中文摘要：

目的构建白细胞介素-7（interleukin-7,IL-7）基因慢病毒表达载体,并在CHO-K1细胞中稳定表达,为生产试剂级的IL-7奠定基础。方法参照Gen Bank中公布的人IL-7基因核苷酸序列（J04156.1）,人工合成该条基因,亚克隆至表达载体pLV-CMV-EF1-GP上,构建重组慢病毒表达质粒pLV-CMV-EF1-GP-r IL-7,在HEK293T细胞中进行病毒包装,检测病毒滴度后,收获细胞毒液,感染CHO-K1细胞,经嘌呤霉素筛选,获得稳定表达IL-7的CHO-K1-r IL-7细胞。荧光显微镜观察感染细胞绿色荧光蛋白的表达;RT-PCR法检测IL-7基因m RNA的转录;ELISA法检测IL-7的含量;Western blot法检测IL-7的表达。结果重组慢病毒表达质粒pLV-CMV-EF1-GP-r IL-7经双酶切及测序结果证明构建正确。慢病毒的滴度为3×10~8 TU/ml。慢病毒感染CHO-K1细胞24、48和72 h后,均可见特异性绿色荧光蛋白表达,且随着培养时间的延长,荧光强度明显增强;CHO-K1-r IL-7细胞可扩增出约540 bp的IL-7基因片段;经ELISA检测,样品中IL-7含量为0.6 ng/ml;表达产物主要存在于CHO-K1-r IL-7细胞上清中,且具有良好的反应原性。结论成功构建了IL-7基因慢病毒重组表达质粒,并实现了IL-7在CHO-K1细胞上的稳定表达。

英文摘要：

Objective To construct the lentivirus vector for expression of interleukin-7（IL-7）, stably express in CHO-K1 cells and lay a foundation of production of IL-7 of reagent grade. Methods IL-7 gene was synthesized artificially according to the published nucleotide sequence in Gen Bank（J04156. 1）and subcloned to vector pLV-CMV-EF1-GP. The constructed recombinant lentivirus expression vector pLV-CMV-EF1-GPL-r IL-7 was transfected to HEK293 T cells for packaging. The obtained bulk of virus was determined for titer, then harvested and infected to CHO-K1 cells. The CHOK1-r IL-7 cells for stable expression of IL-7 were screened with puromycin. The expression of green fluorescent protein（GFP）in infected cells was observed by fluorescent microscopy. The transcription of IL-7 m RNA was determined by RTPCR, while the IL-7 content by ELISA, and the IL-7 expression by Western blot. Results Restriction analysis and sequencing proved that recombinant plasmid pLV-CMV-EF1-GP-r IL-7 was constructed correctly. The titer of lentivirus was3 × 10~8TU/ml. The expression of GFP was observed in CHO-K1 cells 24, 48 and 72 h after infection with lentivirus, and the fluorescence intensity increased significantly with the increasing culture time. The IL-7 gene fragment at a length of about 540 bp was amplified from CHO-K1-r IL-7 cells. ELISA proved that the IL-7 content in sample was 0. 6 ng/ml.The expressed product mainly existed in th e supernatant of CHO-K1-r IL-7 cells, which showed good reactogenicity.Conclusion Th e lentivirus vector for expression of IL-7 was constructed successfully, and IL-7 was stably expressed in CHO-K1 cells.