体内电穿孔在质粒介导的基因转移及DNA免疫中的应用

目的 研究体内电穿孔技术对于质粒DNA介导的体内基因表达以及HIV-1 Env DNA疫苗免疫效果的影响.方法 将携带荧光素酶Luciferase基因的表达质粒p1.0-Luc和携带HIV-1 CN54 env基因的DNA疫苗质粒p1.0-gp1455m通过单独肌肉注射或肌肉注射后加电穿孔两种不同方法 注射小鼠.用IVIS(R)活体成像系统实时检测Luciferase报告基因在体内的表达情况.用ELISA检测HIV-1 Env特异的抗体反应,用IFN-γ ELISPOT检测HIV-1 Env特异的T细胞免疫反应.结果 体内电穿孔技术可以显著提高Luciferase在小鼠体内的表达水平,幅度达35倍.HIV-1 Env DNA疫苗免疫结果 显示,8μg质粒剂量电穿孔途径诱导的体液和细胞免疫应答强于40μg质粒剂量单纯肌肉注射组;体内电穿孔途径免疫2次与单纯肌肉注射途径免疫3次诱导的体液和细胞免疫应答水平相当.结论 体内电穿孔技术可以大幅度提高报告基因在体内的表达水平和DNA疫苗的免疫应答.     

体内电穿孔在质粒介导的基因转移及DNA免疫中的应用

Objective To investigate the effects of in vivo electroporation on plasmid mediated reporter gene expression and immunogenicity of DNA vaccine. Methods Luciferase expression plasmid was administered intramuscularly to BALB/c mice at 8μg and 40μg dosage level through injection with or without eletroporation Luciferase expression level in murine muscle was detected by IVIS imaging system 24 h after injection. DNA vaccine plasmid p1.0-gp1455m carrying codon-optimized env gene of CN54 strain ( HIV-1 CRF07_BC) was administered to mice at dosages of 8μg and 40μg through the two approaches mentioned above. Mice were immunized at week 0,2 and 4. Env-specific immune responses were detected at two weeks post the second and the third vaccinations. Env-specific antibody immune responses were determined by ELISA. Euv-specific cellular immune responses were determined by IFN-γ ELISPOT. Results Luciferase expression level in murine muscle was significantly increased as much as 35 folds through in vivo eletroporation. Results of ELISA and ELISPOT revealed that in vivo eletroporation could significantly enhance both the humoral and cellular immune responses induced by DNA vaccination. The responses induced by electrodelivered p1.0-gp1455m at 8 μg dosage were better than those induced by simple intramuscular injection with 40 μg of plasmid DNA. On the other hand, 2 injections followed by electroporation elicited comparable level of humoral and cellular immune responses with those induced by 3 injections without electroporation. Conclusion In vivo electroporation was capable of enhancing both the plasmid-mediated gene expression and immunogenicity of DNA vaccine.     

体内电穿孔对报告基因表达和HIV GagDNA疫苗诱导的免疫反应的影响

目的 研究体内电穿孔递送途径在小鼠模型中对于报告基因表达水平及HIV Gag DNA疫苗所诱导的免疫反应的影响。方法 构建荧光素酶（1uciferase）表达质粒p1．0-1ue，将其通过直接肌肉注射、肌注后以双针电极进行电穿孔、肌注后以钳式电极进行电穿孔等3种不同方法注射小鼠，72h后取注射部位的肌肉测定luciferase的表达情况。同时构建携带密码子优化的HIV-1 B′／C重组亚型CN54株gag基因的DNA疫苗质粒p1．0-gag，在10μg、100μg两个剂量水平上通过以上3种不同的方法免疫BALB／c雌性小鼠，ELISA方法检测Gag特异的抗体反应，ELISPOF方法和细胞内因子染色（intracellular cytokine staining，ICS）技术检测细胞免疫应答。结果 通过体内电穿孔可以使luciferase在小鼠肌肉中的表达水平显著提高，最大提高幅度达到66倍。Gag DNA疫苗免疫结果显示，电穿孔可以显著提高Gag特异的体液免疫应答，其中使用双针电极的效果要显著好于钳式电极，前者所诱导的抗体滴度比不使用电穿孔组提高可达28倍。但体内电穿孔对于Gag特异的细胞免疫应答并没有显著影响。结论 体内电穿孔（尤其是使用双针电极）可以大幅度提高报告基因在体内的表达水平和DNA疫苗诱导的抗原特异性体液免疫应答。     

