CAJ | 학술논문

目的构建小干扰唾液酸黏附素(sialic acid-binding immunoglobulin-like lectin 1,Siglec-1)的慢病毒载体,并体外筛选出具有干扰效果的病毒载体.方法利用软件设计3条Siglec-1 siRNA片段,并克隆到慢病毒pGCSIL-GFP质粒载体,再与pHelper 1.0载体和pHelper 2.0载体共转染293T细胞,培养48 h后,收集并浓缩富含慢病毒颗粒的细胞上清液.逐孔稀释法测定慢病毒滴度并转导原代培养的骨髓巨噬细胞(BMM),流式细胞术和实时荧光定量PCR筛选具有干扰效果的病毒载体.结果成功构建3个vshRNA质粒载体和一个阴性对照质粒载体,并经测序验证序列正确.包装的慢病毒滴度介于1×108 ～ 1×109 TU/ml之间,适合体外转导及体内试验.体外转导BMM筛选出Lv-1能靶向抑制Siglec-1表达.结论成功构建并筛选出具有体外干扰效果的小干扰Siglec-1慢病毒载体,为体内应用该病毒载体进行基因敲除实验奠定了基础.
목적 구건소간우타액산점부소(sialic acid-binding immunoglobulin-like lectin 1,Siglec-1)적만병독재체,병체외사선출구유간우효과적병독재체.방법 이용연건설계3조Siglec-1 siRNA편단,병극륭도만병독pGCSIL-GFP질립재체,재여pHelper 1.0재체화pHelper 2.0재체공전염293T세포,배양48 h후,수집병농축부함만병독과립적세포상청액.축공희석법측정만병독적도병전도원대배양적골수거서세포(BMM),류식세포술화실시형광정량PCR사선구유간우효과적병독재체.결과 성공구건3개vshRNA질립재체화일개음성대조질립재체,병경측서험증서렬정학.포장적만병독적도개우1×108 ～ 1×109 TU/ml지간,괄합체외전도급체내시험.체외전도BMM사선출Lv-1능파향억제Siglec-1표체.결론 성공구건병사선출구유체외간우효과적소간우Siglec-1만병독재체,위체내응용해병독재체진행기인고제실험전정료기출.
Objective To construct lentiviral vectors containing small interfering RNA (siRNA) sequence of Siglec-1 and to screen the effective vector.Methods Three fragments of Siglec-1 siRNA were designed and cloned into pGCSIL-GFP lentiviral plasmid.And then the plasmid was cotransfected into 293T cells with pHelper 1.0 and pHelper 2.0 plasmids.Forty-eight hours later,culture supernatant with virus particles was collected and concentrated.Virus titer was determined by 10-fold serial dilution method and virus was transduced into primary cultured mouse bone marrow-derived macrophages (BMM).Flow cytometry and QRT-PCR were used to screen effective vector with inhibition ability.Results Three vshRNA lentiviral plasmids and a control plasmid were constructed successfully and verified by DNA sequencing.Virus titer was between 1×10s TU/ml and 1×109 TU/ml,which was suitable for in vitro and in vivo experiments.The Lv-1 could inhibit Siglec-1 expression effectively in vitro transduction of BMM.Conclusion Lentiviral vectors containing siRNA sequence of Siglec-1 were constructed successfully and an effective vector was screened,which may lay the foundation for using the vector in gene knockdown experiment in vivo.