摘要
目的构建ITIH4基因重组慢病毒干扰系统。方法选取干扰效果最佳的siRNA片段，设计shRNA结构，退火反应形成双链后采用T4DNA连结酶与线性化慢病毒载体Psico连接，转化大肠杆菌后提取质粒，并经质粒DNA测序和PCR鉴定重组质粒构建是否成功，转染293T细胞并测定培养上液的病毒滴度。结果测序结果显示，重组慢病毒干扰载体Psico／ITIH4测序结果与设计的shRNA序列完全一致，转染293T细胞后，其病毒滴度为9．5×10^3 IU／mL。结论成功地构建了ITIH4基因的重组慢病毒系统，为今后深入研究ITIH4奠定了基础。
Objective To construct a lentiviral vector for RNA interference of the inter-α-trypsin inhibitor H4 （ITIH4）gene. Methods A short hairpin RNA（shRNA）targeting the ITIH4 gene was designed based on the optimal siRNA sequence.The double-stranded DNAs obtained afler annealing were ligated with the linearized lentiviral vector Psico using T4 DNA ligase and transformed into E. coli.The recombinant plasmid was extracted and identified by PCR and sequencing.The plasmid DNA of Psico-shRNA-lTIH4 was packaged into 293 T cells,and the fluid virus titer in the culture medium was determined. Results The sequence of recombinant lentiviral plasmid Psico-shRNA-ITIH4 was consistent with the sequence of the designed shRNA.The virus titer was approximately 9.5×103IU/ml. Conclusion The recombinant plasmid Psico-shRNA-ITIH4 was constructed successfully and provides an experimental basis for the further study of ITIH4.
出处
《中国癌症防治杂志》
CAS
2012年第3期242-246,共5页
CHINESE JOURNAL OF ONCOLOGY PREVENTION AND TREATMENT
基金
广西科技厅开发项目资助(2010GXNSFD013053)