表达HIV-1 gag-gp120嵌合基因的DNA疫苗在小鼠体内的免疫应答

目的 :检测表达HIV 1gag gp12 0嵌合基因的DNA疫苗在小鼠体内的免疫应答效果。方法 :将真核表达质粒pVAXGE肌肉注射BALB C小鼠 ,观察免疫小鼠脾T淋巴细胞亚群的数量、特异性CTL杀伤率及小鼠免疫后不同时间点血清中IgG抗体滴度。结果 :重组质粒pVAXGE免疫组小鼠脾淋巴细胞进行了增殖 ,脾特异性CTL杀伤率显著高于对照组 (P <0 0 1) ;小鼠免疫后于第 8周血清抗体达到最高。结论 :表达HIV 1gag gp12 0嵌合基因的DNA疫苗质粒可诱导BALB C小鼠发生免疫应答     

DNA介导免疫的研究进展

ＤＮＡ介导免疫是指将质粒ＤＮＡ导入动物组织后，质粒ＤＮＡ原位表达并产生特异的体液和细胞免疫应答，目前主要应用的基因导入方法是质粒ＤＮＡ直接注射骨骼肌，并可在质粒ＤＮＡ注射前应用再生诱导剂提高导入基因的蛋白表达水平以增强免疫效果，ＤＮＡ介导免疫多被应用于抗感染免疫研制成核酸疫苗。     

DNA介导免疫的研究进展

DNA介导免疫是指将质粒DNA导入动物组织后，质粒DNA原位表达并产生特异的体液和细胞免疫应答。目前主要应用的基因导入方法是质粒DNA直接注射骨骼肌，并可在质粒DNA注射前应用再生诱导剂提高导入基因的蛋白表达水平以增强免疫效果。DNA介导免疫多被应用于抗感染免疫研制成核酸疫苗。     

重组乙肝表面抗原真核表达质粒pVAX-S2S的构建及DNA免疫

目的为探索更安全的乙型肝炎病毒(HBV)DNA疫苗,构建编码乙肝表面抗原中蛋白的卡那霉素抗性真核表达质粒,并观察其诱导BALB/c小鼠产生体液免疫应答情况.方法采用限制性内切酶从重组的真核表达质粒pcDNA-S2S中分离出乙肝表面抗原中蛋白(preS2+S)基因片段,将其亚克隆于pVAX1真核表达载体,酶切鉴定,按不同剂量一次性肌内注射免疫小鼠,ELISA法检测小鼠血清抗-HBs.结果酶切鉴定重组质粒pVAX-S2S为正向插入的阳性克隆.HBVDNA疫苗(pVAX-S2S)高(100μg/只)、中(50μg/只)、低(10μg/只)三组剂量一次性免疫健康BALB/c小鼠,均能在2 w诱导抗-HBs产生,抗体效价随时间延长而增长.血清抗体水平比较,高剂量组(97.83±38.78)mU/ml较中剂量组(45.13±21.12)mU/ml、低剂量组(19.74±11.92)mU/ml差异均具显著性(P＜0.05),以后的4,8 w高、中剂量组间差别缩小,但两者较低剂量组差异均具显著性(P＜0.05)和非常显著性(P＜0.01).结论卡那霉素抗性的重组质粒pVAX-S2S能有效诱导正常小鼠产生体液免疫应答.     

