【作者】 王丹；

【导师】 陈吉祥； 胡国斌；

【作者基本信息】 中国海洋大学 ， 遗传学， 2007， 硕士

【摘要】 近年来由于对虾病害的威胁,对虾养殖产业的发展受到了严重的影响。为了深入研究对虾病毒和发展有效的疾病防治策略,建立对虾细胞系的要求日益迫切。本文采用逆转录病毒介导的方法将细胞转化基因-猿猴病毒大T抗原基因(SV40LT)导入培养的中国对虾(Penaeus Chinensis)淋巴细胞(lymphoid cells)中,尝试对其进行转化培养。本研究旨在探索对虾细胞体外培养的可行技术,为对虾细胞的建系工作奠定基础。从携带SV40LT基因的质粒pLLTSN中高保真PCR扩增出2.1 Kb的SV40LT基因,将其插入到泛嗜性逆转录病毒载体(pantropic retroviral vector)pLXRN的BamHⅠ和XhoⅠ位点之间,构建重组载体pLXRN-SV40LT。酶切分析和测序验证结果表明,载体插入基因的位置、大小、序列均正确,成功构建重组泛嗜性逆转录病毒表达载体pLXRN-SV40LT。采用磷酸钙沉淀法将重组载体pLXRN-SV40LT与包装质粒pVSV-G共转染GP2-293细胞,病毒包装48小时后收集上清。经NIH3T3细胞测定,重组病毒的滴度为4×106 cfu/ml。以重组病毒感染原代培养的中国对虾淋巴细胞,经G418筛选阳性细胞后传代培养。细胞培养结果表明,转基因细胞株具有贴壁能力下降,从壁上脱落的细胞可悬浮生长等转化细胞的特征,传至10代时,其生长状态和分裂势头仍很好,而对照细胞只能传至3代;转基因细胞株传至12代时因真菌污染而丢失。PCR法检测第10代转基因细胞株基因组中的SV40LT基因,结果表明成功扩增出270 bp SV40LT片段,证实转基因细胞株基因组中较稳定的含有SV40LT基因;采用端粒重复序列扩增法(TRAP)分析转基因细胞株的端粒酶活性,聚丙烯酰胺凝胶电泳结果显示,转基因细胞株的端粒酶活性明显高于原代对照细胞,这从一个侧面反映了转基因对虾细胞株获得了转化的特性。本研究有希望发展成为建立对虾永生化细胞系的可行技术,同时为探索其他海洋无脊椎动物细胞的建系方法提供了有益的借鉴。更多还原

【Abstract】 Recently, the shrimp aquaculture industry has been seriously hampered by shrimp viral diseases. In order to understand the cellular biology, immune response, and pathogenesis of viral infections in penaeid shrimp, an immortalized cell line is urgently needed. The present studies were carried out to transfer the Simian Virus 40 Large T antigen gene (SV40LT) into penaeus Chinensis lymphoid cells with recombinant pantropic ritrovirals. The aim of our studies was to transform shrimp cells and develop techniques for the in vivo culture of shrimp cells.The recombinant vector was constructed by subcloning SV40LT gene (2.1 Kb) from SV40LT gene-carrying plasmid pLLTSN into the BamH I and Xho I sites of pantropic retroviral expression vector pLXRN. The results of enzyme digestion and DNA sequencing indicated that the recombinant retroviral expression vector pLXRN-SV40LT was constructed correctly.The recombinant vector and pVSV-G plasmid were co-transfected into a packaging cell line GP2-293 by calcium phosphate precipitation method. The viral supernatants were collected at 2 days post-transfection. Subsequently the viral titer was determined by NIH3T3, with the result of 4×106 cfu/ml.The recombinant virals were used to infect lymphoid cells of Penaeus chinensis. The positive population was selected by G418-resistant and then subcultured. Cell culture results showed the transduced cells had transformative features and were subcultured for more than 10 passages. In contrast, the untransduced cells could be subcultured only for 3 passages. The transduced cells were contaminated by fungi when subcultured for 12 passages and had to be discarded. The SV40LT gene in the genome of transduced cells was detected by PCR. The telomerase activities of transduced cells and untransduced cells were assayed respectively using the telomeric repeat amplification protocol (TRAP). PCR results suggested that the genomic DNA of the transduced cells contained SV40LT gene. Moreover, telomerase activity in transduced cells was detected to be distinctly higher than that in untransduced cells.更多还原