Bax基因重组腺病毒表达载体的构建及其对包装细胞HEK293的毒性作用
摘要
Oltvai等于1993年用Bcl-2基因蛋白产物Bcl-2特异性单克隆抗体免疫沉淀等方法，从人和鼠B细胞中发现一种与Bcl-2共沉淀的(21 KD)蛋白质，命名为Bax(Bcl-associated X protein)，与Bcl-2促细胞存活的作用相反，Bax促细胞死亡。细胞内Bax和Bcl-2的比值是影响细胞凋亡的关键。胶质瘤细胞中Bcl-2的高表达，Bax的相对低表达，Bcl2／Bax比例的失调与胶质瘤的组织学级别，肿瘤转移能力，肿瘤生长速度，肿瘤复发以及肿瘤对于放疗，化疗的反应性均具有密切关系。如果能够在肿瘤细胞中提高Bax基因的表达，将促进肿瘤细胞的凋亡，提高肿瘤细胞对于放疗、化疗的敏感性。人类腺病毒是双链DNA病毒，具有转染靶细胞后以附加体(episome)形式存在，极少发生插入突变，载体操作方便、容易繁殖，宿主范围广，既可感染分裂细胞，又可感染非分裂细胞，携带外源基因容量大，可表达接近翻译后成熟水平蛋白质等优点，复制缺陷性腺病毒由于其在非包装细胞中不能复制，避免了对周围正常细胞的感染，成为现阶段最好的基因治疗载体之一。我们以复制缺陷性腺病毒为基因转移载体，以人类巨细胞病毒早期启动子为真核基因表达启动子，以Baxα基因为治疗基因，构建Baxα-pShuttle-Adeno重组腺病毒表达载体，并且证明其具有较高的目的基因转录和表达活性，同时发现由于重组病毒载体Baxα的表达先于病毒本身复制和包装的发生，以及Bax蛋白对于包装细胞HEK293的毒性作用，导致包装细胞的死亡，而病毒本身尚未完成复制和包装，致使病毒扩增受阻，不能达到基因治疗所需的病毒浓度。这提示我们在将来的实验中可以通过以可控的启动子代替CMV启动子，使含Baxα基因的重组腺病毒在包装细胞HEK293细胞中不表达或低表达，避免Bax
浙江大学硕士研究生学位论文
蛋白对于HEK293细胞的致死性毒性，使重组体得以包装和扩增，从而得到足够的病
毒量进行胶质瘤的基因治疗。
实验材料和仪器
1.主要试剂:Baxa基因全序列由浙江大学生命科学学院潘杰教授惠赠，TGI菌
为浙江大学生命科学学院生物化学研究所保存，Adeno一xTM表达载体(美国BD
Biosciences公司)，HEK293细胞(中国科学院上海生物化学与细胞生物学研究所)，
DMEM培养液、1640培养液、胎牛血清(美国玩vitrogen公司)，，DosPER阳离子
脂质体(瑞士Roche公司)。X一gal试剂、MTT试剂(美国A刀甘esco公司)，Bax(B一9)
鼠抗人单克隆抗体、Western Blotting荧光显影试剂盒(美国Santa cruz Biotechnology
公司)，辣根酶标记山羊抗小鼠IgG(H一L)(北京中山生物技术有限公司)，Pacl限制性
内切酶(美国NEw ENGLAND Biotechnology公司)。限制性内切酶Xbal、勒nl、
BajnHI、pstl，DNA Marker DL2000，DNA Marker DL15o0O(大连宝生物工程有限公
司)。
2.试验仪器:352型酶标仪(芬兰Labsystems Multiskan公司)，TC2323二氧化
碳生化培养箱(美国SHEL LAB公司)，UV-24OIPC紫外分光光度计(日本岛津公司)，
X一22R冷冻离心机(德国Beckman公司)，PCR扩增仪(杭州宝诚科技有限公司)，YJ一875
型医用净化工作台(苏州长桥净化设备厂)。
实验方法
用一次性塑料培养瓶，含10%胎牛血清的高糖DMEM，按常规方法培养HEK293
细胞，每3天传代1次。以xba 1/ KPnl双酶切pshuttle质粒，将双酶切的pshuttle
质粒与潘杰教授惠赠的Baxa基因进行连接反应，以Balnl硕1和Pstl酶切鉴定。以
Pl一sce工压ceul双酶切Baxa一pshuttle重组体，电泳并回收含Baxa的片断，连接回
收的Bax片断和已经Pl一Scel压Ceul双酶切的Adeno一X基因， PCR鉴定。以同样
浙江大学附属第二医院
浙江大学硕士研究生学位论文
方法构建LacZ一pshuttle一Adeno重组腺病毒载体，PcR鉴定。提纯Bax。一pshuttle一Aden。
重组腺病毒质粒后(3 .07协岁时)，以Pacl酶切，苯酚:氯仿:异戊醇(25:24:1)提纯酶
切产物，提纯后浓度为1 .68。留协1。以DosPer阳离子脂质体转染重组载体进HEK293
细胞，5天和8天后换液，10天后传代，离心收集每次换液时脱落的细胞。以同样的
方法转染LacZ一pshuttle一Adeno重组腺病毒载体进入HEK293细胞。Westem Blot检测
Bax。一Pshuttle一Adeno重组腺病毒载体在HEK293细胞中的表达。X一gal底物显色法检
测LacZ一pshuttle一Aden。重组腺病毒载体在HEK293细胞中的表达。MTT法测定Bax
。一pshuttle一Adeno重组腺病毒载体对HEK293细胞的毒性作用。试验数据利用The
SAS System for Windows VS统计软件包进行统计学分析。
结果
第1组(HEK293细胞转染Bax。一pshuttle~Adeno重组腺病毒载体后换液时离心
收集的脱落细胞)细胞裂解液通过W七stem Blot可检测到相对分子质量为2 1 O00D的
蛋白，而第2组(转染BaxQ一pshuttle一Adeno重组载体后存活并传代培养的细胞)和
对照组(未转染Bax。一pshuttle一Adeno重组载体的HEK293细胞)无可检测到的Bax
蛋白表达。
转染了LacZ一shuttle一Adeno重组腺病毒载体的现K293细胞在加入染色底物
X一咧4h后，细胞培养液变成蓝色，倒置显微镜下见甩K293细胞亦蓝染。
酶标仪(570run波长)测量结果见表z。经The SAS System for w
