SOX17过表达慢病毒载体和稳定转染细胞系的构建

doi:10.13481/j.1671-587X.20230603

摘要/Abstract

摘要：

目的构建性别决定区Y盒17（SOX17）过表达慢病毒载体，使用SOX17过表达慢病毒感染PC12细胞并建立稳定过表达SOX17的细胞系。方法在NCBI数据库中查找、设计并合成SOX17过表达序列，将其与经BamHⅠ和AgeⅠ双酶切的慢病毒GV492载体连接，构建GV492-SOX17过表达重组质粒。琼脂糖凝胶电泳鉴定PCR产物，筛选携带GV492-SOX17过表达重组质粒的阳性菌，克隆后测序。将GV492空载质粒和GV492-SOX17过表达重组质粒分别转染至人胚肾HEK 293T细胞中，转染48 h后收集GV492对照慢病毒和GV492-SOX17过表达慢病毒进行包装并测定病毒滴度。将PC12细胞分为空白组、GV492对照组和GV492-SOX17组，空白组不作处理，GV492对照组和GV492-SOX17组分别采用相应慢病毒感染细胞（感染复数＝100），10 mg·L^－1嘌呤霉素筛选成功感染慢病毒的PC12细胞，荧光显微镜观察各组PC12细胞生长状态及绿色荧光表达情况。采用实时荧光定量PCR（RT-qPCR）法检测各组PC12细胞中SOX17 mRNA表达水平，Western blotting 法检测各组PC12细胞中SOX17蛋白表达水平。结果 GV492-SOX17过表达重组质粒的基因片段长度约为744 bp， GV492-SOX17过表达重组质粒基因序列与设计合成的SOX17过表达序列一致。GV492对照慢病毒和GV492-SOX17过表达慢病毒滴度均为2.5×10⁸TU·mL^－1。各组PC12细胞生长状态良好且有绿色荧光表达。RT-qPCR法检测，与空白组和GV492对照组比较，GV492-SOX17组PC12细胞中SOX17 mRNA表达水平明显升高（P<0.01）。Western blotting法检测，各组细胞在相对分子质量44 000处出现特异性条带，提示PC12细胞中SOX17蛋白表达成功；与空白组和GV492对照组比较，GV492-SOX17组PC12细胞中SOX17蛋白表达水平升高（P<0.05）。结论成功构建了GV492-SOX17慢病毒表达载体，建立了稳定过表达GV492-SOX17的PC12细胞系。

关键词: 性别决定区Y盒17, 慢病毒载体, PC12细胞, 实时荧光定量PCR, 感染复数

Abstract:

Objective To construct the sex-determined region Y-box 17 （SOX17） over-expression lentiviral vector， and to construct the cell line stably over-expressing SOX17 by using the PC12 cells infected with SOX17 over-expression lentivirus. Methods The over-expression sequence of SOX17 was designed and synthesized based on the National Center for Biotechnology Information （NCBI） Database， and was connected to the GV492 lentiviral vector after being doubly digested with BamHⅠ and AgeⅠ enzymes to construct the GV492-SOX17 over-expression recombinant plasmid. The agarose gel electrophoresis was used to identify the PCR products， and the positive bacteria carrying the GV492-SOX17 over-expression recombinant plasmid were selected， cloned and sequenced. The GV492 empty plasmid and GV492-SOX17 over-expression recombinant plasmid were transfected into the human embryonic kidney HEK 293T cells， respectively. After transfected for 48 h， the GV492 control lentivirus and GV492-SOX17 over-expression lentivirus were packaged and the viral titer was detected. The PC12 cells were divided into blank group， GV492 control group， and GV492-SOX17 group. The cells in blank group was not treated， and the cells in GV492 control and GV492-SOX17 groups were infected with the corresponding lentivirus （multiplicity of infection = 100）， and the PC12 cells successfully infected with lentivirus were selected with 10 mg·L^－1 puromycin. The growth state and green fluorescence expression of the PC12 cells were observed under fluorescence microscope；real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression level of SOX17 mRNA in the PC12 cells in various groups；Western blotting method was used to detect the expression level of SOX17 protein in the PC12 cells in various groups. Results The gene fragment length of GV492-SOX17 over-expression recombinant plasmid was about 744 bp， and the sequence of GV492-SOX17 over-expression recombinant plasmid gene was identical to the designed and synthesized SOX17 over-expression sequence. The titers of GV492 control lentivirus and GV492-SOX17 over-expression lentivirus were both 2.5×108 TU·mL^－1. The growth state of the PC12 cells in various groups was good and the green fluorescence was expressed. The RT-qPCR results showed that the expression level of SOX17 mRNA in the cells in GV492-SOX17 group was significantly higher than those in blank group and GV492 control group（P<0.01）. The Western blotting results showed that there was a specific band appeared at a relative molecular mass of 44 000， suggesting the SOX17 protein was successfully expressed in the PC12 cells； compared with blank group and GV492 control group， the expression level of SOX17 protein in the cells in GV492-SOX17 group was increased （P<0.05）. Conclusion The GV492-SOX17 lentiviral expression vector is successfully constructed and the PC12 cell line stably over-expressing GV492-SOX17 is established.

