人血管生成素1和VEGF基因腺相关病毒共表达系统的构建

目的 :建立一个可同时表达人血管生成素 1(Angiopoietin 1)和血管内皮生长因子 16 5 (VEGF16 5 )的重组腺相关病毒 (AAV)载体基因转移系统 ,为下一步的研究做准备工作。方法 :利用PCR技术 ,获取目的基因Angiopoietin1和VEGF16 5 ,Angiopoietin 1的上游含有HindⅢ位点 ,下游含有BamHⅠ位点 ,VEGF16 5的上游含有BglⅡ位点 ,下游含有BamHⅠ位点。通过重组DNA技术 ,利用过渡质粒pZero 中含有的BamHⅠ、BglⅡ、HindⅢ、NotⅠ、XhoⅠ、XbaⅠ、SalⅠ、BspHⅠ、KspⅠ酶切位点以及pAAV MCS中含有的相应粘端互补的酶切位点 ,将Angiopoietin 1和VEGF16 5亚克隆进入pAAV MCS。结果 :测序证实Angiopoietin 1和VEGF16 5与GeneBank提供的原始序列完全一致。酶切鉴定与PCR筛选证实重组质粒和预期结果完全一致。在新的重组质粒中 ,Angiopoietin 1和VEGF16 5各有一套CMV启动子和PolyA终止子系统。结论 :Angiopoietin 1和VEGF基因AAV共表达系统的构建成功 ,为严重缺血性疾病基因治疗的研究奠定了基础。     

Construction of recombinant adeno-associated virus vector co-expressing hVEGF_(165) and hBMP_7 gene

Objective: To construct recombinant adeno-associated virus co-expressing human vascular epithelial growth factor 165(hVEGF165) and bone morphogenetic protein 7(hBMP7),measure the virus titer and verify the recombination. Methods:The AAV helper-free system was used as basis to generate recombinant AAV. The IRES sequence of plasmid pIRES was cut down and subcloned into ITR/MCS containing vector pAAV-MCS to construct recombinant plasmid pAAV-MCSa-IRES-MCSb. The hVEGF165 and hBMP7 gene was amplified by PCR and inserted into upstream MCSa and downstream MCSb respectively. Then,recombinant plasmid pAAV-hVEGF165-IRES-hBMP7,pAAV-RC and pHelper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hBMP7 packaging. The GFP labeled rAAV-IRES-GFP was simultaneously packaged by using the parallel plasmid pAAV-IRES-hrGFP. The efficiency of AAV packaging was monitored under fluorescent microscope and recombinant viral particles were harvested from infected AAV-293 cells. The virus titer was measured by infecting AAV-HT1080 cells,and the recombinant AAV-hVEGF165-IRES-hBMP7 was verified by PCR of the exogenous interest genes. Results:Recombinant pAAV-hVEGF165-IRES-hBMP7 was verified by double digestion. GFP expression in AAV-293 could be observed under fluorescent microscope 72 h after transfection and the system provided a high packing ratio of 95%. The recombinant adeno-associated virus has a high titer of 5.5×1011vp/ml,and AAV-HT 1080 was infected at a ratio of 90%. The recombinant virus was confirmed by PCR of exogenous hBMP7 and hVEGF165 gene. Conclusion:Recombinant rAAV-hVEGF165-IRES-hBMP7 was successfully constructed with a high virus titer,which may offer foundation for in vitro and in vivo experiments of hVEGF165 and hBMP7 co-expression and provide a new method for gene therapy of bone regeneration.     

CONSTRUCTING ADENO-ASSOCIATED VIRUS-TGFβ3 AND COMPARING ITS BIOLOGICAL EFFECT ON PROTEOGLYCAN SYNTHESIS IN DEDIFFERENTIATED NUCLEUS PULPOUS CELLS WITH ADENOVIRUS-TGFβ1

