表达人单核细胞趋化蛋白-1基因重组腺病毒的制备

目的制备表达人单核细胞趋化蛋白-1(MCP-1)复制缺陷型重组腺病毒。方法RT-PCR法获得人肝组织中MCP-1cDNA片段,与T载体连接后亚克隆至腺病毒穿梭载体的巨细胞病毒启动子下游,构建重组穿梭载体pAdTrack-CMV-MCP-1,将其线性化后与pAdEasy-1共转化大肠杆菌B J5183,进行同源重组得到重组腺病毒载体。将重组腺病毒载体转染入包装细胞HEK293内制备复制缺陷型重组腺病毒,PCR扩增鉴定病毒颗粒。结果MCP-1重组腺病毒载体能有效转染HEK293细胞并在细胞内成功包装,5 d后可以观察到绿色荧光蛋白(GFP)明显表达,搜集的病毒经过PCR扩增得到特定MCP-1基因片段。结论MCP-1重组腺病毒载体及相应重组腺病毒颗粒的成功构建和制备,为进一步研究MCP-1的作用及应用MCP-1进行基因治疗奠定了基础。     

重组人Bcl-2腺病毒载体的构建与鉴定

目的　构建人bcl-2重组腺病毒载体。方法　通过酶切分析获得正向插入目的基因的真核表达载体pcDNA/bcl -2 ,然后将bcl -2定向克隆到腺病毒穿梭质粒pAdCMV中 ,通过体内同源重组 ,重组质粒与质粒pJM17共转染 2 93细胞 ,获得复制缺陷型重组腺病毒AdCMV/bcl -2 ,进行扩增、纯化。通过PCR检测病毒DNA ,空斑实验检测病毒滴度。结果　腺病毒穿梭质粒与pJM17体内同源重组后获得含目的基因的重组腺病毒 ,病毒滴度为 2 0× 10 1 0 pfu/ml。结论　基因重组腺病毒载体能成功获得高滴度含目的基因的腺病毒     

肿瘤相关基因hyrdC基因重组腺病毒的构建和鉴定

目的利用AdEasy腺病毒载体系统构建携带hyrdC基因的高滴度重组腺病毒，为进一步研究hyrdC基因的功能及机制做准备。方法以逆转录聚合酶链反应（RT—PCR）法扩增hyrdC基因，构建腺病毒重组质粒pShutth—CMV—hyrdC，线性化后采用电穿孔法与腺病毒骨架质粒pAdEasy-1共同转化BJ5183感受态大肠杆菌，挑选重组质粒将其转染293细胞，获得高滴度重组腺病毒Ad—hyrdC。结果成功地制备了高滴度携带hyrdC基因的重组腺病毒。结论采用AdEasy腺病毒载体系统能够构建携带hyrdC基因的重组腺病毒，为进一步研究hyrdC基因的功能及机制奠定了基础。     

表达人JNK基因重组腺病毒的构建和鉴定

目的 制备表达人c-jun氨基末端激酶(JNK)复制缺陷型重组腺病毒.方法 将重组穿梭载体pAdTrack-CMV-WT-JNK线性化后,与pAdEasy-1共转化大肠杆菌BJ5138,进行同源重组得到重组腺病毒载体.将重组腺病毒载体转染入包装细胞HEK293内制备复制缺陷型重组腺病毒,并经PCR及DNA测序鉴定.结果 JNK重组腺病毒载体能有效转染HEK293细胞并在细胞内成功包装,5 d后可以观察到绿色荧光蛋白(GFP)明显表达,搜集的病毒经过PCR扩增得到特定JNK基因片段并测序鉴定.动物实验证实构建的Ad-WT-JNK能有效在肝组织表达.结论 该研究成功构建了JNK重组腺病毒载体及相应重组腺病毒颗粒,为进一步研究JNK的作用及应用JNK进行相关疾病的基因治疗奠定了基础.     