Oltvai et al identified an associated 21 kD protein partner, Bax, that has extensive amino acid homology with Bcl-2 in 1993. Bax homodimerizes and forms heterodimers with Bcl-2 in vivo. Overexpressed Bax accelerates apoptotic death and also counters the death represser activity of Bcl-2. The ratio of Bcl-2 to Bax determines survival or death following an apoptotic stimulus. There are high expression of Bcl-2 and relatively low expression of Bax in glioma. There is relation between unbalance of ratio of Bcl-2 to Bax and histological grade of glioma, capacity of metastasis of glioma, growth rate of glioma, relapse of glioma as well as response of glioma to chemotherapy and radiotherapy. If we can enhance the expression of bax gene in glioma, we'll promote the apoptosis of glioma and enhance the response of glioma to chemotherapy and radiotherapy. Human adenovirus is a kind of double strains DNA (dsDNA) virus. The adenoviral genome exists as an episome in the host cell's neucleus, dose not integrate into host c
ell's genome, and rarely causes host's gene insert mutation. As a vector, Adenovirus has the advantages of easy operation, easy propagation, wide variety of host cell spectrum, noncell-cycle dependent infectious ability, capacity of ligating large foreign DNA to adenoviral vector. Replication-incompetent(AEl/AE3) human adenovirus is one of the best vector at present because it neither replicate nor actively transcribe in non-package cell science the cell lacks the necessary transcription factors-the El genes. We made replication-incompetent
adenovirus as a vector, human cytomegalovirus immediate early promoter as a mammalian expression cassette promoter, Baxa gene as therapeutic gene, to recombine a Bax a -pShuttle-Adeno recombinant adenoviral expression vector. We demonstrated that the recombinant adenoviral expression vector has a high activity of transcription and expression of gene inserted. We also found that the expression of Bax precede the replication and package of adenovirus and overexpression of Bax is cytotoxic in HEK293 cell line, which would impede the propagation of recombinant adenoviral expression vector in sensitive packaging cells-HEK293 by killing the HEK293 cells. This implies that we can replace a controllable promoter to MCV IE promoter, which do not express downstream Bax gene or lowly express in HEK293 cells in order to avoiding its toxicity to HEK293 cell. This make recombinant Bax-spshuttle-adeno expression vector propagate itself enough to proceed the gene therapy to glioma.
