CD74-ROS1-L1951R点突变质粒的构建与鉴定
|
摘要
目的通过重叠延伸PCR技术构建CD74-ROS1-L1951R点突变质粒。方法根据CD74-ROS1融合基因序列和真核表达质粒p CM V-C-Flag上的多克隆位点设计引物,利用重叠延伸PCR技术对CD74-ROS1融合基因进行基因定点突变得到CD74-ROS1-L1951R点突变融合基因,随后将其酶切定向连入真核表达质粒p CMV-C-Flag启动子下游,构建p CMV-C-Flag-CD74-ROS1-L1951R重组质粒。重组质粒经菌落PCR、双酶切鉴定和测序,鉴定目的基因是否插入p CM V-CFlag质粒及定点突变是否成功。结果通过双酶切鉴定和测序结果显示,质粒的方向及序列正确,阅读框正确无误,表明CD74-ROS1-L1951R基因成功插入p CMV-C-Flag质粒,且CD74-ROS1-L1951R基因定点突变正确。结论成功构建了p CM V-C-Flag-CD74-ROS1-L1951R真核表达重组质粒,为研究CD74-ROS1点突变导致的耐药机制奠定了基础,可用于后续研究。
Objective To construct the recombinant plasmid carrying CD74-ROS1-L1951 R fusion gene.Methods Design primers based on the CD74-ROS1 fusion gene sequence and the multiple cloning site on the eukaryotic expression plasmid p CMV-C-Flag. The L1951 R point mutation was site-directed mutation which was constructed by overlapping extension PCR. Then CD74-ROS1 fusion gene was inserted into the carrier promoter downstream of vector p CMV-C-Flag to build the p CMV-C-Flag-CD74-ROS1-L1951 R recombinant plasmid. The recombinant plasmid was verified by restriction enzyme digestion and gene sequencing. Results The inserted part of pCMV-C-Flag-CD74-ROS1-L1951 R plasmid showed that the direction and sequence pCMV-C-Flag-CD74-ROS1-L1951 R plasmid and the reading frame were correct. The results proved that the CD74-ROS1-L1951 R point mutation was correct. Conclusion pCMV-C-Flag-CD74-ROS1-L1951 R eukaryotic expression vector was successfully constructed,which can be used for the following-up study. Our experiment laid a foundation for further mechanism study on CD74-ROS1 mutation-leaded resistance.
Objective To construct the recombinant plasmid carrying CD74-ROS1-L1951 R fusion gene.Methods Design primers based on the CD74-ROS1 fusion gene sequence and the multiple cloning site on the eukaryotic expression plasmid p CMV-C-Flag. The L1951 R point mutation was site-directed mutation which was constructed by overlapping extension PCR. Then CD74-ROS1 fusion gene was inserted into the carrier promoter downstream of vector p CMV-C-Flag to build the p CMV-C-Flag-CD74-ROS1-L1951 R recombinant plasmid. The recombinant plasmid was verified by restriction enzyme digestion and gene sequencing. Results The inserted part of pCMV-C-Flag-CD74-ROS1-L1951 R plasmid showed that the direction and sequence pCMV-C-Flag-CD74-ROS1-L1951 R plasmid and the reading frame were correct. The results proved that the CD74-ROS1-L1951 R point mutation was correct. Conclusion pCMV-C-Flag-CD74-ROS1-L1951 R eukaryotic expression vector was successfully constructed,which can be used for the following-up study. Our experiment laid a foundation for further mechanism study on CD74-ROS1 mutation-leaded resistance.
引文
[1] DAVIDSON M R,GAZDAR A F,CLARKE B E. The pivotal role of pathology in the management of lung cancer[J]. J Thorac Dis,2013,55(4):63-78.
[2] WIESWEG M,EBERHARDT W E,REIS H,et al.High prevalence of concomitant oncogene mutations in prospectively identified patients w ith ROS1-positive metastatic lung cancer[J]. J Thorac Oncol,2017,12(1):54–64.
[3] RUSSO A,FRANCHINA T,RICCIARDI G R,et al.A decade of EGFR inhibition in EGFR-mutated non small cell lung cancer(NSCLC):old successes and future perspectives[J]. Oncotarget,2015,6(29):26814-26825.
[4] GOLDING B,LUU A,JONES R,et al. The function and therapeutic targeting of anaplastic lymphoma kinase(ALK)in non-small cell lung cancer(NSCLC)[J]. Mol Cancer,2018,17:52-66.
[5] DUGAY F,LLAMAS-GUTIERREZ F,GOURNAY M,et al. Clinicopathological characteristics of ROS1-and RET-rearranged NSCLC in caucasian patients:Data from a cohort of 713 non-squamous NSCLC lacking KRAS/EGFR/HER2/BRAF/PIK3CA/ALK alterations[J]. Oncotarget,2017,8(32):53336-53351.