DN—JNK基因重组腺病毒的构建和鉴定

目的制备表达人c-jun氨基末端激酶（JNK）复制缺陷型重组腺病毒（Ad—DN—JNK）,并通过动物实验进行功能鉴定。方法将重组穿梭载体pAdTrack—CMV—DN—JNK线性化后,与pAdEasy—1共转化大肠杆菌BJ5138,进行同源重组得到重组腺病毒载体。将重组腺病毒载体转染入包装细胞HEK293内制备复制缺陷型重组腺病毒,并经PCR及DNA测序鉴定。Western blot检测Ad—DN-JNK在SD大鼠肝组织中JNK1蛋白表达情况,及胰岛素受体底物1丝氨酸307（IRS-1^Ser307）磷酸化水平。结果JNK重组腺病毒载体能有效转染HEK293细胞并在细胞内成功包装,5d后观察到绿色荧光蛋白（GFP）明显表达,搜集的病毒经过PCR扩增得到了特定JNK基因片段并测序鉴定。所制备的Ad—DN—JNK滴度为2．5×10^10pfu／ml,动物实验证实构建的Ad—DN-JNK能有效在肝组织表达。结论该研究成功构建了DN—JNK重组腺病毒载体及相应重组腺病毒颗粒,动物实验证明Ad—DN—JNK能有效介导DN—JNK基因蛋白表达并下调IRS-1^Ser307磷酸化水平。为进一步研究JNK的作用及应用DN—JNK进行相关疾病的基因治疗奠定基础。     

携带丙型肝炎病毒非结构蛋白3基因的重组腺病毒构建研究

目的构建表达丙型肝炎病毒(HCV)非结构蛋白3 (NS3)的复制缺陷型重组腺病毒,为防治HCV感染的基因免疫和基因治疗提供实验基础. 方法将HCV NS3基因定向克隆到穿梭质粒pAdTrack-CMV上,经与腺病毒骨架质粒pAdEasy-1在大肠埃希菌BJ5183内同源重组后,转染293细胞进行包装,并反复感染293细胞进行扩增. 结果通过PCR扩增、酶切、核酸测序及Western杂交法检测鉴定,均证实插入穿梭质粒中的片段为HCV NS3基因(基因型1b);所包装出的复制缺陷型重组腺病毒AdEasy-GFP-NS3具有良好的感染能力,并可在293细胞中表达HCV NS3蛋白. 结论 AdEasy-GFP-NS3携带有HCV NS3基因,能有效地感染293细胞并高效表达,从而为丙型肝炎基因疫苗的进一步研究奠定了基础.     

肿瘤相关基因hyrdC基因重组腺病毒的构建和鉴定

目的 利用AdEasy腺病毒载体系统构建携带hyrdC基因的高滴度重组腺病毒,为进一步研究hyrdC基因的功能及机制做准备.方法以逆转录聚合酶链反应(RT-PCR)法扩增hyrdC基因,构建腺病毒重组质粒pShuttle-CMV-hyrdC,线性化后采用电穿孔法与腺病毒骨架质粒pAdEasy-1共同转化BJ5183感受态大肠杆菌,挑选重组质粒将其转染293细胞,获得高滴度重组腺病毒Ad-hyrdC.结果成功地制备了高滴度携带hyrdC基因的重组腺病毒.结论采用AdEasy腺病毒载体系统能够构建携带hyrdC基因的重组腺病毒,为进一步研究hyrdC基因的功能及机制奠定了基础.     

含HSV-TK基因及IL-18基因复制缺陷型腺病毒载体的构建和鉴定

目的：分别构建含TK及IL-18基因的重组腺病毒载体,并进行鉴定。方法：通过DNA重组技术,构建含有目的基因的复制缺陷型腺病毒Ad CMV-TK及Ad CMV-IL-18;将上述重组腺病毒液感染293细胞,提取DNA,进行PCR鉴定;将病毒液感染293细胞,扩增病毒,用OD260法测定病毒滴度。结果：经PCR鉴定,鉴定正确的腺病毒命名为Ad CMV-TK及Ad CMV-IL-18,测定病毒滴度Ad CMV-TK=1.28×109 PFU/m L;Ad CMV-IL-18=1.28×109 PFU/m L。结论：成功构建了含HSV-TK基因及IL-18基因重组腺病毒载体,为肿瘤的基因治疗提供了理论依据及实验数据。     

