摘要
目的建立稳定表达痘苗病毒D13L基因的细胞系。方法克隆D13L基因并将其插入真核表达质粒pEGFP-C2中构建重组质粒pEGFP-D13L,利用脂质体法转染BHK21细胞,G-418进行筛选,有限稀释法获取亚克隆细胞株。荧光显微镜观察和RT-PCR分析表达效果。结果D13L基因在细胞中稳定表达,连续传代10次后仍然能检测到其表达,荧光显微镜下能看到绿色荧光,RT-PCR可以得到1650bp大小的目的条带。结论筛选到了稳定表达D13L基因的细胞株,为下一步构建缺陷型痘苗病毒以及研究D13L基因的功能奠定基础。
Objective To establish a cell line expressing vaccinia virus D13L gene. Methods The D13L gene of vaccinia virus was cloned and insert into the eukaryotic expression vector pEGFP C2 to generate the recombinant plasmid pEGFP D13L. pEGFP D13L was transfected into the BHK21 cell line by the method of using lipotransfectamine. The stable transfectants were screened by G 4l8. Cell subclones were isolated by utmost dilution. The expression of D13L gene was determined by fluoroscopy and RT PCR. Results D13L gene was stably expressed in BHK21 cells. Green fluorescence can be observed under fluorescence microscope and a 1 650 bp cDNA fragment of the expressed RNA was detected by RT PCR. Conclusion A cell line stably expressing vaccinia virus D13L gene was established.This cell line can be used for the construction of D13L defective vaccinia virus and the further study of the function of D13L gene.
出处
《热带医学杂志》
CAS
2004年第3期230-233,共4页
Journal of Tropical Medicine
基金
国家高技术研究发展计划(863计划)(No.2001AA215171)
广东省科技攻关项目(No.A1090206)基金资助。
