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本实验设计和构建了人促红细胞生成素（Epo）重组逆转录病毒载体（LXSN-Epo）,载体中小鼠白血病病毒长末端调控序列翻译调控人Epo基因的表达。通过重组逆转录病毒,将Epo基因转导到大鼠T和B淋巴细胞中,DNA-PCR分析证明带有人Epo基因的逆转录病毒已整合到T和B淋巴细胞基因中,RT-PCR分析显示了在两种细胞中均表达了 Epo mRNA。体外生物学活性测定显示,在转导的T淋巴细胞培养上清中Epo表达水平高达243mU/ml,在感染的B淋巴细胞培养上清中Epo活性达43mU/ml。在感染的B细胞中,Epo表达在mRNA和蛋白水平均证明了逆转录病毒能有效地和迅速地转导Epo基因到新培养的B细胞中,而不需要药物选择。这些研究开始了逆转录病毒介导的基因转移走向体内Epo基因治疗研究的过程。
In this study, an Epo recombinant retroviral vector (LXSN-Epo) was designed and constructed. The translational regulation of human Epo gene by long terminal regulatory sequences of mouse leukemia virus in the vector was carried out. Epo genes were transduced into rat T and B lymphocytes by recombinant retroviruses and DNA-PCR analysis demonstrated that retroviruses with human Epo genes have been integrated into T and B lymphocyte genes and RT-PCR analysis revealed Epo mRNA was expressed in both cells. In vitro biological activity assays showed that Epo expression was as high as 243 mU / ml in transduced T cell supernatants and 43 mU / ml in infected B lymphocyte culture supernatants. In infected B cells, Epo expression at mRNA and protein levels demonstrates that retroviruses transduce Epo genes efficiently and rapidly into newly cultured B cells without the need for drug selection. These studies began the process of retrovirus-mediated gene transfer toward in vivo Epo gene therapy research.