HBsAg腺病毒疫苗载体的构建及其免疫学研究

目的 构建含HBsAg基因的重组复制缺陷型腺病毒疫苗载体,观察小鼠抗HBsAg免疫反应. 方法以pEcob-6为模板,PCR扩增目的 基因HBsAg,腺病毒穿梭载体PAd-track-cmv与HBsAg经T4连接酶连接,重组为PAd-track-cmv-HBs穿梭质粒;腺病毒骨架载体质粒PAd-Easy-1再与PAd-track-cmv-HBs在大肠杆菌BJ-5183中同源重组PAd-Easy-1-HBs质粒,PAd-Easy-1-HBs脂质体介导转染293细胞,包装HBsAg重组腺病毒.30只小鼠随机分成3组,分别肌肉注射PAd-Easy-1(载体组)、HBsAg重组腺病毒(实验组)、PBS(正常对照组),免疫2次.2月后取小鼠血清检测抗HBsAg抗体反应. 结果 PAd-track-cmv-HBs穿梭质粒经上海基康生物技术公司进行DNA测序,结果与预期结果相同,含有完整的HBsAg目的 基因.PAd-Easy-1-HBs用Pac-1酶切,Pac-1能将载体切成两个片段,大片段约为30 kb,小片段约为3 kh.HBsAg重组腺病毒组抗HBsAg的OD值明显升高,与正常对照组比较,差异有统计学意义(P＜0.05).与PAd-Easy-1载体对照组比较,差异有统计学意义(P＜0.05). 结论 HBsAg腺病毒疫苗载体构建成功,且能够诱导小鼠产生抗HBsAg.     

Role of enhanced radiosensitivity and the tumor-specific suicide gene vector in gene therapy of nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is one of the common malignant tumors in China. Radiotherapy and chemotherapy are the main therapy methods for NPC. To enhance the specific antitumor effect, a novel vector with radiosensitivity and tumor specificity was constructed in this study, which enables the reduction of dosage of radiation and chemotherapeutic drugs due to its double killing effect. Four DNA elements, Egr-1 promoter, Cytosine deaminase (CD) gene, hTERT promoter, Survivin antisense oligonucleotides were amplified and constructed in pcDNA3.1 vector. CD and Survivin gene expression in CNE-2 cells were detected by RT-PCR. High performance liquid chromatography (HPLC) was employed to determine the transformation from the prodrugs 5-FC to 5-FU. Hoechst33258 staining of the nuclei and methylthiazolyl tetrazolium(MTT) assay were applied to detect apoptosis and cell survivability, respectively. In addition, the anti-tumor effects were examined in vivo by injecting cells with different vectors into nude mice. Our results revealed a notable killing effect of combined treatment with 5-FC and radiation on CNE-2 cells transfected with vectors in vitro. This effect was especially notable on pEC-TS transferred cells, which showed 57% of cells were killed. In vivo, an obvious suppression of tumor was displayed in pEC-TS group, which was significantly different from other groups (p < 0.05). Consequently, this expression cassette may have a great therapeutic potential for the treatment of NPC.     

腺病毒载体介导hTERT特异性RNAi诱导乳腺癌细胞凋亡的研究

[目的]构建hTERT特异性RNAi腺病毒载体,并研究该载体对乳腺癌MCF-7细胞增殖的影响,为针对端粒酶的乳腺癌基因治疗提供新的途径。[方法]设计针对人端粒酶逆转录酶催化亚基(hTERT)的干扰靶序列GGAACACCAAGAAGTTCATCT,构建RNAi腺病毒载体rAd-shRNA-hTERT。同时构建不针对任何基因的shRNA阴性对照rAd-shRNA-HK和只含EGFP的rAd-EGFP对照。转染人乳腺癌MCF-7细胞后48h,以荧光定量PCR和免疫印迹法检测MCF-7细胞hTERT基因的表达、流式细胞仪检测细胞凋亡情况。[结果]病毒载体经酶切、电泳分析表明插入序列正确,构建成功;转染MCF-7细胞后,rAd-shRNA-hTERT组可明显抑制hTERT基因表达,并抑制细胞增殖、诱导细胞凋亡。[结论]通过腺病毒介导的RNAi技术可有效抑制MCF-7细胞中hTERT基因表达进而诱导肿瘤细胞凋亡,有望用于肿瘤的基因治疗。     

