摘要
目的构建CXCR4基因慢病毒过表达载体并对其进行包装、鉴定。方法首先设计合成引物,采用PCR法扩增目的基因CXCR4,将目的基因与酶切线性化的载体进行定向交换,成功构建重组载体慢病毒表达载体p GC-FU-CXCR4,将其产物转化大肠杆菌感受态细胞。对培养出的克隆先进行菌落PCR鉴定,再对PCR鉴定阳性的克隆进行测序和比对分析,最后将获得的病毒载体共同转染293T细胞,收集病毒颗粒,并采用Real time PCR法测定病毒滴度。结果 PCR鉴定结果显示扩增的目的基因已成功插入p GC-FU载体,Western Blot结果显示得到的片段与理论大小相符,阳性克隆测序结果与目的基因序列一致,说明重组慢病毒载体的插入序列完全正确。结论本实验成功构建了CXCR4慢病毒表达载体,并成功对慢病毒进行了包装及病毒滴度测定。
Objective To construct and identify lentiviral vector p GC-FU-CXCR4 gene. Methods CXCR4 gene amplification was used by real-time polymerase chain reaction. The target gene fragments with the digested plasmids were exchange. Then the lentiviral vector p GC-FU-CXCR4 was constructed successfully. Use the constructed lentiviral vector to infect the competent escherichia coli cells. Polymerase chain reaction analysis was used to identify the cultural clones and DNA sequencing and comparative analysis were used to positive fragments.The successfully constructed plasmids had the same sequence with the target gene. Results Polymerase chain reaction tests showed that amplified target genes were inserted in p GC-FU vectors. The electrophoresis results,digestion showed that the reconstructed plasmid was consistent with the theoretical fragment and the sequence result of the positive fragments were exactly the same with the target gene. Conclusion Lentiviral vectors of CXCR4 gene over-expression were successfully constructed.
出处
《局解手术学杂志》
2015年第5期473-476,共4页
Journal of Regional Anatomy and Operative Surgery
基金
国家自然科学基金(30900669)
北京市科技新星计划基金(ZXKTYANLI001)