Objective To construct adeno-associated virus (AAV) expression system for transforming growth factor β3 (TGFβ3) and detect its biological effect on proteoglycan synthesis of the earlier and later dedifferentiated rabbit lumbar disc nucleus pulpous (NP) cells, which was compared with that of adenovirus (AV) expression system for TGFβ1. Methods TGFβ3 gene was obtained using PCR. Its upstream contained restriction enzyme site Kpn Ⅰ, and its downstream contained restriction enzyme site SalⅠ. Using the restriction enzyme sites of PCR product of TGFβ3 and the corresponding multiple cloning site (MCS) in plasmid AAV, TGFβ3 was subcloned into AAV. The recombinant plasmid AAV-TGFβ3 was transfected into H293 cells with LipofectamineTM 2000, and the expression of TGFβ3 gene was detected using immunofluorescent analysis. After AAV-TGFβ3 virus particle with infectious activity was packaged, TGFβ3 expression in NP cells was detected by immunoblotting, and its biological effect on proteoglycan synthesis was detected by antonopulos method and compared with that of AV-TGFβ1 in the earlier and later dedifferentiated NP cells. Results For the earlier dedifferentiated NP cells, AAV-TGFβ3 slowly and stably enhanced proteoglycan synthesis, but AV-TGFβ1 rapidly and transiently enhanced its synthesis. For the later dedifferentiated NP cells, AAV-TGFβ3 stably enhanced proteoglycan synthesis, but AV-TGFβ1 inhibited its synthesis. Conclusion AAV expression system can mediate TGFβ3 gene to be expressed stably, and AAV-TGFβ3 can enhance proteoglycan synthesis of the earlier and later dedifferentiated NP cells.     

表达UGT1A1重组腺相关病毒载体的构建

目的 获得表达胆红素尿苷二磷酸葡萄糖醛酸酶（UGT1A1）基因；构建UGT1A1重组腺相关病毒载体，制备腺相关病毒。方法 设计、合成引物，通过逆转录聚合酶链反应（RT—PCR）从HepG2细胞总RNA中扩增UGT1A1基因，最终连接于pAAV—MCS．双酶切、PCR鉴定阳性重组质粒。并在HEK293T细胞表达，制备高滴度重组腺相关病毒。结果 成功扩增了人类UGT1A1基因．构建表达UGT1A1基因重组腺相关病毒载体。并成功表达于HEK293T细胞。结论 本实验构建的人类UGT1A1重组腺相关病毒将为因UGT1A1活性不足导致的高胆红素血症的基因治疗奠定基础。     

tPA和VEGF165基因共表达质粒的构建及其在血管平滑肌细胞中的表达

目的 构建携带人组织纤溶酶原激活物(tPA)和血管内皮细胞生长因子165(VEGFl65)基因的真核表达质粒pBudCE4．1／tPA-VEGF165,并观察其在血管平滑肌细胞(VSMC)中的表达。方法从人心脏组织中克隆tPA和VEGF165基因。将tPA和VEGF165基因克隆至真核表达质粒pBudCE4．1中,构建共表达质粒pBudCE4．1／tPA-VEGF165。将pBudCE4．1／tPA-VEGF165转染VSMC,用RT-PCR法检测转染的VSMC中tPA和VEGFl65mRNA的表达,用ELISA法检测转染的VSMC培养液中tPA和VEGF165蛋白质。结果人tPA和VEGF165基因的RT-PCR产物分别为1．9kb和576bp。经酶切鉴定证实,tPA和VEGF165基因已克隆至真核表达质粒pBudCE4．1中。以其转染VSMC后,经RT-PCR和ELISA检测发现,tPA和VEGF165在mRNA和蛋白质水平均有表达。结论成功地构建了真核共表达质粒pBudCE4．1／tPA-VEGF165,并在VSMC中表达tPA和VEGF165。     

构建及在血管内皮细胞中的表达

目的:构建人血管内皮细胞生长因子165(VEGF165)及绿色荧光蛋白报告基因的融合蛋白真核表达质粒,并检测其在血管内皮细胞中的表达.方法:采用PCR扩增VEGF165基因全长,并将其定向克隆入pEGFP-N1的多克隆位点,构建pEGFP/VEGF165重组质粒,经酶切、PCR及序列分析鉴定,脂质体介导转染体外培养的血管内皮细胞,荧光显微镜、RT-PCR及Western免疫印迹等方法检测EGFP/VEGF融合蛋白的表达.结果:PCR、酶切及测序证实目的基因VEGF165正确连接至pEGFP-N1的多克隆位点,pEGFP/VEGF165重组质粒转染血管内皮细胞后,荧光显微镜、RT-PCR及Western免疫印迹检测均显示EGFP/VEGF蛋白在血管内皮细胞中表达.结论:成功构建了携带人VEGF165及EGFP报告基因的融合蛋白真核表达质粒,pEGFP/VEGF165可在血管内皮细胞中表达,此为进一步研究VEGF基因治疗缺血性血管疾病奠定了实验基础.     