[6] YOSHIDA A,TSUTA K,WAKAI S,et al. Immunohistochemical detection of ROS1 is useful for identifying ROS1 rearrangements in lung cancers[J]. M od Pathol,2014,27:711-720.
[7] GAO Q Q,MA H H,WANG B,et al. Comparison of ALK status betw een primary and corresponding lymph node metastatic tumors in lung cancer patients[J].Oncotarget,2017,8(65):108840-108847.
[8] LENG L,METZ C N,FANG Y,et al. MIF signal transduction initiated by binding to CD74[J]. J Exp M ed,2003,197(11):1467-1476.
[9] TAKEUCHI K,SODA M,TOGASHI Y,et al. RET,ROS1,and ALK fusions in lung cancer[J]. Nat M ed,2012,18(3):378-381.
[10]ZHAN S,WANG F,KEATS J,et al. Crizotinib-resistant mutants of EM L4-ALK identified through an accelerated mutagenesis screen[J]. Chem Biol Drug Des,2011,78(6):999-1005.
[11]WEI J,VAN DER WEKKEN A J,SABER A,et al.M utations in EM T-related genes in ALK positive crizotinib resistant non-small cell lung cancers[J].Cancers,2018,10(1):10.
[12]ZHU Y C,LIAO X H,WANG W X,et al. Patients harboring ALK rearrangement adenocarcinoma after acquired resistance to crizotinib and transformation to small-cell lung cancer:a case report[J]. OncoTargets Ther,2017,10:3187-3192.
[13]AWAD M M,KATAYAMA R,Mc TIGUE M,et al.Acquired resistance to crizotinib from a mutation in CD74-ROS1[J]. N Eng J M ed,2013,369(12):2395-2401.
[14]DRILON A,SOMWAR R,WAGNER J P,et al. A novel crizotinib-resistance solvent-front mutation responsive to cabozantinib therapy in a patient w ith ROS1-rearranged lung cancer[J]. Clin Cancer Res,2016,22(10):2351-2358.
[15] FACCHINETTI F,LORIOT Y,KUO M S,et al. Crizotinib-resistant ROS1 mutations reveal a predictive kinase inhibitor sensitivity model for ROS1-and ALKrearranged lung cancers[J]. Clin Cancer Res,2016,22(24):5983-5991.
[16]KATAYAMA R,KOBAYASHI Y,FRIBOULET L,et al. Cabozantinib overcomes crizotinib resistance in ROS1 fusion positive cancer[J]. Clin Cancer Res,2015,21(1):166–174.
[17]STELLA G M,LUISETTI M,POZZI E,et al. Oncogenes in non-small-cell lung cancer:emerging connections and novel therapeutic dynamics[J]. Lancet Respir M ed,2013,1(3):251-261.
[18]DOEBELE R C,PILLING A B,AISNER D L,et al.M echanisms of resistance to crizotinib in patients w ith ALKgene rearranged non-small cell lung cancer[J].Clin Cancer Res,2012,18(5):1472-1482.
[19] YOSHIDA R,SASAKI T,MINAMI Y,et al. Activation of Src signaling mediates acquired resistance to ALK inhibition in lung cancer[J]. Int J Oncol,2017,51(5):1533-1540.
[20]DAVIES K D,MAHALE S,ASTLING D P,et al. Resistance to ROS1 inhibition mediated by EGFR pathw ay activation in non-small cell lung cancer[J]. PLoS One,2013,8(12):e82236-e82248.
[21]CHOI S H,KIM D H,CHOI Y J,et al. Multiple receptor tyrosine kinase activation related to ALK inhibitor resistance in lung cancer cells w ith ALK rearrangement[J]. Oncotarget,2017,8(35):58771-58780.
[22] LIN J J,SHAW A T. Recent advances in targeting ROS1 in lung cancer[J]. J Thorac Oncol,2017,12(11):1611-1625.
[23]RIKOVA K,GUO A,ZENG Q,et al. Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer[J]. Cell,2007,131(6):1190-1203.
[2] WIESWEG M,EBERHARDT W E,REIS H,et al.High prevalence of concomitant oncogene mutations in prospectively identified patients w ith ROS1-positive metastatic lung cancer[J]. J Thorac Oncol,2017,12(1):54–64.
[3] RUSSO A,FRANCHINA T,RICCIARDI G R,et al.A decade of EGFR inhibition in EGFR-mutated non small cell lung cancer(NSCLC):old successes and future perspectives[J]. Oncotarget,2015,6(29):26814-26825.
