Dong Yuhang, Zhang Xiaoxiao, Wang Fang, Ying Qikang, Feng Yunan, Liu Rongrong, Wu Xingan
Department of Microbiology, School of Basic Medicine, Air Force Medical University, Xi'an 710032, China.
Department of Microbiology, School of Basic Medicine, Air Force Medical University, Xi'an 710032, China. *Corresponding author, E-mail:
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2020 Feb;36(2):152-156.
Objective To construct nucleotide-binding oligomerization domain-like receptors family member X1 (NLRX1) lentivirus plasmid and stable NLRX1-overexpressing A549 cell line. Methods Full-length cDNA encoding human NLRX1 was amplified from pCMV3-NLRX1 plasmid and then was inserted into pCDH-CMV-MCS-EF1-Puro vector to obtain NLRX1-overexpressing plasmid pCDH-NLRX1. pCDH-NLRX1 lentivirus particles were obtained by co-transfecting pCDH-NLRX1 with packaging plasmids (pMD2 and pAX2) into HEK293T cells. A549 cells were infected with concentrated pCDH-NLRX1 lentivirus particles and then were screened by puromycin to obtain stable NLRX1-overexpressed A549 cell line. The mRNA transcription and protein expression of NLRX1 were detected by real-time quantitative PCR, Western blot analysis and immunofluorescence. Results The lentivirus plasmid pCDH-NLRX1 was successfully constructed. After pCDH-NLRX1 and packaging plasmids were co-transfected into HEK293T cells, we obtained NLRX1 lentivirus particles with the titer of 1×10 TU/mL. After A549 cells were infected with lentivirus particles and screened by puromycin, a stable over-expression cell line of NLRX1 was obtained. NLRX1 was obviously expressed in the lentivirus-infected A549 cells and its mRNA and protein levels significantly increased. Conclusion NLRX1 lentivirus plasmid and stable NLRX1-overexpressing A549 cell line have been successfully constructed.
目的 构建核苷酸结合寡聚化结构域样受体家族成员X1（NLRX1）慢病毒质粒及稳定过表达NLRX1的A549细胞系。方法 从pCMV3-NLRX1质粒中扩增出编码人NLRX1的全长cDNA，然后将其插入pCDH-CMV-MCS-EF1-Puro载体中，获得过表达NLRX1的质粒pCDH-NLRX1。通过将pCDH-NLRX1与包装质粒（pMD2和pAX2）共转染至HEK293T细胞中，获得pCDH-NLRX1慢病毒颗粒。用浓缩的pCDH-NLRX1慢病毒颗粒感染A549细胞，然后用嘌呤霉素进行筛选，获得稳定过表达NLRX1的A549细胞系。通过实时定量PCR、蛋白质免疫印迹分析和免疫荧光检测NLRX1的mRNA转录和蛋白表达情况。结果 成功构建了慢病毒质粒pCDH-NLRX1。将pCDH-NLRX1与包装质粒共转染至HEK293T细胞后，获得了滴度为1×10 TU/mL的NLRX1慢病毒颗粒。用慢病毒颗粒感染A549细胞并经嘌呤霉素筛选后，获得了稳定过表达NLRX1的细胞系。NLRX1在慢病毒感染的A549细胞中明显表达，其mRNA和蛋白水平显著升高。结论 成功构建了NLRX1慢病毒质粒及稳定过表达NLRX1的A549细胞系。