人血管内皮细胞生长因子165重组腺病毒的构建及其在真核细胞中的表达

目的:体外构建人血管内皮生长因子165(hVEGF165)的腺病毒表达载体,并检测其在HEK293细胞中的表达。方法:从重组质粒pcDNA3/hVEGF165中获得hVEGF165,经酶切及测序鉴定,将hVEGF165基因亚克隆到穿梭质粒pAdTrack-CMV,重组穿梭质粒经酶切线性化后,与pAEdasyl质粒在大肠杆菌BSJ183中进行同源重组,线性化后转染HEK293细胞进行包装扩增获取重组病毒上清,同时应用RT-PCR及ELISA法检测hVEGF165的表达。结果:目的基因的测序结果与人VEGF165序列(Genbank)相符。转染后经RT-PCR和ELISA检测证实转染重组质粒组hVEGF165 mRNA及蛋白表达,而转染空质粒组及未转染组没有检测到hVEGF165的表达。结论:本研究构建了人VEGF165重组腺病毒表达载体pAd-hVEGF165,并能成功地在HEK293细胞中表达。     

高滴度表达抑制转录因子的重组腺相关病毒的制备

目的 获得表达小鼠抑制转录因子ROG(repressor of GATA-3)基因；构建表达ROG的重组腺相关病毒载体，制备腺相关病毒。方法 设计、合成引物，通过逆转录聚合酶链反应(RT-PCR)从CTLL-2细胞总RNA中扩增ROG基因，测序成功后，最终连接于pAAV-MCS，双酶切、PCR鉴定阳性重组质粒，并在Hela细胞中表达；制备高滴度重组腺相关病毒。结果 成功扩增了小鼠ROG基因，构建表达ROG基因的重组腺相关病毒表达载体，并成功表达于Hela细胞。结论 本实验构建的小鼠ROG重组腺相关病毒将为哮喘的基因治疗研究奠定物质基础。     

人TAT-PDX-1融合蛋白原核表达载体的构建

目的构建人TAT-PDX-1融合蛋白原核表达载体。方法以人胰腺癌(PANC)-1细胞株cDNA为模板,采用反转录聚合酶链式反应(RT-PCR)扩增胰腺十二指肠同源异型盒基因-1(PDX-1)蛋白编码的全部序列,克隆入原核表达载体pET28a中,经限制性内切酶HindIII和BamHI双酶切及DNA序列分析重组质粒的目的基因。结果 RT-PCR扩增产物的特异性片段长度为885 bp,以此构建的重组质粒pET28a-TAT-PDX-1经HindIII和BamHI双酶切后显示5 900 bp和885 bp左右的2条片段,测序结果与Genbank中的人PDX-1基因cDNA序列一致。结论成功构建了人TAT-PDX-1融合蛋白原核表达载体。     

腺病毒介导的血管内皮生长因子体外转染心肌细胞的研究

目的:构建携带人血管内皮生长因子(VEGF)基因的重组腺病毒载体,并转染体外培养的心肌细胞,检测VEGF的表达.方法:将人源性的VEGF165cDNA正向插入到腺病毒载体PDC315,构建重组质粒,通过脂质体共转染293细胞,经同源重组获得携带人VEGF165基因的重组腺病毒,通过PCR扩增法鉴定所构建的腺病毒,扩增并测定滴度后,体外转染培养的心肌细胞,利用ELISA、Western印迹分析等方法检测VEGF在心肌细胞中的表达.结果:人VEGF165cDNA成功地正向插入到PDC315载体中,以重组病毒基因组DNA为模板,同时扩增出了610bp的VEGF165cDNA基因片段,证实了所构建病毒的正确性,病毒滴度为2.8×108 pfu/ml,Ad VEGF165体外转染心肌细胞3 d后,在培养细胞的上清液及细胞内检测到了VEGF的表达.结论:成功构建了表达人VEGF 165基因的腺病毒载体,体外转染心肌细胞后能够满意表达VEGF,为基因治疗心肌缺血奠定基础.     

