论文标题:hGM-CSF在鱼腥藻7120中的表达及其表达系统的改进
Human Granulocyte-Macrophage Colony Stimulating Factor (hGM-CSF) Gene Expression in Anabaena sp.PCC7120 and the Reconstruction of Its Expression System
论文作者
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 474,论文页数 109页File Size5294K
2006-05-01论文网 http://www.lw23.com/lunwen_3962512/
Human Granulocyte-Macrophage Colony Stimulating Factor;Anabeaba sp. PCC 7120;triparetal conjugative transfer; transgenic cyanobacteria;Shine-Dalgarno(SD) sequence;homology recombination method
人粒细胞—巨噬细胞集落刺激因子(hGM-CSF)是第一个被克隆和利用重组DNA技术生产的造血刺激因子，是一个具有多项潜能的造血生长因子，具有十分重要的临床应用价值，在国际生物技术制药市场上占有重要的位置。目前市场上销售的hGM-CSF多是由大肠杆菌作为宿主生产的，由于其过表达及含内毒素的缺点，人们曾尝试在酵母、哺乳细胞、昆虫细胞、植物细胞中表达hGM-CSF，但或多或少都有一些缺陷。而蓝藻作为新兴的制备基因工程药物的宿主具有遗传背景简单，便于外源基因转化、蛋白酶活性低、培养基低廉、不含内毒素等优点，是制备基因工程药物的很有潜力的宿主。 本文在利用鱼腥藻7120作为宿主成功表达hGM-CSF基因的基础上，对克隆到的hGM-CSF进行修饰，在基因的5’端添加了可以在蓝藻中高效表达的SD序列，并将SD序列与起始密码子ATG之间的距离调整为相差6个碱基，然后将hGM-CSF基因插入到中间载体pRL-439强启动子PpsbA的下游，得到中间表达载体pRL-GMs，进一步插入到穿梭载体pDC-08的相应位点，构建成穿梭表达载体pDC-GMs。利用三亲接合转移法转化鱼腥藻7120，通过相应抗生素筛选并经继代培养后得到含有目的基因的转基因藻GMs藻株。并对转基因藻的最适生长条件进行了优化筛选。同时本文还针对我们前期所构建的穿梭表达载体系统存在稳定性欠佳，质粒易丢失的特点，所以又克隆了鱼腥藻7120中编码谷氨酰胺合成酶(GS)的glnA基因，将其作为整合平台，插入到pRL-721中的hGM-CSF基因和强启动子PpsbA的上游，构建成了整合载体pRG-GM。为提高hGM-CSF基因在鱼腥藻7120中的表达效率和提高外源基因在鱼腥藻7120中的稳定性提供了资料。 本论文的主要内容及结果如下： 1．克隆hGM-CSF基因，在5’端添加SD序列，并调整SD序列与ATG间距离，然后将其插入强启动子PpsbA的下游，克隆到穿梭载体pDC-08的相应位点，构建成穿梭表达载体pDC-GMs，然后利用三亲接合转移转化鱼腥藻7120，通过相应抗生素筛选并经继代培养后得到含有目的基因的转基因藻GMs藻株；转基因藻DNA水平检测，表明hGM-CSF已经转入GMs藻株中；Western Blot分析表明，在转基因藻中表达了具有免疫活性的hGM-CSF。 2．通过对转基因藻的最适生长条件测定发现，转基因藻株的最适生长温度为30℃，最适pH值为9.5～10.5，在调整了环境因子的基本培养基中，GMs藻株与野生
Human Granulocyte-Macrophage Colony Stimulating Factor (hGM-CSF) is the first haematopoietic cytokines that has been cloned and been utilized by recombinant DNA technology. It has potent effects in stimulating proliferation, maturation and function of haematopoietic cell. It occupied large percent of recombinantion medicine market. In the meantime, commercial produced hGM-CSF usually use E. coli to be the host. For its disadvantages of overexpression and endotoxin, people try to express hGM-CSF in many other hosts just as yeast, mammal, hexapod, etc. But the results have not been optimized. Cyanobactreria as the potential candidate host to produce genetic engineering medicine have simple structure and makes them be a potentially powerful model system of modern biotechnology, and they provide advantageous hosts to produce organic substances. Based on these advantages, we attempt to express hGM-CSF gene in cyanobacterial cell.In this article, base on successfully expressed hGM-CSF in Anabaena sp. PCC 7120, we modified hGM-CSF gene by add shine-dalgarno(SD) sequence in N-terminal and adjusted the distance between it and ATG which affects the efficiency of translation. Then inserted it into downstream of the promoter PpsbA, ligated it with pDC-08 to contruct the shuttle expression vector, pDC-GMs. Then transformed it into Anabaena sp. PCC 7120 by the triparental conjugation transfer method. Gained transgenic sp. PCC 7120 harboring pDC-GMs by screening with neomycine. Then optimized the culture conditions to found the restrict curver factors. And for the reason of the prophase shuttle vectors which we constructed have the unstable disadvantage, we cloned glnA gene which encoding glutamine synthetase by TD PCR in Anabeaba sp. PCC 7120 as a integrative platform. Then, an integrative expression vector pRG-GM which contained hGM-CSF gene and endogenous promoter PpsbA was constructed. It provides reference to improve the expression level and the stability of foreign hGM-CSF gene in Anabeaba sp. PCC 7120.The contents and results of this experiment were as follows:1. We add -GGAGAG- SD sequence in hgm-CSF N-terminal and adjusted the sequence between SD and ATG into 6bp by PCR. Then inserted it into downstream of the strong promoter, PpsbA of shuttle vector pRL-439, then ligated it with pDC-08 to contruct the shuttle expression vector, pDC-GMs. Then transformed it into Anabaena sp. PCC 7120, by the triparental conjugation transfer method. Screening of positive clones were performed on BG-11(-N) agar plates supplemented with neomycine. PCR amplification of
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