增殖诱导配体基因短发夹RNA慢病毒表达载体的构建

Construction of lentiviral vectors of shRNA targeting human APRIL gene

目的构建针对人的增殖诱导配体（a proliferation-inducing ligand，APRIL）基因的短发夹RNA（small hairpin RNA，shRNA）慢病毒表达载体，并在感染293T细胞后检测病毒滴度及适合的感染复数（multiplicity of infection，MOI）值。方法以人APRIL基因为靶点，筛选出3条shRNA靶序列：shAPRILl210、shAPRILl754、shAPRILl604；各自合成靶序列的Oligo DNA，退火形成双链DNA后分别与经酶切的pGCL-GFP载体连接，构建3个慢病毒表达载体LV-shAPRILl210、LV-shAPRILl754、LV-shAPRILl604；将连接产物分别转化到DH5α感受态细胞，经PCR及DNA测序鉴定。再用LV-shAPRIL与包装质粒pHelper1．0、pHelper2．0共转染293T细胞，包装产生慢病毒，以293T细胞绿色荧光蛋白的表达水平确定病毒滴度及适合的MOI值。结果PCR和测序结果与构建的3个慢病毒载体LV-shAPRILl210、LV-shAPRILl754、LV-shAPRILl604的预期结果一致，经包装产生的慢病毒滴度分别为5×10^7、6×10^7、4×10^7转导单位（TU）／ml；适合高效感染的MOI值为5。结论成功构建人APRIL基因3个慢病毒表达载体LV-shAPRIL，为后继的在相关靶细胞中诱导特异性的APRIL基因沉默奠定基础。

Objective To construct small hairpin RNA（shRNA） lentiviral vectors targeting human a proliferation-inducing ligand（APRIL） gene and detect the titer of virus and suitable multiplicity of infection （MOI） after 293T cells were infected by the lentival vectors. Methods Three RNA interference targeting sequences of APRIL gene were screened including shAPRIL1210, shAPRIL1754 and shAPRIL1604. Both sense and antisense Oligo DNA of the targeting sequences were synthesized and cloned into the pGCL-GFP vector, respectively. The resulting lentiviral vectors containing shAPRIL were named LV-shAPRIL1210, LV-shAPRIL1754, LV-shAPRIL1604. Then they were confirmed by PCR and DNA sequencing. 293T cells were co-transfected with LV-shAPRIL, pHelper 1.0 and pHelper 2.0 to product lentivirus, respectively. The titer of virus and suitable MOI were tested according to the expression level of GFP in the 293T cells. Results PCR analysis and DNA sequencing confirmed that three shAPRIL DNA were successfully inserted into the lentiviral vectors. The titers of concentrated virus were 5× 10^7 , 6 ×10^7 and 4 ×10^7 （transduction units ）TU/ml, respectively, and the suitable MOI was 5. Conclusions Three shRNA lentiviral vectors targeting human APRIL gene have been successfully constructed, which lays a foundation for future studying APRIL＇s gene silencing in related target cells.