摘要
【目的】克隆绵羊IGF1基因CDS区，构建绵羊IGF1基因慢病毒表达载体，分析IGF1基因在绵羊成肌细胞增殖过程中的作用。【方法】提取绵羊肝组织总RNA，通过RT-PCR方法获得IGF1全长CDS序列，经测序鉴定后与慢病毒载体pLEX-mcs连接，构建pLEX-IGF-1慢病毒重组表达质粒，并在293T细胞中进行病毒包装和生产。分离培养绵羊原代成肌细胞，并用重组慢病毒使其感染，建立稳定转染pLEX-IGF-1的绵羊成肌细胞系。通过绘制细胞生长曲线，分析过表达IGF1对绵羊成肌细胞生长的影响；采用流式细胞仪检测细胞周期变化。【结果】克隆获得长518 bp的IGF1 CDS区序列。成功构建了pLEX-IGF-1载体，其可在绵羊细胞系中进行稳定表达。通过Western blotting检测显示，IGF1在293T细胞以及绵羊原代成肌细胞中获得表达，细胞生长曲线显示，稳定表达IGF1的成肌细胞比对照细胞生长速度显著加快（第1天差异显著（P＜0.05），第2～6天差异极显著（P＜0.01））；细胞周期测定结果显示，稳定表达IGF1的成肌细胞比对照细胞较早进入S期，且在S期的细胞数目比对照细胞多29.35%。【结论】绵羊IGF1能够有效促进绵羊成肌细胞的生长。
【Objective】Aiming to investigate the role of ovine IGF1 gene in ovine myoblast growth,the lentiviral vector of IGF1 was constructed and its effects on ovine myoblast proliferation were investigated.【Method】The coding sequence of IGF1 gene was amplified by RT-PCR and confirmed by sequencing.It was cloned into pLEX－mcs－lentiviral vector to obtain pLEX-IGF-1 and the recombinant lentivirus was packaged and produced in 293T cells.The ovine myoblast cells were isolated and cultured before being transfected by pLEX-IGF-1 to establish stable cell lines.Cell growth curve was used to study the effects of IGF1 gene on cell growth and flow cytometry was used to detect changes in the cell cycle.【Result】Sequence of IGF1 CDS with 518 bp was cloned and the pLEX-IGF1 vector that could express stably in sheep cell lines was established.The Western blotting showed that IGF1 could stably express in 293T cells and ovine myoblast cell lines.Cell growth curve showed that the ovine IGF1 gene could enhance ovine myoblast proliferation significantly compared to control cells （P〈0.05 at the first day and P〈0.01 from the second day to the sixth day）.Cell cycle determination showed that the myoblasts with express of IGF1 entered S phase earlier than control cells and the number of cells in S phase was 29.35% more than the control.【Conclusion】Ovine IGF11 gene could enhance the growth of ovine myoblasts.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2014年第4期15-21,共7页
Journal of Northwest A&F University(Natural Science Edition)
基金
国家高技术研究发展计划(863计划)项目(2011AA100307)