RyR2-shRNA重组慢病毒载体的构建及其对离体心肌细胞RyR2基因表达的影响

目的构建携带RyR2-shRNA的慢病毒载体,并检测其对小鼠离体心肌细胞中RyR2的基因沉默效果。方法设计并合成针对小鼠RyR2基因的shDNA,克隆入含U6启动子和EGFP报告基因的慢病毒载体psiHIV-U6-eGFP,构建表达RyR2-shRNA的慢病毒载体,通过将慢病毒转移质粒和包装质粒共转染293T细胞,得到重组慢病毒载体颗粒,然后离体感染小鼠心肌细胞,采用Real-timePCR检测RyR2在mRNA水平的沉默效应。结果经测序证实RNAi慢病毒载体构建成功并成功包装。流式细胞仪测得重组慢病毒感染小鼠心肌细胞的阳性率达80%以上。Real-timePCR证实,重组慢病毒Lenti-siRyR2感染的小鼠心肌细胞中RyR2在mRNA水平基因抑制率达90%以上。结论成功构建了表达RyR2-shRNA的重组慢病毒表达载体,并在小鼠心肌细胞中实现有效的基因沉默效应。     

Egr-1基因启动子介导肿瘤基因放疗的研究进展

Egr-1基因是一种即刻早期基因，其启动子可感受自由基、电离辐射等理化刺激，继而诱导Egr-1基因或其下游基因表达。对转染了Egr-1基因启动子启动的肿瘤杀伤基因的肿瘤细胞．实施局部照射可诱导基因表达，通过射线与基因的双重作用杀伤肿瘤。此方法既解决了外源基因靶向表达的难题，又降低了照射剂量．减少了正常组织的损伤。     

Heterologous HIV-based lentiviral/adenoviral vectors immunizations result in enhanced HIV-specific immunity

Viral vectors are considered as one of the major means for the induction of strong immune responses against recombinant antigens by genetic immunization. Among these, lentiviral vectors are particularly attractive vehicles, as they can infect a wide variety of cells and can transduce replicating as well as non-replicating cells. We have engineered VRX1023, an HIV-1-based lentiviral vector (LV) vaccine candidate, to deliver HIV-1 Gag, Pol and Rev antigens under control of the native LTR promoter. While VRX1023 has been shown to elicit strong cell-mediated and humoral immunity as a stand-alone vaccine, we report here its combination in a heterologous prime–boost approach. Its combination with an adenovirus serotype 5 (Ad5)-based vector in the mouse model increased the frequency and polyfunctionality of HIV-specific CD4+ and CD8+ T cells. Homologous prime–boost regimens induced high levels of anti-vector neutralizing antibodies in Ad5-immunized mice, whereas the VSV-G-pseudotyped VRX1023 LV elicited low levels of anti-lentiviral vector neutralization. In addition, the heterologous prime–boost strategy resulted in a 5-fold reduction in Ad5-specific vector neutralization as compared to Ad5 homologous immunization.     

Novel adenovirus vaccine vectors based on the enteric-tropic serotype 41

Replication-defective adenovirus vectors, primarily developed from serotype 5 (Ad5) viruses, have been widely used for gene transfer and vaccination approaches. Vectors based on other serotypes of adenovirus could be used in conjunction with, or in place of, Ad5 vectors. In this study, Ad41, an enteric adenovirus usually described as 'non-cultivable' or 'fastidious,' has been successfully cloned, rescued and propagated on 293-ORF6 cells. The complementation capabilities of this cell line allow generation of Ad41 vectors at titers comparable to those obtained for Ad5 vectors. Mice immunized with an Ad41 vector containing an HIV envelope (Env) gene mounted anti-Env cellular and humoral immune responses. Ad41-Env vectors appear to be particularly attractive when used in heterologous prime-boost regimens, where they induce significantly higher cellular immune responses to HIV-Env than Ad5-based regimens. Ad41-based constructs are attractive vaccine vectors alone or in combination with Ad5 adenovectors, since each vector type can provide circumvention of pre-existing immunity to the other.     