钩端螺旋体DNA疫苗pcD-flaB在豚鼠体内的免疫保护性研究

目的观察钩端螺旋体DNA疫苗peD-flaB是否具有抗钩体交叉感染的保护作用。方法将纯化后的钩体DNA疫苗pcD-flaB100μg200μl肌肉注射豚鼠，共免疫2次，间隔7d，初次免疫后的第21天分别攻击感染赖型、临海型和波摩那型钩体，观察保护作用。结果该疫苗对3型钩体的保护率分别是88．89％、100％和100％，总保护率为96．3％，与对照组差异具有显著性。结论DNA疫苗pcD-flaB在豚鼠体内不但具有抗同型钩体(临海型)感染的作用，也具有较好的交叉保护作用。     

电穿孔质粒DNA转染人膀胱癌细胞系的应用研究

本文报道了用电穿孔方法将质粒ＤＮＡ导入人膀胱癌细胞的方法及效率，并对电穿孔过程中各参数与转化克隆形成率、细胞存活率以及某些与细胞转染有关的因素进行了研究与分析。     

电穿孔法提高恶性疟疾DNA疫苗免疫原性

利用体内电穿孔的免疫途径来研究不同的真核表达载体和免疫剂量对DNA疫苗M.RCAg-1和D10的免疫应答的影响.分别将M.RCAg-1和D10基因克隆到VR1012和pVAX1表达载体中,用质粒免疫小鼠,采用ELISA和IFA法比较这两种载体的免疫效果,并对基因疫苗免疫小鼠的剂量进行摸索.D10基因免疫大白兔,检测纯化...     

The effects of agouti-related protein gene transfer in vivo by electroporation in mice

In the present study, the cDNA encoding agouti-related protein (AGRP) gene known as an orexigenic factor was transferred in vivo to test whether food intake and body weight gain is improved in mice. When the expression plasmid of AGRP gene driven by mouse β-actin, pActAGRP, was transferred into leg muscle by electroporation, body weight of gene-transferred mice was significantly increased at 14 days and afterwards compared with that of control counterparts (p < 0.05). Likewise, daily food intake was also significantly higher in the AGRP gene-transferred mice than in the control mice at 4 days and afterwards (p < 0.05). A significant increase in serum AGRP concentration of the AGRP gene-transferred group was detected compared with the control group at 1 week (p < 0.01), but the difference quickly disappeared at 3 weeks. However, the hypothalamic NPY mRNA abundance of AGRP gene-transferred mice was significantly higher than that of the control mice at 3 weeks (p < 0.05). These results suggested that instead of hormone administration per se, in vivo AGPR gene transfer into skeletal muscle was found to mimic hormonal effects. The present methodology of in vivo gene transfer by electroporation might be useful to promote growth and food intake in farm livestock as well as experimental animals.     

一种自制电极鸡胚视顶盖转基因技术

目的建立一种高效鸡胚视顶盖电转基因技术,对自制电极进行评估。方法在鸡胚发育的第5天(E5),将0.5 g/L的p CAGGS-绿色荧光蛋白(GFP)质粒0.25~0.5μl准确地注射到视顶盖内,每组60个鸡胚,在电压18V、每次脉冲60ms、间隔100ms、电脉冲6次的条件下,以原装电极为对照,自制电极为实验组进行定时定位活体电转基因,电转后在E6~E12时观察胚胎成活率,取材后在体视荧光显微镜下观察报告基因GFP表达结果以此判断转染的效率。结果在鸡胚模型中,通过自制电极转染鸡胚视顶盖,24h后鸡胚成活率达到89%,在鸡胚发育到E12时存活率达到35%,与原装电极相比分别提高48.3%和600%个百分点。结论自制电极在鸡胚视顶盖电转过程对胚胎的损伤小,转染后胚胎成活率高,为鸡胚视顶盖电转基因提供一种高效的转染技术。     

体内电穿孔法转基因技术的应用

电穿孔法转基因是将外源基因通过电场作用导入动物目标组织或器官细胞中的技术.Neumann等[1]首次成功应用该技术,把外源DNA导人小鼠成纤维细胞.随后大最的实验证明,几乎所有类型的细胞,包括植物原生质体、动物初生细胞,以及不能用其他方法转染的细胞和组织,都可以成功地使用电穿孔技术进行基因转移.     

