CAJ | 학술논문

目的：构建含大鼠降钙素相关基因肽（CGRP）基因的慢病毒表达载体，为后续转染目的细胞并研究 CGRP 的功能奠定基础。方法通过基因工程技术将 CGRP 基因克隆到穿梭质粒中，构建 Puc57-CGRP 质粒，用双酶切法构建慢病毒表达载体（pLenO-DCE-CGRP），将该质粒载体与4种辅助包装质粒共转染293T 细胞，转染的293T 细胞继续培养48 h 后，收集其上清液，浓缩得到高滴度的慢病毒液，然后采用倍比稀释法和流式细胞术检测病毒滴度，并通过实时定量聚合酶链反应（PCR）检测293T细胞中 CGRP 基因的表达。结果成功构建了 pLenO-DCE-CGRP 的重组慢病毒载体，滴度为5．1×108 TU /mL 。结论成功构建了含 CGRP 基因高滴度的慢病毒载体，为后续转染间充质干细胞（MSC）并研究 CGRP 的功能奠定了基础。
목적：구건함대서강개소상관기인태（CGRP）기인적만병독표체재체，위후속전염목적세포병연구 CGRP 적공능전정기출。방법통과기인공정기술장 CGRP 기인극륭도천사질립중，구건 Puc57-CGRP 질립，용쌍매절법구건만병독표체재체（pLenO-DCE-CGRP），장해질립재체여4충보조포장질립공전염293T 세포，전염적293T 세포계속배양48 h 후，수집기상청액，농축득도고적도적만병독액，연후채용배비희석법화류식세포술검측병독적도，병통과실시정량취합매련반응（PCR）검측293T세포중 CGRP 기인적표체。결과성공구건료 pLenO-DCE-CGRP 적중조만병독재체，적도위5．1×108 TU /mL 。결론성공구건료함 CGRP 기인고적도적만병독재체，위후속전염간충질간세포（MSC）병연구 CGRP 적공능전정료기출。
Objective To construct lentiviral vector carrying rat′s calcitonin gene-related peptide(CGRP) gene for the following-up study on the function of CGRP .Methods CGRP gene segment was subcloned into shuttle plasmid ,become Puc57-CGRP .The pLenO-DCE-CGRP expression vector was be constructed by double digests .The pLenO-DCE-CGRP and 4 auxiliary packaging plas-mids were co-transfected into 293T cells .Cells were cultured for 48 hours .The supernant was collected and concentrated ,and then the viral titers were tested by multiple proportions dilution method and flow cytometer .The expression levels of CGRP were detec-ted in CGRP-modified 293T cells by Real-time PCR .Results The results of digestion and sequencing show that the pLenO-DCE-CGRP vector was constructed successfully .The titer of the lentiviral particles was 5 .1 × 108 TU /mL .Conclusion The high-titer lentvirus vector containing CGRP gene is constructed successfully ,which lay a foundation for transfecting mesenchymal stem cell (MSC) and studying the function of CGRP .