腺相关病毒介导的人血管内皮生长因子体外转染兔骨髓内皮祖细胞

目的:探讨腺相关病毒-2(AAV-2)介导人血管内皮生长因子-165(hVEGF165)转染兔骨髓内皮祖细胞(EPCs)及其对EPCs增殖能力的影响。方法:构建携带hVEGF165基因的重组腺相关病毒(rAAV)载体pAAV-hVEGF165;制备携带hVEGF165基因的rAAV(rAAV-2-hVEGF165)和携带β-半乳糖苷酶(-βgal)基因的rAAV(rAAV-2-LacZ);体外培养和鉴定兔骨髓EPCs;分别用rAAV-2-hVEGF165和rAAV-2-LacZ体外转染EPCs(AAV/VEGF-EPC和AAV/LacZ-EPC);用RT-PCR、免疫细胞化学、流式细胞术等法检测hVEGF165和-βgal的表达,MTT法检测AAV/VEGF-EPC的增殖能力。结果:经酶切鉴定和DNA测序,得到了序列正确的重组质粒pAAV-hVEGF165;AAV/VEGF-EPC能够表达hVEGF165蛋白,AAV/LacZ-EPC能够表达-βgal;rAAV-2-hVEGF165对于EPCs的转染率为64.4%;AAV/VEGF-EPC的增殖能力明显强于AAV/LacZ-EPC和EPCs。结论:EPCs可以被rAAV-2-hVEGF165高效转染,其增殖能力明显增强。本文为进一步探讨AAV/VEGF-EPC用于缺血性血管疾病治疗的可能性奠定了基础。     

人血管内皮生长因子基因cDNA的克隆及其在COS-7细胞中的表达

目的：克隆和在COS－7细胞中表达人血管内皮生长因子165（VEGF165）。方法：利用RT－PCR方法，从新鲜卵巢癌组织中扩增到人VEGF165的全长cDNA，并测定其核酸序列；利用基因重组技术构建VEGF165的真核表达载体pcDNA3.1-VEGF165；应用脂质体介导的基因转移术将这一表达载体导入COS－7细胞后，免疫组化（SP法）检测瞬时表达的产物。结果：经酶切鉴定和基因测序证实，克隆的基因片段为人VEGF65cDNA，重组质粒pcDNA3．1－VEGF165转染COS－7细胞后，免疫组化检测有VEGF表达，结论：成功克隆人VEGF165基因，并在COS－7细胞获得表达。     

hIL-2与ZsGreen双基因共表达腺相关病毒的包装及鉴定

[目的]包装并鉴定带有人白介素2（hIL-2）及ZsGreen的双基因共表达的重组5型腺相关病毒（recombinant adeno-associated virus 5,rAAV5）,为今后利用5型重组腺相关病毒载体进行胰腺癌基因治疗提供研究基础。[方法]以5型腺相关病毒包装系统（helper-free system）为模版,PCR法扩增pLVX-IRES-ZsGreen和PES-IL-2模板上的IRES-ZsGreen和IL-2基因,利用酶切位点插入重组腺相关病毒骨架质粒,获得重组质粒pAAVhIL-2-IRES-ZsGreen。经三质粒共转染AAV293细胞包装重组腺相关病毒rAAV5-hIL-2-IRES-ZsGreen。荧光显微镜下检测病毒包装效率,超速离心纯化、浓缩重组病毒,qPCR测定病毒的滴度。重组病毒转导细胞经荧光显微镜检测、外源基因经PCR检测证实病毒包装结果。[结果]5型重组腺相关病毒骨架质粒pAAV-IL-2-IRES-ZsGreen经双酶切和测序鉴定,确定其序列正确。荧光显微镜下观察三质粒共转染AAV293细胞72h后,病毒包装效率达90%以上。5型重组病毒感染HEK 293细胞48h有荧光出现,重组病毒基因组成功扩增出外源基因IL-2,证明有感染力的重组病毒包装成功。[结论]成功包装带有hIL-2及ZsGreen标记的双基因共表达重组腺相关病毒,滴度达到2.62×1012。     