Experimental materials
Full sequence of Baxa gene was donated by Pro. Jie Pan, College of Life Sciences, Zhejiang University. E coli-PGl was obtained from Biochemical Institute of College of Life Sciences, Zhejiang University. Adeno-X鈩?expression system was bought from BD Biosciences Company, America. HEK293 cell line was bought from Institute of Biochemistry and Cell Biology, SIBS, CAS, China. DMEM, 1640 Medium, and FBS were bought form Introgen Co., America. DOSPER Liposomal Transfection Reagent was bought from Roche Co., Switzeland. MTT and X-gal reagent were bought from Amresco Inc., America. Western Blotting Luminal Reagent was bought from Santa Cruz Biotechnology Co., America. Peroxidase-Conjugated Goat Anti-Mouse IgG was bought from Beijing Zhongshan Biotechnology Co. Ltd., China. Pac I was bought from New England Biotechnology Co., America. Xba I , Kpn I , BamH I , Pst I , DNA Marker DL2000 and DNA Marker DL15000 were bought from TaKaRa Biotechnology(Dalian) Co.
Ltd.,China.
Experimental Methods
Culture HEK293 cells in high glucose DMEM supplemented with 100 units/ml penicillin G sodium, 100 g/ml streptomycin, and 10% fetal bovine serum in plastic flasks under optimum growth conditions(37 ,5% CO2). Split every 3 days when they reach about 80% confluency. Double-digest pShuttle plasmid DNA with Xba I and Kpn I . Combine the Xba I / Kpn I -digested pShuttle plasmid DNA with the Baxa gene donated by Pro. Jei Pan. Identify putative recombinant Baxa-pShuttle DNA by BamH I or Pst I restrict
浙江大学硕士研究生学位论文
蛋白对于HEK293细胞的致死性毒性，使重组体得以包装和扩增，从而得到足够的病
毒量进行胶质瘤的基因治疗。
实验材料和仪器
1.主要试剂:Baxa基因全序列由浙江大学生命科学学院潘杰教授惠赠，TGI菌
为浙江大学生命科学学院生物化学研究所保存，Adeno一xTM表达载体(美国BD
Biosciences公司)，HEK293细胞(中国科学院上海生物化学与细胞生物学研究所)，
DMEM培养液、1640培养液、胎牛血清(美国玩vitrogen公司)，，DosPER阳离子
脂质体(瑞士Roche公司)。X一gal试剂、MTT试剂(美国A刀甘esco公司)，Bax(B一9)
鼠抗人单克隆抗体、Western Blotting荧光显影试剂盒(美国Santa cruz Biotechnology
公司)，辣根酶标记山羊抗小鼠IgG(H一L)(北京中山生物技术有限公司)，Pacl限制性
内切酶(美国NEw ENGLAND Biotechnology公司)。限制性内切酶Xbal、勒nl、
BajnHI、pstl，DNA Marker DL2000，DNA Marker DL15o0O(大连宝生物工程有限公
司)。
2.试验仪器:352型酶标仪(芬兰Labsystems Multiskan公司)，TC2323二氧化
碳生化培养箱(美国SHEL LAB公司)，UV-24OIPC紫外分光光度计(日本岛津公司)，
X一22R冷冻离心机(德国Beckman公司)，PCR扩增仪(杭州宝诚科技有限公司)，YJ一875
型医用净化工作台(苏州长桥净化设备厂)。
实验方法
用一次性塑料培养瓶，含10%胎牛血清的高糖DMEM，按常规方法培养HEK293
细胞，每3天传代1次。以xba 1/ KPnl双酶切pshuttle质粒，将双酶切的pshuttle
质粒与潘杰教授惠赠的Baxa基因进行连接反应，以Balnl硕1和Pstl酶切鉴定。以
Pl一sce工压ceul双酶切Baxa一pshuttle重组体，电泳并回收含Baxa的片断，连接回
收的Bax片断和已经Pl一Scel压Ceul双酶切的Adeno一X基因， PCR鉴定。以同样
浙江大学附属第二医院
浙江大学硕士研究生学位论文
方法构建LacZ一pshuttle一Adeno重组腺病毒载体，PcR鉴定。提纯Bax。一pshuttle一Aden。
重组腺病毒质粒后(3 .07协岁时)，以Pacl酶切，苯酚:氯仿:异戊醇(25:24:1)提纯酶
切产物，提纯后浓度为1 .68。留协1。以DosPer阳离子脂质体转染重组载体进HEK293
细胞，5天和8天后换液，10天后传代，离心收集每次换液时脱落的细胞。以同样的
方法转染LacZ一pshuttle一Adeno重组腺病毒载体进入HEK293细胞。Westem Blot检测
Bax。一Pshuttle一Adeno重组腺病毒载体在HEK293细胞中的表达。X一gal底物显色法检
测LacZ一pshuttle一Aden。重组腺病毒载体在HEK293细胞中的表达。MTT法测定Bax
。一pshuttle一Adeno重组腺病毒载体对HEK293细胞的毒性作用。试验数据利用The
SAS System for Windows VS统计软件包进行统计学分析。