Key words: Sex-determined region Y box 17, Lentiviral vector, PC12 cells, Real-time fluorescence quantitative PCR, Multiplicity of infection

中图分类号:

Q254

黄少婷,李友,吴钊淳,何嘉文,廖科棋,李胜男. SOX17过表达慢病毒载体和稳定转染细胞系的构建[J]. 吉林大学学报(医学版), 2023, 49(6): 1424-1430.

Shaoting HUANG,You LI,Zhaochun WU,Jiawen HE,Keqi LIAO,Shengnan LI. Construction of SOX17 over-expression lentiviral vector and stably transfected cell line[J]. Journal of Jilin University(Medicine Edition), 2023, 49(6): 1424-1430.

图/表 5

图1

图2

图3

图4

图5

参考文献 23

1	SARKAR A， HOCHEDLINGER K. The sox family of transcription factors：versatile regulators of stem and progenitor cell fate［J］.Cell Stem Cell，2013，12（1）：15-30.
2	SCHEPERS G E， TEASDALE R D， KOOPMAN P. Twenty pairs of sox： extent， homology， and nomenclature of the mouse and human sox transcription factor gene families［J］. Dev Cell， 2002， 3（2）： 167-170.
3	CAI J L， LIANG J Y， ZHANG Y T， et al. Cyclo-（Phe-Tyr） as a novel cyclic dipeptide compound alleviates ischemic/reperfusion brain injury via JUNB/JNK/NF-κB and SOX5/PI3K/AKT pathways［J］. Pharmacol Res， 2022， 180： 106230.
4	SUN X G， SUN B L， BABICHEVA A， et al. Direct extracellular NAMPT involvement in pulmonary hypertension and vascular remodeling. transcriptional regulation by SOX and HIF-2α［J］. Am J Respir Cell Mol Biol， 2020， 63（1）： 92-103.
5	LAARMAN M D， GEEVEN G， BARNETT P， et al. Chromatin conformation links putative enhancers in intracranial aneurysm-associated regions to potential candidate genes［J］.J Am Heart Assoc，2019，8（9）：e011201.
6	GARCÍA-RAMÍREZ M， MARTÍNEZ-GONZÁLEZ J， JUAN-BABOT J O， et al. Transcription factor SOX18 is expressed in human coronary atherosclerotic lesions and regulates DNA synthesis and vascular cell growth［J］. Arterioscler Thromb Vasc Biol，2005，25（11）：2398-2403.
7	LU P F， WANG P， WU B R， et al. A SOX17-PDGFB signaling axis regulates aortic root development［J］. Nat Commun， 2022， 13（1）： 4065.
8	CORADA M， ORSENIGO F， BHAT G P， et al. Fine-tuning of Sox17 and canonical Wnt coordinates the permeability properties of the blood-brain barrier［J］. Circ Res， 2019， 124（4）： 511-525.
9	MONTANI D， LECHARTIER B， GIRERD B， et al. An emerging phenotype of pulmonary arterial hypertension patients carrying SOX17 variants［J］. Eur Respir J， 2022， 60（6）： 2200656.
10	SABATER-LLEAL M， HUFFMAN J E， DE VRIES P S， et al. Genome-wide association transethnic meta-analyses identifies novel associations regulating coagulation factor Ⅷ and von willebrand factor plasma levels［J］. Circulation， 2019， 139（5）： 620-635.
11	LANGEN U H， AYLOO S， GU C H. Development and cell biology of the blood-brain barrier［J］. Annu Rev Cell Dev Biol， 2019， 35： 591-613.
12	LEE S H， LEE S， YANG H， et al. Notch pathway targets proangiogenic regulator Sox17 to restrict angiogenesis［J］. Circ Res， 2014， 115（2）： 215-226.
13	XU Z， RUI Y N， HAGAN J P， et al. Intracranial aneurysms： pathology， genetics， and molecular mechanisms［J］.Neuromolecular Med， 2019，21（4）： 325-343.
14	KARSCHNIA P， NISHIMURA S， LOUVI A. Cerebrovascular disorders associated with genetic lesions［J］. Cell Mol Life Sci， 2019， 76（2）： 283-300.
15	LIU M L， ZHANG L H， MARSBOOM G， et al. Sox17 is required for endothelial regeneration following inflammation-induced vascular injury［J］. Nat Commun， 2019， 10（1）： 2126.
16	NATARELLI L， GEIßLER C， CSABA G， et al. miR-103 promotes endothelial maladaptation by targeting lncWDR59［J］. Nat Commun， 2018， 9（1）： 2645.
17	RITZ K， DENSWIL N P， STAM O C G， et al. Cause and mechanisms of intracranial atherosclerosis［J］. Circulation， 2014， 130（16）： 1407-1414.
18	RIVAS-URBINA A， RULL A， ORDÓÑEZ-LLANOS J，et al. Electronegative LDL：an active player in atherogenesis or a by-product of atherosclerosis？［J］.Curr Med Chem，2019，26（9）： 1665-1679.
19	WANG J Y， LAI C L， LEE C T， et al. Electronegative low-density lipoprotein L5 impairs viability and NGF-induced neuronal differentiation of PC12 cells via LOX-1［J］. Int J Mol Sci， 2017， 18（8）： 1744.
20	SHAO C L， WANG J J， TIAN J， et al. Coronary artery disease： from mechanism to clinical practice［J］. Adv Exp Med Biol， 2020， 1177： 1-36.
21	KONG P， CUI Z Y， HUANG X F， et al. Inflammation and atherosclerosis： signaling pathways and therapeutic intervention［J］. Signal Transduct Target Ther， 2022， 7（1）： 131.
22	ZHANG Y， WU H K， HE R， et al. Dickkopf-2 knockdown protects against classic macrophage polarization and lipid loading by activation of Wnt/ β-catenin signaling［J］. J Cardiol， 2021， 78（4）： 328-333.
23	VIECELI DALLA SEGA F， FORTINI F， AQUILA G，et al. Notch signaling regulates immune responses in atherosclerosis［J］. Front Immunol， 2019， 10： 1130.

