标题：Satb2基因慢病毒载体的构建及其鉴定

      作者：陈晓霞 1，2，颜世果 2，李玉兰 1    1.陕西省康复医院口腔科，陕西 西安 710065；2.山东大学口腔医学院牙周科 山东省口腔生物医学重点实验室，山东 济南 250012

      卷次： 2020年31卷23期

      【摘要】 目的 构建特殊富含AT序列结合蛋白2 (Satb2)基因慢病毒表达载体并通过感染293T细胞检测目的基因的表达。方法 采用PCR技术扩增目的基因Satb2，并在目的基因的上下游分别添加酶切位点PacⅠ和AscⅠ，将其PCR产物和目的载体用PacⅠ和AscⅠ分别进行双酶切，通过T4 DNA ligase连接酶切后的PCR产物及目的载体，构建pLenti6.3-Satb2-IRES-EGFP慢病毒表达载体，通过菌液PCR、酶切及测序鉴定其结果；将阳性克隆质粒与包装质粒Mix共转染293T细胞，收集、浓缩病毒液并测定其浓度；将重组病毒液感染293T细胞，观察其荧光表达情况，通过qPCR检测目的基因表达。结果 菌液PCR结果得到一条约2 202 bp的亮带，与目的基因大小相符；重组载体经过双酶切得到一条 9.1 kb大片段及一条 2 202 bp小片段；通过测序验证重组载体中 Satb2基因序列与GenBank报道的序列完全一致，表明慢病毒表达载体构建成功。经过包装，重组慢病毒滴度达到7.9×107 ifu/mL，该病毒液感染293T细胞后可观察到大量荧光，感染率约为70%，通过qPCR检测结果显示实验组Satb2 mRNA的表达量增加了约106倍(P<0.05)。结论 本实验成功构建pLenti6.3-Satb2-IRES-EGFP慢病毒表达载体并包装出具有感染能力的慢病毒，为进一步研究该基因在牙周支持组织再生中的作用奠定基础。
      【关键词】 慢病毒；特殊富含AT序列结合蛋白2；牙周炎；牙周组织再生；293T细胞
      【中图分类号】 R781.4 【文献标识码】 A 【文章编号】 1003—6350（2020）23—2993—05

Construction and identification of Satb2 lentiviral expression vector.
CHEN Xiao-xia 1, 2, YAN Shi-guo 2, LI Yu-lan 1. 1.Department of Stomatology, Shanxi Rehabilitation Hospital, Xi'an 710065, Shaanxi, CHINA; 2. Department of Periodontics,College of Stomatology, Shandong University/Shandong Provincial Key Laboratory of Oral Biomedicine, Jinan 250012,Shandong, CHINA
【Abstract】 Objective To construct a lentiviral eukaryotic expression vector with special AT-rich sequence-bind-ing protein 2 (Satb2) gene and test the expression of the target gene by infecting 293T cells. Methods The aim geneSatb2 was amplified from plasmids by PCR. PacⅠ and AscⅠ were added in the upstream and downstream of the targetfragment, and the target gene PCR product and vector were digested by PacⅠ and AscⅠ, respectively. Then the T4 DNAligase was used to connect the PCR product and the target vector after enzyme digestion, and an pLen-ti6.3-Satb2-IRES-EGFP lentivirus expression vector was constructed, and the results were identified by PCR, enzyme di-gestion and sequencing. The positive clone plasmid and package plasmid Mix were transfected into 293T cells, and thevirus venom was collected, concentrated, and the concentration was determined. 293T cells were infected with recombi-nant virus venom and their fluorescence expression was observed. The expression of the target gene was detected by qP-CR. Results A bright band of 2 202 bp was obtained by PCR, which was consistent with the size of the target gene.The recombinant vector was digested by double enzyme to obtain the large fragment vector (9.1 kb) and the desired tar-get band (2 202 bp). The sequence of Satb2 gene in the recombinant vector was verified to be the same as that reportedby GenBank, indicating that the lentivirus expression vector was successfully constructed. After packaging, the virus ti-ter reached 7.9×107 ifu/mL. A large amount of fluorescence was observed after 293T cells were infected with virus, andthe infection rate was about 70%. The results of qPCR test showed that the expression of Satb2 mRNA in the experimen-tal group increased by about 106 times (P<0.05). Conclusion In this study, pLenti6.3-Satb2-IRES-EGFP lentivirus ex-pression vector was successfully constructed and the lentivirus with infective ability was packaged, laying a foundationfor further study on the function of this gene in periodontal tissue regeneration.
      【Key words】 Lentivirus; Special AT-rich sequence-binding protein 2 (Satb2); Periodontist; Periodontal regenera-tion; 293T celldoi:10.3969/j.issn.1003-6350.2020.23.001基金项目：国家自然科学基金(编号：81200790)；山东省重点研究开发计划(公益类)(编号：2017GSF218063)