结果
第1组(HEK293细胞转染Bax。一pshuttle~Adeno重组腺病毒载体后换液时离心
收集的脱落细胞)细胞裂解液通过W七stem Blot可检测到相对分子质量为2 1 O00D的
蛋白，而第2组(转染BaxQ一pshuttle一Adeno重组载体后存活并传代培养的细胞)和
对照组(未转染Bax。一pshuttle一Adeno重组载体的HEK293细胞)无可检测到的Bax
蛋白表达。
转染了LacZ一shuttle一Adeno重组腺病毒载体的现K293细胞在加入染色底物
X一咧4h后，细胞培养液变成蓝色，倒置显微镜下见甩K293细胞亦蓝染。
酶标仪(570run波长)测量结果见表z。经The SAS System for w
Oltvai et al identified an associated 21 kD protein partner, Bax, that has extensive amino acid homology with Bcl-2 in 1993. Bax homodimerizes and forms heterodimers with Bcl-2 in vivo. Overexpressed Bax accelerates apoptotic death and also counters the death represser activity of Bcl-2. The ratio of Bcl-2 to Bax determines survival or death following an apoptotic stimulus. There are high expression of Bcl-2 and relatively low expression of Bax in glioma. There is relation between unbalance of ratio of Bcl-2 to Bax and histological grade of glioma, capacity of metastasis of glioma, growth rate of glioma, relapse of glioma as well as response of glioma to chemotherapy and radiotherapy. If we can enhance the expression of bax gene in glioma, we'll promote the apoptosis of glioma and enhance the response of glioma to chemotherapy and radiotherapy. Human adenovirus is a kind of double strains DNA (dsDNA) virus. The adenoviral genome exists as an episome in the host cell's neucleus, dose not integrate into host c
ell's genome, and rarely causes host's gene insert mutation. As a vector, Adenovirus has the advantages of easy operation, easy propagation, wide variety of host cell spectrum, noncell-cycle dependent infectious ability, capacity of ligating large foreign DNA to adenoviral vector. Replication-incompetent(AEl/AE3) human adenovirus is one of the best vector at present because it neither replicate nor actively transcribe in non-package cell science the cell lacks the necessary transcription factors-the El genes. We made replication-incompetent
adenovirus as a vector, human cytomegalovirus immediate early promoter as a mammalian expression cassette promoter, Baxa gene as therapeutic gene, to recombine a Bax a -pShuttle-Adeno recombinant adenoviral expression vector. We demonstrated that the recombinant adenoviral expression vector has a high activity of transcription and expression of gene inserted. We also found that the expression of Bax precede the replication and package of adenovirus and overexpression of Bax is cytotoxic in HEK293 cell line, which would impede the propagation of recombinant adenoviral expression vector in sensitive packaging cells-HEK293 by killing the HEK293 cells. This implies that we can replace a controllable promoter to MCV IE promoter, which do not express downstream Bax gene or lowly express in HEK293 cells in order to avoiding its toxicity to HEK293 cell. This make recombinant Bax-spshuttle-adeno expression vector propagate itself enough to proceed the gene therapy to glioma.