hTRX-PR39重组腺相关病毒载体的构建

目的 探讨hTRX-PR39融合基因在脑缺血疾病中的治疗作用。方法 将已经构建好的hTRX-PR39融合基因插入到具有相应酶切位点的pSSCMV病毒载体质粒中,得到重组腺相关病毒载体质粒,酶切电泳鉴定。将已构建的重组腺相关病毒载体质粒、腺病毒辅助质粒PFG140和包装质粒pAAV/Ad,三质粒磷酸钙共沉淀法转染293细胞系,通过同源重组获得hTRX-PR39重组腺相关病毒载体, 收集病毒, 斑点杂交（Dot blot）法测定病毒滴度。结果 成功构建重组腺相关病毒质粒pSSCMV/ hTRX-PR39。包装、回收病毒后,Dot blot法测定重组病毒滴度为3.46×(1012~1013)PFU/mL。结论 成功构建pSSCMV/ hTRX-PR39重组腺相关病毒载体,并包装较高浓度的重组病毒。     

人TAT-Survivin融合蛋白原核表达载体的构建

目的:构建人TAT-Survivin融合蛋白原核表达载体。方法:以人胸腺细胞瘤cDNA为模板,采用RT-PCR扩增survivin基因成熟蛋白编码的全部序列,克隆入原核表达载体pET28a(+)中,经限制性内切酶双酶切及DNA序列分析鉴定目的基因。结果:PCR扩增的特异性片段长度为462bp,以此构建的重组质粒pET28a-TAT-survivin,经HindIII和BamHI双酶切后显示5.9kb和462bp左右的两条片段,测序结果与Genbank中的人survivin基因cDNA(Genbank序列号)序列一致。证明人survivin已成功克隆到了原核细胞表达载体pET28a(+)中。结论:成功构建了pET28a-TAT-survivin重组原核表达载体。     

携带信号肽NT4-GFP-穿膜肽Ant融合基因的重组腺相关病毒载体的构建及意义

目的：构建携带具有穿膜功能的融合报告基因NT4-GFP-Ant基因的重组腺相关病毒载体。方法：应用PCR技术和T载体克隆法克隆绿色荧光蛋白（GFP）基因;用T4 DNA连接酶将测序正确的GFP与已构建成功的Ant基因片段及PBV220/NT4线性质粒载体定向连接,构建PBV220/NT4-GFP-Ant融合载体,酶切获取融合基因,并将其连入腺相关病毒载体PSSHG中,构建PSSHG/NT4-GFP-Ant重组腺相关载体并进行酶切鉴定。结果: T-easy/GFP经EcoRⅠ酶切后,可得到730 bp左右的片段,GFP基因经DNA测序证实与GenBank序列一致;pBV220/NT4-GFP-Ant经BamHI/EcoRⅠ联合双酶切后,得到约为 1 000 bp的基因片段; PSSHG /NT4-GFP-Ant经 BamHI/EcoRⅠ联合双酶切后,得到大小约为1 000 bp长度的基因片段。结论：成功克隆GFP基因,成功构建NT4信号肽-GFP-Ant穿膜肽融合基因和PSSHG/NT4-GFP-Ant重组腺相关病毒载体。     

Construction of recombinant adenovirus co-expression vector carrying the human transforming growth factor-β1 and vascular endothelial growth factor genes and its effect on anterior cruciate ligament fibroblasts

Background  Remodeling of the anterior cruciate ligament (ACL) graft usually takes longer than expected. Gene therapy offers a radical different approach to remodeling of the graft. In this study, the internal ribosome entry site (IRES) sequence was used to construct a new recombinant adenovirus which permits co-expression of transforming growth factor-β1 (TGFβ1) and vascular endothelial growth factor 165 (VEGF165) genes (named Ad-VEGF165-IRES-TGFβ1). We investigated the effects of the new adenovirus on the migration of and matrix synthesis by ACL fibroblasts.
Methods  Adenoviral vector containing TGFβ1 and VEGF165 genes was constructed. ACL fibroblasts were obtained from New Zealand white rabbits. After ACL fibroblasts were exposed to Ad-VEGF165-IRES-TGFβ1, the expression of VEGF165 and TGFβ1 proteins were assessed by enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis. Bioassay of VEGF165 and TGFβ1 proteins were assessed by Western blotting analysis. Proliferation and migration of ACL fibroblasts were assessed by in vitro wound closure assay. Gene expression of collagen type I, collagen type III, and fibronectin mRNA among matrix markers were assessed by real-time PCR.
Results  The results showed the successful construction of a recombinant co-expression adenovirus vector containing TGFβ1 and VEGF165 genes. Co-expression of TGFβ1 and VEGF165 can induce relatively rapid and continuous proliferation of ACL fibroblasts and high gene expression of collagen type I, collagen type III, and fibronectin mRNA among matrix markers.
Conclusion  Co-expression of TGFβ1 and VEGF165 genes has more powerful and efficient effects on the migration of and matrix synthesis by ACL fibroblasts.

