20%的速度递增。因此,生物工程药物的开发将是国内外今后很长一段时期的研究热点。人粒-巨噬细胞集落刺激因子(hGM-CSF)作为一种主要的造血生长因子,能够刺激T 细胞和巨噬细胞增殖、成熟和分化,具有极其重要的免疫调解功能,在造血调控和免疫调节中发挥着重要作用。但是天然hGM-CSF 因制备量少,纯度不够高等原因,远不能满足市场需求。因此,用基因工程来生产hGM-CSF 有其重要经济意义和独特的优越性。蓝藻作为基因工程重要的受体系统之一,有其独特的优点:(1)不形成包涵体;(2)通常不含内毒素;(3)蛋白酶活性低;(4)培养基低廉;(5)生长速度快;(6)含有对人体有益的营养保健成分。因此,本研究选择鱼腥藻7120 作为受体系统,利用蓝藻基因工程来制备重组的hGM-CSF。外源基因在蓝藻细胞中表达效率较低一直以来可能都是制约蓝藻基因工程长足发展的一个重要原因。而hGM-CSF 的cDNA 5’端起始序列中G、C 含量较高并存在一些不偏好的密码子等使其不利于在蓝藻细胞中进行高效地表达。因此本研究根据密码子偏好性,运用PCR 手段,在不改变氨基酸序列的前提下对hGM-CSF 的cDNA5’端进行插入和定点突变,并在其5’端添加利于在蓝藻细胞中高效表达的SD 序列及启始密码子ATG,然后将hGM-CSF 编码区插入到穿梭载体(pRL-439)强启动子PpsbA的下游,进一步与穿梭表达载体pDC-08 相连构建成穿梭表达载体pDC-GM_1。为了研究密码子偏好性对外源基因在蓝藻中表达效率的影响,我们利用未修饰的hGM-CSF 基因构建了穿梭表达载体pDC-GM_0。利用三亲接合转移将该穿梭表达载体(pDC-GM_1 和pDC-GM_0)转入丝状鱼腥藻7120,通过相应抗生素筛选并经继代培养后得到能稳定遗传的转pDC-GM_0基因鱼腥藻7120(GM_0藻株)和转pDC-GM_1基因的鱼腥藻7120(GM_1藻株)。以两种转基因藻基因组DNA 为模板进行PCR 检测,结果表明hGM-CSF 基因已分别转入GM_0 藻株和GM_1 藻株中。Western Blot 分析表明,在两种转基因藻中均表达了具有免疫活性的hGM-CSF。并且,修饰后的hGM-CSF 基因与未修饰的相比,在GM_1藻株中的表达量比在GM_0藻株中提高了136%。对两种转基因藻的生长情况测定表明,在常规培养条件下,GM_0 藻株、GM_1 藻株与野生藻的细胞密度和叶绿素含量类似。光合放氧测定的结果显示,在添加10mM的NaHCO3后,GM_0藻株的净光合和真实光合均与野生藻类似;而GM_1藻株的净光
It is well known that since the first recombination medicine, human insulin, had beenappeared in America in 1982, more and more recombination medicines were come intomarket. Further investigations are found that recombination medicines will be a hotspot inthe medicine fields either in nowadays or in the future.Human granulocyte-macrophage colony stimulating factor (hGM-CSF) is one of afamily glycoprotein cytokines that have the potent effects in stimulating proliferation,maturation and function of hematopoietic cell. In 1977, GM-CSF was purified from lungtissues of mice that had been pre-injected with endotoxin; however, due to its strongbiological activity at minute amounts, starting material from 100,000 mice was required.Thus, it suggested that a potential autocrine role for hGM-CSFwould overexpress theprotein in host cell. Experimental overexpression of the hGM-CSF protein has beenachieved using two strategies: establishment of transgenic mice, and insertion of retroviralvectors expressing GM-CSF into the bone marrow of mice, but both of them are notsuccessful.Cyanobacteria are autotrophic prokaryotes with perform oxygenic photosynthesissimilar to that of higher plants, and they provide advantageous hosts to produce organicsubstances, since cyanobacteria use solar energy, CO2, H2O, and inorganic substances veryefficiently. Consequently, we can obtain useful organic products with little expenditure ofenergy and resources. It is quite possible that recombinant protein products accumulated incyanobacteria, in some instance, may not require further processing and purification,offering the option of oral delivery. Based on these advantages, we attempt to expresshGM-CSF gene in cyanobacterial cell.Although many expression vectors of cyanobacteria have been reported, expressionlevels of the foreign genes have not been optimized. The upstream sequence of hGM-CSFgene includes a GC-rich region and some codons that are discriminated in prokaryoticorganism. A pair of oligo-nucleotide primers was design to modify at the matureN-terminal cDNA sequence of hGM-CSF according to the degeneracy codon rules, selectfor prokaryotic usage codons, as well as add cyanobacterial SD sequence to promotehigh-level expression by PCR. The DNA fragment enoding mature hGM-CSF was insertedinto downstream of the strong promoter, PpsbA of shuttle vector pRL-439, then ligatedwith pDC-08 to produce the shuttle expression vector, pDC-GM_1. At the same time, theshuttle expression vector, pDC-GM_0 was constructed according to pDC-GM_1 method as anexperimental control without any modifications. Then pDC-GM_0 and pDC-GM_1 were
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