Experimental materials
Full sequence of Baxa gene was donated by Pro. Jie Pan, College of Life Sciences, Zhejiang University. E coli-PGl was obtained from Biochemical Institute of College of Life Sciences, Zhejiang University. Adeno-X鈩?expression system was bought from BD Biosciences Company, America. HEK293 cell line was bought from Institute of Biochemistry and Cell Biology, SIBS, CAS, China. DMEM, 1640 Medium, and FBS were bought form Introgen Co., America. DOSPER Liposomal Transfection Reagent was bought from Roche Co., Switzeland. MTT and X-gal reagent were bought from Amresco Inc., America. Western Blotting Luminal Reagent was bought from Santa Cruz Biotechnology Co., America. Peroxidase-Conjugated Goat Anti-Mouse IgG was bought from Beijing Zhongshan Biotechnology Co. Ltd., China. Pac I was bought from New England Biotechnology Co., America. Xba I , Kpn I , BamH I , Pst I , DNA Marker DL2000 and DNA Marker DL15000 were bought from TaKaRa Biotechnology(Dalian) Co.
Ltd.,China.
Experimental Methods
Culture HEK293 cells in high glucose DMEM supplemented with 100 units/ml penicillin G sodium, 100 g/ml streptomycin, and 10% fetal bovine serum in plastic flasks under optimum growth conditions(37 ,5% CO2). Split every 3 days when they reach about 80% confluency. Double-digest pShuttle plasmid DNA with Xba I and Kpn I . Combine the Xba I / Kpn I -digested pShuttle plasmid DNA with the Baxa gene donated by Pro. Jei Pan. Identify putative recombinant Baxa-pShuttle DNA by BamH I or Pst I restrict
引文
1 Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, bax, that accelerates programmed cell death. Cell, 1993, 74(4): 609-619.
2 Tsuijimoto Y, Gorham J,Cossman J et al.The t(14,18)chromosome translocations involved in B-cell neoplasms result from mistakes in VDJ joining. Science, 1985, 229: 1390-1398.
3 Vaux D,Cory S,Adams J et al.Bcl-2 gene promotes heamopoitic cell survival and cooperates with C myc to immortalize pre-B cell. Nature, 1988, 335: 440-445.
4 Mcpake CR, Tillman DM, Poquette CA, et al. Bax is an important determinant of chemosensitivity in pediatric cell lines independent of Bcl-2 expression and P53 status. Oncol Res, 1998, 10: 235-245
5 Hara A, Hirose Y, Yoshimi N et al. Expression of Bax and Bcl-2 proteins, regulators of programmed cell death, in human brain tumors. Neurol Res, 1997, 19: 623-634
6 王成林，潘德业，陈峰等。Bcl-2和Bax蛋白在人脑星型细胞瘤中的表达及意义。中华神经外科杂志，1999，15：222-225．
7 方胜，袁先厚，裴永恩等，Bcl-2和Bax蛋白在人脑胶质瘤中的表达及意义。中国临床神经外科杂志，2001，6：109-111．
8 Kaufmann SH, Gores GJ. Apoptosis in cancer: cause and cure. Bioessays, 2000, 22: 1007-1010.
9 Lee A, De Jong G, Guo J, et al, Bax expressed froom a herpes viral vector enhances the efficacy of N,N'-bis(2-hydroxyethyl)-N-nitrosourea treatment in a rat glioma model. Cancer Gene Ther, 2000, 7: 1113-1119.