[1]	马亚楠,谭兵,徐磊,张健东. 实时荧光定量PCR法对ICU患者痰液标本中鲍曼不动杆菌耐药基因的检测及其评价[J]. 吉林大学学报(医学版), 2022, 48(6): 1623-1628.
[2]	黄笑尘,李浩,王保华,李凯. 利多卡因对帕金森模型PC12细胞的保护作用及其机制[J]. 吉林大学学报(医学版), 2022, 48(3): 638-647.
[3]	巩培, 刘竟然, 赵世敏, 王玉珍, 谢基明. MCM10沉默对乳腺癌MDA-MB-231细胞的增殖抑制作用及其机制[J]. 吉林大学学报(医学版), 2021, 47(3): 652-659.
[4]	陈少凤, 李胜男, 邓福, 朱佩仪, 李友. RHBDF2基因过表达慢病毒载体的构建和稳定表达RHBDF2细胞系的建立[J]. 吉林大学学报(医学版), 2020, 46(03): 444-450.
[5]	李胜男, 王梦旭, 胡伟东, 陈少凤, 陈杏兰, 李友. miR-186过表达慢病毒载体的构建及鉴定[J]. 吉林大学学报(医学版), 2019, 45(05): 997-1002.
[6]	朱广伟, 黄金胜, 黄永建, 郑炜, 杨树钢, 林春霖, 陈恒恺, 叶建新. 内质网蛋白29过表达慢病毒载体的构建及其对胃癌细胞生物学行为的影响[J]. 吉林大学学报(医学版), 2019, 45(02): 244-250.
[7]	杨麒巍, 杜珍武, 于杉, 高素洁, 赵冠杰, 张琳, 卢佳, 任明, 张桂珍. 孕妇血浆DNA中内参基因拷贝数稳定性的评价[J]. 吉林大学学报(医学版), 2016, 42(03): 523-529.
[8]	邢洪源, 赵婧, 刘纯岩, 李贺, 刘扬, 王珍琦. LPL基因mRNA在精神分裂症模型大鼠脑组织中的表达[J]. 吉林大学学报(医学版), 2016, 42(01): 11-14.
[9]	徐姗姗, 朴美花, 王艳姝, 刘楠, 裴爱月, 冯春生. 异氟醚对Aβ25-35诱导大鼠PC12细胞氧化应激损伤的影响及海藻糖的保护作用[J]. 吉林大学学报(医学版), 2015, 41(02): 207-212.
[10]	刘畅, 高薇, 冯欣. RNA干扰沉默STAT3对胶原诱导大鼠类风湿性关节炎的治疗作用[J]. 吉林大学学报(医学版), 2015, 41(01): 77-82.
[11]	华兴，潘文海. DAB2IP基因慢病毒载体构建及其在前列腺癌PC3细胞中的表达[J]. 吉林大学学报(医学版), 2014, 40(05): 938-942.
[12]	刘换新,白义凤,王炜,郭琳琅. LRIG1和 EGFR mRNA在非小细胞肺癌患者外周血中的表达及其临床意义[J]. 吉林大学学报(医学版), 2014, 40(02): 363-368.
[13]	曹鹤，李薇，钱磊，王雪梅，崔久嵬. RNA干扰COPS3基因对肺癌细胞A549增殖的抑制作用及其机制[J]. 吉林大学学报(医学版), 2014, 40(02): 238-242.
[14]	邹泳国，周春燕，朴美花，刘楠，岳云，冯春生. 异氟醚对β淀粉样蛋白诱导的大鼠PC12细胞凋亡和内质网应激的影响及其机制[J]. 吉林大学学报(医学版), 2014, 40(02): 224-228.
[15]	王伟，朴美花，王艳姝，刘楠，岳云，冯春生. 异氟醚对Aβ25-35诱导大鼠PC12细胞自噬和凋亡的影响[J]. 吉林大学学报(医学版), 2014, 40(01): 49-54.