趋化因子受体3 RNA干扰慢病毒载体的构建及对嗜酸粒细胞作用的初步研究

Construction and identification of mouse eosinophils CCR3 gene RNA interference lentiviral vector

目的构建趋化因子受体3（chemokine receptor3，CCR3）基因RNA干扰（RNA interference，RNAi）慢病毒表达载体，了解其对嗜酸粒细胞的作用。方法针对小鼠CCR3基因序列，设计出RNAi的靶序列，退火形成双链DNA，与经MluI、SacI、EcoRI、HindIll、BamHI和XhoI进行酶切后的pLVX—shRNA2一m载体连接产生短发夹RNA（shoa hairpin RNA，shRNA）慢病毒载体。应用shRNA慢病毒载体转染293T细胞及嗜酸粒细胞，测定病毒滴度，实时定量聚合酶链反应检测嗜酸粒细胞中CCR3基因mRNA的表达情况。结果成功构建了shRNA—mCCR3慢病毒载体，经测序与设计合成的靶向链完全一致。荧光显微镜下观察293T细胞感染效率大于90％，病毒滴度为4×10。TU／ml；对嗜酸粒细胞CCR3基因沉默效率为86．7％。结论成功构建CCR3基因RNAi慢病毒表达载体，为后续感染嗜酸粒细胞，探索其在变应性鼻炎发生和发展中的作用奠定了基础。

Objective Through construction of a lentiviral expression vector of chemokine receptor 3 （CCR3）RNA interference （RNAi） of mouse, to further study the function of CCR3 gene on eosinophils. Methods Focused on the CCR3 gene sequences, RNAi target sequences were designed, then the target sequences of Oligo DNA were synthesized and annealed to double stranded DNA, which was subsequently connected to pLVX-shRNA2-m vector digested by MluI, Sac I , EcoR I , Hind lll, BamH I and Xho I , short hairpin RNA lentiviral vectors were constructed. Short hairpin RNA lentiviral vectors were constructed. 293T cells and eosinophils were transfeeted by shRNA lentiviral vector, and virus titer was determined. The expression of the CCR3 gene in eosinophils was identified by quantitative-PCR. Results The lentiviral vector of shRNA-mCCR3-oligonucleotide chain was inserted correctly. Infection efficiency of 293T cells observed under fluorescence microscope was more than 90%, the virus titer was 4 x 108TU/ml. CCR3 interference rate was 86. 7%. Conclusion A lentiviral vector of CCR3-gene RNAi was constructed successfully by the genetic engineering technology, and it provides a condition for further research in vitro and vivo.