摘要
目的:构建不同长度的高迁移率族蛋白1(HMGB1)基因调控序列的真核表达载体及其稳定表达的Jurkat细胞株,为研究HMGB1基因在白血病发病机制建立基础。方法:以Jurkat细胞基因组为模板,PCR扩增3'端固定的8个不同长度的HMGB1调控基因(-83 bp～+83 bp、-383 bp～+83 bp、-504 bp～+83 bp、-688 bp～+83 bp、-975 bp～+83 bp、-1 163 bp～+83 bp、-1 327 bp～+83 bp、-1 520 bp～+83 bp),均将其连入pMD18-T载体,并转化大肠杆菌DH5a。对氨苄青霉素筛选的阳性克隆进行扩增、质粒抽提,应用KpnⅠ/HindⅢ、BamHⅠ/HindⅢ双酶切鉴定和DNA测序获得序列正确的阳性克隆,然后再经酶切连入pGL3-neo-luc质粒,成功构建HMGB1基因调控序列报告基因pGL3-HMGB1-luc(按由短至长顺序依次命名为1～8号质粒)。利用脂质体介导的方法将不同长度的pGL3-HMGB1-luc质粒和pGL3-neo-luc质粒分别转染至Jurkat细胞,48小时后加入终浓度600μg/ml的G418进行药物加压筛选,20天后得到有效转染pGL3-HMGB1-luc及pGL3-neo-luc载体的Jurkat细胞株(按有无HMGB1调控基因及其由短至长顺序,依次命名为NEO细胞和1～8号细胞)。通过检测荧光素酶活性,验证构建的Jurkat稳定细胞株(Jurkat-HMGB1)。结果:HMGB1调控基因成功插入到pGL3-neo-luc的XhoⅠ和HindⅢ位点之间构建出3号质粒;HMGB1调控序列1016 bp定向克隆至3号质粒的KpnⅠ和XhoⅠ位点之间,成功构建出8号质粒;将166、466、771、1 058、1 246、1 410 bp长度的HMGB1调控基因定向克隆至3号质粒的KpnⅠ和HindⅢ位点之间,分别构建出1号、2号、4号、5号、6号、7号质粒。8个质粒均经KpnⅠ/HindⅢ、BamHⅠ/HindⅢ双酶切,电泳显示条带与扩增的HMGB1调控序列长度一致。HMGB1-T载体的DNA测序结果与NCBI提供序列完全匹配。成功构建了不同长度的3'端固定的pGL3-HMGB1-luc报告基因。pGL3-neo-luc和pGL3-HMGB1-luc稳定转染至Jurkat细胞后,成功构建了NEO细胞和稳定细胞株HJ1～8,其荧光素酶发光值分别为:123、151 288、136 057、110 623、100 874、214 523、147 597、161 348、145 490。结论:构建的HMGB1调控序列报告基因和HJ稳定细胞株为找寻有意义的HMGB1调控区域,以及后期研究HMGB1基因在成人T淋巴细胞白血病中的发病机制奠定了基础。
Objective：To construct and identify luciferase reporter gene vectors containing different-length human high mobility group box 1（HMGB1） gene regulatory sequence and stable expression cell strains of Jurkat cells（Jurkat-HMGB1）,so as to lay a foundation for the further exploring the role of HMGB1 regulatory gene in the pathogenesis of adult T cell leukemia（ALT）.Methods：HMGB1 gene regulatory sequences（-83 bp~＋83 bp,-383 bp~＋83 bp,-504 bp~＋83 bp,-688 bp~＋83 bp,-975 bp~＋83 bp,-1 163 bp~＋83 bp,-1 327 bp~＋83 bp,-1 520 bp~＋83 bp） were amplified by PCR with DNA as template from Jurkat cells,in which 3＇-flanking region were fixed.HMGB1 regulatory genes were ligated into pMD18-T vector respectively and then transformed into E.coli strain DH5a,grown on LB agar plate supplemented with 100 μg/ml ampicillin overnight.The single ampicillin-selected DH5a clone was picked for plasmid extraction.The extracted plasmids were digested with the desired restriction enzymes of KpnⅠ/HindⅢ or BamHⅠ/HindⅢ.The correctness of HMGB1 regulatory sequence was confirmed with DNA sequencing.The insert of HMGB1 contained in pMD18-T vector was cut out with restriction enzymes（KpnⅠ/HindⅢ） and then ligated into vector pGL3-neo-luc to form pGL3-HMGB1-lucs,named according to the length of HMGB1 from short to long in order.PGL3-HMGB1-lucs and pGL3-neo-luc were transiently transfected into Jurkat cells mediated by liposome for 48 hours,the cells were then cultured with G418 at a final concentration of 600 μg/ml for over 20 days.Finally stably transfected sublines of Jurakt cells were obtained after 20 days.And efficiency and validity of stable sublines of HMGB1-Jurkat were testified by luciferase reporter gene activity.Results：587 bp-HMGB1 regulatory sequence was firstly ligated into XhoⅠand HindⅢ sites of pGL3-neo-luc to get plasmid 3,1016bp-HMGB1 were then ligated into KpnⅠand XhoⅠsites of plasmid 3 to obtain plasmid 8;and 166 bp-,466 bp-,771 bp-,1 058 bp,1 246 bp,1 410 bp-HMGB1 genes were ligated into KpnⅠand HindⅢ sites of plasmid 3 to construct recombinant plasmid 1,2,4,5,6 and 7,respectively.PGL3-HMGB1-lucs were confirmed correctly by digesting of KpnⅠ/HindⅢ or BamHⅠ/HindⅢ and DNA sequencing of HMGB1-T vectors,consistent with the sequence in NCBI to a large extent.And pGL3-hgmb1-lucs were constructed successfully.Following,Jurkat cells were stablely transfected with plasmid pGL3-hgmb1-lucs and pGL3-neo-luc to construct cell HJ 1-8 and cell NEO successfully validated by luciferase luminescence value,which were 151 288,136 057,110 623,100 874,214 523,147 597,161 348,145 490 and 123,respectively.Conclusion：pGL3-hgmb1-lucs and stable sublines of HMGB1-Jurkat established successfully provide a basis for looking for meaningful HMGB1 regulatory region and exploring the roles and mechanisms of HMGB1 used in ATL.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2012年第10期926-929,共4页
Chinese Journal of Immunology
基金
河南省教育厅重点项目(12A310006)
新乡医院重点研究领域招标课题(ZD2011-13、ZD2011-15)