10 Arafat WO, Buchsbaum D J, Gomez-Navarro J, et al. An adenovirus encoding proapoptotic bax synergistically radiosensitizes malignant glioma. Int J Radiation Ontology Biol Phys, 2003, 55: 1037-1050.
11 Shinoura N, Saito K, Yoshida Y, et al. Adenovirus-mediated transfer of bax with caspase-8 controlled by myelin basic protein promoter exerts an enhanced cytotoxic effect in gliomas. Cancer Gene Ther, 2000, 7: 739-748.
12 Gridley DS, Timiryasova TM, Miller GM, et al. Evaluation of TNF-alpha/Bax gene therapy and radiation against C6 glioma xenografts. Techol Cancer Res Treat, 2003, 2: 41-50.
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14 Sanding V, Youil R, Bett AJ, Franlin LL, et al. Optimization of the helper-dependent adenovirus system for production and potency in vivo. Proc Natl Acad Sci, 2000, 97: 1002-1007.
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17 Havenga M J, Lemckert AA, Ophorst OJ, et al. Exploting the natural diversity in adenovirus tropism for therapy and prevention of disease. J Virol, 2002, 76: 4612-4620.
18 Wang G, Berk AJ. In vivo association of adenovirus large E1 A protein with the human mediator complex in adenovirus-infeeted and -Transformed cells. J Viro, 2002, 76: 9186-9193.
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20 Boshart M, Wever F, Jahn G; et al. A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus. Cell, 1985, 41: 521-527
21 Sanchez TA, Habib I, Leland Booth J, et al. Zinc finger and carboxyl regions of adenovirus E1A 13S CR3 are important for transactivation of the cytomegalovirus major immediate early promoter by adenovirus. Am J Respir Cell Mol Biol, 2000, 23: 670-677.
22 Habib NA, Hodgson H J, Lemoine N, et al. A phase Ⅰ/Ⅱ study of hepatic artery infusion with wtp53-CMV-Ad in metastatic malignant liver tumours. Hum Gene Ther, 1999, 81: 2019-2034.
23 Stevens JL, Cantin GT, Wang G; et al. Transcription control by E1 A and MAP kinase pathway via Sur2 mediator subunit. Science, 2002, 296: 755-758.
24 Nada S, Trempe JP, Characterization of adeno-assoeiated virus rep protein inhibition of adenovirus E2a gene expression.Virology, 2002, 293: 345-355.
25 Jing XJ, Kalman-Maltese V, Cao X, et al Inhibition of adenovirus cytotoxicity, replication, and E2a gene expression by adeno-associated virus. Virology, 2001: 91: 140-151.
2 Tsuijimoto Y, Gorham J,Cossman J et al.The t(14,18)chromosome translocations involved in B-cell neoplasms result from mistakes in VDJ joining. Science, 1985, 229: 1390-1398.
3 Vaux D,Cory S,Adams J et al.Bcl-2 gene promotes heamopoitic cell survival and cooperates with C myc to immortalize pre-B cell. Nature, 1988, 335: 440-445.
4 Mcpake CR, Tillman DM, Poquette CA, et al. Bax is an important determinant of chemosensitivity in pediatric cell lines independent of Bcl-2 expression and P53 status. Oncol Res, 1998, 10: 235-245
5 Hara A, Hirose Y, Yoshimi N et al. Expression of Bax and Bcl-2 proteins, regulators of programmed cell death, in human brain tumors. Neurol Res, 1997, 19: 623-634
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