摘要
目的:利用慢病毒载体建立稳定表达猪载脂蛋白B mRNA编辑酶催化多肽样蛋白3F(pAPOBEC3F,简称pA3F)的PK15细胞系。方法:以实验室前期构建的pBPLV-flag-pA3F质粒为模板,PCR扩增pA3F基因,克隆到pLenti-Puro-3Flag载体构建成pLenti-Puro-pA3F-3Flag质粒,Western印迹鉴定其在HEK293T细胞中的表达;将pLenti-Puro-pA3F-3Flag质粒与包装载体共转染HEK293T细胞,包装成Lenti-pA3F慢病毒并测定病毒滴度;将Lenti-pA3F慢病毒感染PK15细胞,通过嘌呤霉素压力筛选并结合有限稀释法,筛选稳定表达pA3F的细胞单克隆株,最终Western印迹检测细胞单克隆株中pA3F的表达。结果:菌落PCR和序列测定表明pLenti-Puro-pA3F-3Flag质粒构建正确,Western印迹显示该质粒可在HEK293T细胞中表达pA3F蛋白;包装了Lenti-pA3F慢病毒,滴度为1.32×10~8TU/mL,慢病毒感染PK15细胞后可检测到pA3F蛋白的表达;经Western印迹鉴定,筛选得到的单克隆细胞可稳定表达pA3F蛋白。结论:建立了稳定表达pA3F的PK15细胞单克隆株,为进一步深入研究猪内源性反转录病毒在pA3F作用下的免疫逃逸机制奠定了基础。
Objective: To generate a stable PK15 monoclonal cell line expressing pig apolipoprotein B mRNAediting enzyme catalytic polypeptide-like 3 F(pAPOBEC3 F, pA3 F) using a lentivirus vector. Methods: The pA3 F gene was amplified from previously prepared plasmid pBPLV-flag-pA3 F using PCR and cloned into pLenti-Puro-3 Flag vector. The resulting plasmid pLenti-Puro-pA3 F-3 Flag was subsequently transfected into HEK293 T cells and expression of pA3 F protein was identified by Western blot. Then, the constructed plasmid and lentivial packaging mix were co-transfected into HEK293 T cells to obtain packaged Lenti-pA3 F particles and viral titers were determined. PK15 cells were transduced with Lenti-pA3 F. After puromycin selection, single cell clones were isolated by limiting dilution. Finally, the expression of pA3 F protein in the PK15 monoclonal cell lines was identified by Western blot. Results: The pLenti-Puro-pA3 F-3 Flag was identified by colony PCR and sequencing and the expression of pA3 F in HEK293 T cells was confirmed by Western blot. Recombinant lentivirus Lenti-pA3 F was produced with high titer( 1.32 ×108 TU/mL). After the selection, a stable PK15 monoclonal cell line overexpressing pA3 F were confirmed by Western blot. Conclusion: The stable PK15 monoclonal cell line overexpressing pA3 F was successfully generated, which will facilitate further research of the mechanisms of porcine endogenous retrovirus escape from the repression of pA3 F in its natural host.
引文
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