     

表达人血管内皮生长因子和血管生成素1的CHO细胞株的建立

目的：克隆人血管内皮生长因子(VEGFm)和血管生成素1(Ang1)基因，建立可分别表达VEGFm和Ang1的中国仓鼠卵巢细胞(CHO)株。方法：用PCR技术，分别从人心脏cDNA库中扩增VEGF165和Ang1 cDNA，并定向克隆人真核表达载体pSecTag2B中。用Lipofectamine2000将重组质粒转化入CHO细胞中，用Zeocin筛选并维持已转化重组质粒的CHO细胞株。在大肠杆菌DH10B中扩增转化入CHO细胞中的重组质粒，行DNA测序鉴定。用Westem-blot鉴定CHO细胞中重组VEGFm和Ang1的表达。结果：从人心脏cDNA库中扩增出VEGFⅢ和Ang1 cDNA片段，酶切和测序结果显示，已正确构建重组质粒pSecTag-VEGF165和pSecTag-Ang1。用Zeocin筛选到候选的CHO细胞株，DNA测序结果表明，pSecTag-VEGF165和pSecTag-Ang1已分别成功转化入CHO细胞中。Western-blot结果显示，在CHO细胞中表达出可被VEGF和Ang1单抗识别的特异蛋白。结论：克隆了VEGFm和Ang1基因，并建立了表达VEGF165和Ang1的CHO细胞株。     

HSP27基因的克隆与表达

目的构建热休克蛋白27(Heat shock protein 27,HSP27)真核表达质粒以用于肾脏缺血预适应(ischemia Preconditioning,IP)的作用及机制研究.方法利用乳腺癌细胞系MCS-7,用RT-PCR方法扩增HSP27全长基因,将HSP27基因克隆到真核表达载体pAAV-MCS中.重组质粒转染NIH3T3细胞,Western blot法鉴定重组质粒HSP27/pAAV-MCS能在哺乳动物细胞中正确表达.结果RT-PCR方法正确地扩增出全长HSP27基因.限制性内切酶酶切和测序结果证实HSP27基因克隆完全正确.重组质粒转染哺乳动物细胞系NIH3T3后,ECLWestern blot法证实了目的基因能在其中正确表达.结论成功地克隆真核表达重组质粒HSP27/pAAV-MCS,为下一步研究HSP27对肾脏缺血预适应的作用及其机制打下了基础.     

Construction of Adeno-associated Virus System for Human Bone Morphogenetic Protein 7 Gene

To construct the recombinant adeno-associated virus （rAAV） vector with human bone morphogenetic protein 7 （BMP7） and observe the BMP7 mRNA expression in vitro, BMP7 CDS sequence was cloned into expression plasmid pAAV-MCS of AAV Helper Free System. The recombinant plasmid was identified with enzyme digestion and sequencing. The recombinant plasmid, pAAV-RC, pHelper were co-transfected into AAV-293 cells according to the calcium phosphate-based protocol. The viral stock was collected by 4 rounds of freeze/thaw. After purified and concentrated, the recombinant virus titer was determined by dot-blot assay. HEK293 cells were transfected with the recombinant virus at different MOI, and the expression of BMP7 mRNA was detected by RT-PCR. The results showed rAAV-BMP7 was constructed and packaged successfully. The physical particle titer was 2.5×10^11 vector genomes/mL. There was different expression level of BMP7 mRNA after transfecton. These data suggested that recombinant AAV mediated a stable expression of hBMP7 mRNA in 293 cells. The AAV production method may pave the way of an effective strategy for the jaw bone defection around dental implants.