Apoptosis of antigen-specific CTLs contributes to low immune response in gut-associated lymphoid tissue post vaccination

The gut-associated lymphoid tissue (GALT) represents a major reservoir of HIV in infected individuals. Vaccines can induce strong systemic immune responses but these have less impact on CD4 T cells activity and numbers in GALT. In this study, we vaccinated mice with an adenovirus vector that expressed the envelope gene from HIV and observed immune responses in the peripheral blood, spleen, liver, mesenteric lymph nodes, and Peyer's patches. We found that (1) the number of HIV-specific CD8 T cells was dramatically lower in GALT than in other tissues; (2) the programmed cell death protein-1 (PD-1) was expressed at high levels in HIV-specific CD8 T cells including memory T cells in GALT; and (3) high levels of HIV-specific CD8 T cell apoptosis were occurring in GALT. These results suggest that contributing to GALT becoming an HIV reservoir during infection is a combination of exhaustion and/or dysfunction of HIV-specific CTLs at that site. These results emphasize the importance of developing of an effective mucosal vaccine against HIV.     

腺病毒介导的P53基因抗人胃腺癌增殖的研究

目的：研究腺病毒介导的野生型P53基因（Ad-P53）抗人胃腺癌细胞增殖的效果。方法：利用携带P53基因的复制缺陷型腺病毒(Ad-P53)感染人胃腺癌细胞株SGC-7901，以携带报告基因LacZ的同型载体（Ad-LacZ）作对照，MTT法测定及克隆形成实验分析P53基因对胃腺癌细胞的体外抑瘤效应。用流式细胞技术分析P53基因转染后对细胞周期的影响以及引起凋亡情况，并用电镜观察细胞的超微结构变化。结果：野生型P53基因转移对SGC-7901具有明显的抗增殖作用。Ad-P53感染SGC-7901后细胞生长明显被抑制，抑制率达88.73％（MOI为200，7d）。克隆形成能力也明显受到抑制，当MOI分别为50，100，200时，克隆形成数为22，0，0。流式细胞计数分析显示Ad-P53能引起SGC-7901细胞发生周期阻滞和细胞凋亡，凋亡百分率达71.4％（MOI为200，7d）。电镜观察结果也显示细胞出现凋亡的特征性形态改变。结论：腺病毒成功介导野生型P53基因转移入人胃腺癌SGC-7901细胞中，野生型P53基因有显抑制胃腺癌细胞增殖的作用。其表达可以诱导肿瘤细胞的凋亡及细胞周期阻滞。     

Venezuelan equine encephalitis virus vectors expressing HIV-1 proteins: vector design strategies for improved vaccine efficacy

A live virus vaccine vector has been constructed from a molecularly cloned attenuated strain of Venezuelan equine encephalitis virus (VEE). High levels of foreign protein expression are regulated by an additional copy of the 26 S viral subgenomic RNA promoter. The position of this additional promoter and foreign gene in the VEE genome was predicted to have a major influence on expression level of the heterologous protein. Two sites in the genome were tested to determine the optimal site for expression of the matrix/capsid (MA/CA) coding region of human immunodeficiency virus (HIV-1). One vector contained the additional promoter and the MA/CA genes immediately downstream of the VEE E1 gene at the 3' end of the genome. In the second vector, the additional promoter was introduced immediately upstream from the authentic 26 S subgenomic promoter. Significantly higher levels of MA/CA were expressed from the downstream vector compared to the upstream vector. However, the stability of expression for both vectors was similar following passage in baby hamster kidney cells (BHK) cells. In BALB/c mice, the two vectors elicited similar levels of cellular immune responses to MA/CA as determined by bulk cytotoxic T-lymphocyte assays and precursor frequency analysis, but the humoral response induced by the downstream vector was significantly stronger. At 11 months post boosting with the downstream vector, serum antibody levels against HIV MA/CA were undiminished, and MA/CA specific CTLp were detectable in all mice tested. These findings suggest that VEE vectors can be optimized to elicit strong, balanced and long-lived immune responses to foreign viral proteins.