A novel plasmid DNA electroporation method allows transfection of murine DC

Under steady state conditions dendritic cells (DC) exert tolerogenic function, but acquire potent immunogenic function due to strong upregulation of costimulatory molecules and proinflammatory cytokines. In numerous studies the potential of modified DC to induce tolerance or immune reactions towards a distinct antigen has been demonstrated. However, DC are refractory to transfection with plasmid DNA by non-viral methods. In this study we have tested the suitability of a newly developed electroporation device to transfect immature murine bone-marrow derived DC (BM-DC). Transfected BM-DC expressed reporter molecules at considerable extent which renders this method suitable to perform all kinds of promoter studies. While electroporation did not alter the low allostimulatory capacity of immature BM-DC, it impaired the stimulation-associated increase in allostimulatory potency of transfectants. However, stimulated transfected BM-DC pulsed with myelin oligodendrocyte protein (MOG)-derived peptide induced proliferation of MOG-reactive CD4⁺ T cells as potently as did non-transfected MOG peptide-pulsed BM-DC. BM-DC transfected with an expression construct encoding MOG efficiently stimulated MOG peptide-specific T cell proliferation. Transfection of BM-DC with an IL-10 encoding expression construct resulted in high IL-10 expression and strongly diminished allogeneic T cell proliferation. Therefore, this method also allows to study functional properties of genetically altered DC.     

Application of in vivo electroporation to cancer gene therapy

Much intensive research has gone into the development of safe and efficient methods for the delivery of therapeutic genes. In vivo electroporation is a non-viral delivery protocol in which plasmid DNA solutions are injected into targeted tissues, followed by electric pulses (typically 100 V, 50 ms). In general, in vivo electroporation enhances gene expression in targeted tissues by 2-3 orders of magnitude, as compared to the injection of plasmid DNA solutions without electric pulses, and the tissue damage appears to be minimal. Among the other advantages of this technique are that it can safely be administered repeatedly, and it is simpler and more economical to use than viral vectors, especially in clinical cases. Using this approach, highly efficient gene transfer has already been achieved in muscle and liver as well as in tumors. In fact, gene therapies for cancer utilizing in vivo electroporation have been proved effective in a number of experimental murine tumor models. The therapeutic genes delivered in those cases were diverse including, for example, cytokine genes (IL-12) and cytotoxic genes (TRAIL), making possible a wide range of therapeutic strategies. Moreover, systemic antitumor effects were also observed, suggesting that this approach may be effective for the treatment of metastatic as well as primary tumors.     

应用鸡胚活体电转GFP示踪技术观察脊髓左右两侧神经元纤维投射

目的:探索鸡胚发育过程中,脊髓左右侧神经元经纤维之间的联系,为脊髓两侧神经纤维投射提供形态学基础;方法:采用鸡胚带壳开窗培养技术,待胚胎发育至第3天,通过活体电转基因,将pCAGGS-GFP质粒0.1～0.5μl准确注射到脊柱,在电压18 V、每次脉冲60 ms,间隔100 ms,电脉冲6次的条件下进行定时定位活体电转基因,电转后6小时开始到10天,分别收集胚胎,甲醛固定冰冻切片,DAPI染细胞核观察组织形态结构变化。结果:电转后6小时便可以观察到GFP的表达,24小时后可以看到GFP标记细胞纤维的投射,3天可以清晰的观察到转入GFP一侧脊髓的运动神经元纤维束投射到另外一侧脊髓。结论:电转GFP成功标记运动神经元纤维在脊髓左右两侧的投射。