[4] GOLDING B,LUU A,JONES R,et al. The function and therapeutic targeting of anaplastic lymphoma kinase(ALK)in non-small cell lung cancer(NSCLC)[J]. Mol Cancer,2018,17:52-66.
[5] DUGAY F,LLAMAS-GUTIERREZ F,GOURNAY M,et al. Clinicopathological characteristics of ROS1-and RET-rearranged NSCLC in caucasian patients:Data from a cohort of 713 non-squamous NSCLC lacking KRAS/EGFR/HER2/BRAF/PIK3CA/ALK alterations[J]. Oncotarget,2017,8(32):53336-53351.
[6] YOSHIDA A,TSUTA K,WAKAI S,et al. Immunohistochemical detection of ROS1 is useful for identifying ROS1 rearrangements in lung cancers[J]. M od Pathol,2014,27:711-720.
[7] GAO Q Q,MA H H,WANG B,et al. Comparison of ALK status betw een primary and corresponding lymph node metastatic tumors in lung cancer patients[J].Oncotarget,2017,8(65):108840-108847.
[8] LENG L,METZ C N,FANG Y,et al. MIF signal transduction initiated by binding to CD74[J]. J Exp M ed,2003,197(11):1467-1476.
[9] TAKEUCHI K,SODA M,TOGASHI Y,et al. RET,ROS1,and ALK fusions in lung cancer[J]. Nat M ed,2012,18(3):378-381.
[10]ZHAN S,WANG F,KEATS J,et al. Crizotinib-resistant mutants of EM L4-ALK identified through an accelerated mutagenesis screen[J]. Chem Biol Drug Des,2011,78(6):999-1005.
[11]WEI J,VAN DER WEKKEN A J,SABER A,et al.M utations in EM T-related genes in ALK positive crizotinib resistant non-small cell lung cancers[J].Cancers,2018,10(1):10.
[12]ZHU Y C,LIAO X H,WANG W X,et al. Patients harboring ALK rearrangement adenocarcinoma after acquired resistance to crizotinib and transformation to small-cell lung cancer:a case report[J]. OncoTargets Ther,2017,10:3187-3192.
[13]AWAD M M,KATAYAMA R,Mc TIGUE M,et al.Acquired resistance to crizotinib from a mutation in CD74-ROS1[J]. N Eng J M ed,2013,369(12):2395-2401.
[14]DRILON A,SOMWAR R,WAGNER J P,et al. A novel crizotinib-resistance solvent-front mutation responsive to cabozantinib therapy in a patient w ith ROS1-rearranged lung cancer[J]. Clin Cancer Res,2016,22(10):2351-2358.
[15] FACCHINETTI F,LORIOT Y,KUO M S,et al. Crizotinib-resistant ROS1 mutations reveal a predictive kinase inhibitor sensitivity model for ROS1-and ALKrearranged lung cancers[J]. Clin Cancer Res,2016,22(24):5983-5991.
[16]KATAYAMA R,KOBAYASHI Y,FRIBOULET L,et al. Cabozantinib overcomes crizotinib resistance in ROS1 fusion positive cancer[J]. Clin Cancer Res,2015,21(1):166–174.
[17]STELLA G M,LUISETTI M,POZZI E,et al. Oncogenes in non-small-cell lung cancer:emerging connections and novel therapeutic dynamics[J]. Lancet Respir M ed,2013,1(3):251-261.
[18]DOEBELE R C,PILLING A B,AISNER D L,et al.M echanisms of resistance to crizotinib in patients w ith ALKgene rearranged non-small cell lung cancer[J].Clin Cancer Res,2012,18(5):1472-1482.
[19] YOSHIDA R,SASAKI T,MINAMI Y,et al. Activation of Src signaling mediates acquired resistance to ALK inhibition in lung cancer[J]. Int J Oncol,2017,51(5):1533-1540.
[20]DAVIES K D,MAHALE S,ASTLING D P,et al. Resistance to ROS1 inhibition mediated by EGFR pathw ay activation in non-small cell lung cancer[J]. PLoS One,2013,8(12):e82236-e82248.
[21]CHOI S H,KIM D H,CHOI Y J,et al. Multiple receptor tyrosine kinase activation related to ALK inhibitor resistance in lung cancer cells w ith ALK rearrangement[J]. Oncotarget,2017,8(35):58771-58780.
[22] LIN J J,SHAW A T. Recent advances in targeting ROS1 in lung cancer[J]. J Thorac Oncol,2017,12(11):1611-1625.
[23]RIKOVA K,GUO A,ZENG Q,et al. Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer[J]. Cell,2007,131(6):1190-1203.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |