【作者】 王建武；

【导师】 丁家桐；

【作者基本信息】 扬州大学 ， 动物遗传育种与繁殖， 2004， 硕士

【摘要】 重组FSH能够避免天然激素制剂在应用过程中的不足，体外重组表达FSH为新型激素制剂的生产带来了契机。本研究根据已克隆的山羊FSHα、β基因cDNA序列，构建山羊重组FSH真核表达载体，在哺乳动物细胞内进行了体外瞬时和稳定表达，为重组制剂的研制提供必备条件。 构建山羊FSHα、β亚基真核表达载体并在COS-7细胞中瞬时表达。以pGEM-T-FSHβ为模板，PCR扩增FSHβ基因，通过载体与目的基因的双酶切及DNA连接，将FSHβ基因插入到真核表达载体pcDNA3.0，构建成pcDNA-FSHβ。从山羊脑垂体组织中提取总RNA，利用RT-PCR技术成功扩增出FSHα cDNA序列。经与pcDNA3.0的酶切连接，构建成表达载体pcDNA-FSHα。两载体经序列测定表明插入的目的基因序列完全正确。用PolyFect转染试剂将两载体转入COS-7细胞进行瞬时表达，用放射免疫检测表达上清和细胞裂解液，有重组FSH表达，但表达效率不高。提示要获得高分泌重组产物，必须将α、β基因插入同一载体共表达。 构建山羊FSHα、β双表达载体，在CHO细胞中稳定表达。设计含有酶切位点和Kozak序列的PCR特异引物，分别以pcDNA-FSHα、pcDNA-FSHβ为模板，PCR扩增FSHα、β基因。α、β基因与载体pVITRO-2用限制性内切酶酶切、DNA连接酶连接，成功构建山羊FSHα、β双表达载体pVITRO-FSHαβ。基因序列测定表明插入序列完全正确。载体用PolyFect转染试剂转染CHO细胞，经潮霉素B抗性筛选，获得稳定表达山羊FSH的CHO细胞。提取稳定表达的CHO细胞基因组DNA，PCR检测，表明山羊FSHα、β基因稳定整合到CHO基因组中。培养上清FSH放射免疫检测，发现有重组FSH的分泌。 本实验首次成功构建了山羊FSHα、β真核表达载体pcDNA-FSHα、pcDNA-FSHβ及双表达载体pVITRO-FSHαβ；在COS-7和CHO细胞中得到了表达，并获得了稳定表达山羊FSH的CHO细胞。为重组FSH制剂生产及长效激素的研究奠定了基础。更多还原

【Abstract】 Recombinant follicle-stimulating hormone has more advantages than the purified FSH, and would be beneficial to domestic reproduction. Expression of recombinant FSH in vitro might provide a chance to supply commercially. In this study, the eukaryotic expression vectors of goat FSHap were constructed and expressed in COS-7 cells or CHO cells on the basis of the clones of goat FSHa/p cDNA, which would have made preparations for recombinant FSH production.Construct eukaryotic expression vectors of gFSH a/p subunits and transiently express in COS-7 cells.The gene of FSHP was amplified with PCR as a template of a vector pGEM-T-FSHp, then expression vector pcDNAS.O was inserted by FSHP gene through double enzymatic digestions and DNA ligation. So the expression vector pcDNA-FSHp was constructed successfully. The total RNA were extracted from tissues of goat pituitary, and the FSHcc gene was acquied with RT-PCR, then the vector pcDNA-FSHa was constructed by the means of enzymatic digestions and DNA ligation. Both of constructed vectors had been sequenced and were transfected into COS-7 cells by PolyFect transfection reagent. The supernate of culture media and cell lysates were analyzed for recombinant goat FSH a/p subunits and heterodimer by radioimmunoassay. The results showed there were minimal dose of rgFSH, but efficiency of expression was lower. These results would give a hint that FSH a/P must be inserted into a same expression vector in order to obtain higher secretion.Construct dual expression vector of gFSHap and stably express in Chinese hamster ovary (CHO) cells.The process was as follows: specific primers of FSH a/P genes were designed thatcontained unique enzymatic sites and Kozak sequence, and genes of FSH a/P were amplified by PCR with the templates of pcDNA-FSHcc or pcDNA-FSHp, respectively; a/P genes were digested and ligated with dual expression vector pVITRO-2 using restriction endonucleases and DNA ligase. Consequently, FSH a/P genes were inserted into plasmid pVITRO-2. Enzymatic identification and gene sequencing indicated the dual expression vector pVITRO-FSHap was successfully constructed. pVITRO-FSHap were transfected into CHO cells by PolyFect transfection reagent, and stably expressing cells were selected by hygromycin B. The genomic DNA extracted from stable expression CHO cells were identified by PCR, and the result showed genes of gFSH a/P were integrated into genome of CHO cells. There was also secretion of recombinant goat FSH in culture media supernate using radioimmunoassay.In conclusion, we successfully constructed eukaryotic expression vectors of gFSH a/P ?pcDNA-FSHa/pcDNA-FSHp, and dual expression vector ?pVITRO-FSHap. Those vectors were expressed in COS-7 cells or CHO cells respectively. Moreover, CHO cells expressed goat FSH stably were acquired. These results would be the basis of production of recombinant FSH and study of long-acting hormone for the future.更多还原