CpG序列及阳离子脂质体对猪带绦虫抗原基因免疫应答的影响

本实验将猪带绦虫抗原基因VTS76分别与pUC18质粒，重组pUC18-CpG质粒联合及阳离子脂质体包装后免疫小鼠，结果表明：pUC18和pUC18-CpG能显著提高免疫小鼠总IgG和特异性抗体水平，增加小鼠免疫细胞数量，增强淋巴细胞增殖活性及白细胞介素－2的诱生活性，其中，pUC18-CpG的免疫增强效果更显著，阳离子脂质体包裹具有减少质粒用量，提高免疫应答水平的作用。     

CpG序列和猪白细胞介素-6基因转染表达对猪囊尾蚴基因疫苗免疫影响的研究

以猪囊尾蚴VTS76抗原基因和质粒VPIL-6、pUC18-CpG免疫接种小鼠，检测了小鼠的体液及细胞免疫反应，结果发现，VPIL-6或pUC-CpG与VTS76共同免疫小鼠的特异性抗体滴度，IgG含量，淋巴细胞白细胞介素-2诱生活性、脾细胞增殖反应和血液免疫细胞数量显著高于VTS76免疫组和对照组，证明VPIL-6和CpG序列能显著增强动物对DNA疫苗的细胞和体液免疫反应，可作为高效的基因免疫增强剂。     

阳离子PLG纳米颗粒包裹对CpG序列免疫佐剂效应影响的研究

本试验制备了聚乙交酯丙交酯(PLG)纳米颗粒，比较了阳离子PLG纳米颗粒和脂质体作为载体包裹CpG分子对小鼠体液和细胞免疫反应的影响。结果发现，阳离子PLG纳米颗粒包裹CpG能显著提高免疫小鼠总IgG，增加小鼠免疫细胞数量，增强淋巴细胞增殖活性及白细胞介素—2的诱生活性。它同阳离子脂质体作为包裹分子的效应相似或较强，能显著提高裸CpG质粒的免疫增强活性。     

内含子对人凝血因子Ⅸ活体表达的增强作用

为了研究内含子在活体水平对凝血因子Ⅸ的表达增强作用，首先采用lacZ为报告基因，建立了阳离子脂质体SA介导的活体基因转移方法。将带有凝血因子Ⅸ（hFⅨ）cDNA的质粒pCMVIX和带有hFⅨcDNA及其内含子的小基因（minigene）的质粒pCMVi'Ⅸ用SA脂质体包裹，分别经尾静脉注射到Balb／c鼠体内，pCMVIX注射后未在血浆中检测到hFⅨ蛋白；而pCMVi'Ⅸ注射后在Balb／c鼠血浆中检测到hFⅨ蛋白，最高达到10．1ng／ml，持续28天。pCMVi'Ⅸ经二次注射，FⅨ表达水平最高达到43.9ng／ml，持续40天后仍可检测到hFⅨ基因的表达，结果表明，内含子在活体水平对凝血因子Ⅸ的表达有显著的增强作用。     

联合应用凋亡素基因、新城疫病毒HN基因及IL-18基因对黑色素瘤的抑制效应研究

以前的研究结果表明,来自于鸡贫血病毒的凋亡素基因、来自于鸡新城疫病毒的HN基因和IL-18基因具有不同的抗肿瘤效应,但上述基因抗肿瘤的机制不同,本研究在双启动子真核表达质粒pIRES中的CMV启动子下游插入凋亡素基因,IRES启动子下游插入IL-18HN嵌合基因,构建能同时表达三种基因的真核表达质粒pIRVP3IL-18HN。复制C57BL／6小鼠荷黑色素瘤模型,并将质脂体包裹的重组质粒进行瘤内注射,共注射3次,每次100μg质粒,拟观察凋亡素、新城疫病毒HN基因和IL-18基因以核酸疫苗的形式联合应用对小鼠黑色素瘤的抑制效应。结果显示pVIL-18HN、pVlL-18和pVVP3IL-18均有抑制肿瘤作用,但pIRVP3IL-18HN组抑瘤率高于pVIL-18HN组、pVIL-18VP3组、pVIL-18组和Hanks液对照组。研究结果提示,上述3种基因联合应用具有协同抑瘤效应。     

提高猪基因导入效率的研究

采用显微注射的方法，将ＰＯＭＴ／ＰＧＨ，ＰＳＶ４０／ＰＧＨ，ＰＯＭＴ／ＰＧＨ－Ｍｉｃｒｏｓａｔｅｌｌｉｔｅ－ＤＮＡ和ＰＭＨＲ等四种外源基因导和２５９２枚湖北白猪的受精卵，均获得了相应的转基因猪，基因导入的总效率为２．１％，对影响导入效率的几个因素进行了系统的实验，通过超数排卵，可以使供体母猪的获卵数提高１．１倍，在异体移植的情况下，每头受体以移入２０～２９枚注射基因的胚胎为宜；采用自体移植，在移入     

内含子对人凝血因子IX活体表达的增强作用

为了研究内含子在活体水平对凝血因子Ⅸ的表达增强作用，首先采用ｌａｃＺ为报告基因，建立了阳离子脂质体ＳＡ介导的活体基因转移方法。将带有凝血因子Ⅸ（ｈＦⅨ）ｃＤＮＡ的质粒ｐＣＭＶＩＸ和带有ｈＦⅨｃＤＮＡ及其内含子的小基因的质粒ｐＣＭＶｉ＇Ⅸ用ＳＡ脂质体包裹，分别经尾静脉注射到Ｂａｌｂ／ｃ鼠体内，ｐＣＭＶＩＸ注射后未在血浆中检测到ｈＦⅨ蛋白；而ｐＣＭＶｉ＇Ⅸ注射后在Ｂａｌｂ／ｃ鼠血浆中检测到ｈＦⅨ蛋白，     

小鹅瘟病毒VP3基因疫苗的构建及其诱导小鼠和鹅中和抗体的初报

PCR扩增小鹅瘟病毒（GPV）CHv株VP3基因并克隆入pMD 18-T-Simple载体,亚克隆正向插入到真核表达质粒pcDNA3．1（＋）CMV启动子下游多克隆位点,经PCR和Hind与Bandt I双酶切鉴定表明成功构建了GPVVP3基因疫苗（pcDNA3．1．GPV—VP3）。该疫苗以200μg/只肌注免疫Balb／c小鼠和鹅,同时分别设肌注PBS和pcDNA3．1（＋）作为对照,于免疫后第30、45、60、75、90、105天应用鸭胚原代成纤维细胞（DEF）进行微量中和试验检测小鼠和鹅血清抗100TCID50GPV的抗体效价。结果表明,pcDNA3．1-GPV-VP3免疫小鼠第30天可检测到抗GPV的中和抗体,抗体效价缓慢上升至90天达到高峰（平均1：135．8）后下降,105天仍高于75天;免疫鹅的中和抗体效价呈现与小鼠类似的规律,第90天达到高峰（平均1：98．5）。因此,pcDNA3．1-GPV-VV3具有良好的免疫原性,肌注免疫小鼠和鹅后能够诱发产生抗GPV的中和抗体,有进一步开展临床研究和应用的前景。     

猪胎盘冻干粉对小鼠免疫功能的促进作用

为探究猪胎盘冻干粉对小鼠免疫功能的影响,将110只6~8周龄的昆明小鼠随机分为空白对照、阳性对照、高剂量实验组、低剂量实验组,采用经口灌胃给药法,连续给药35 d后,测定各组小鼠的胸腺、脾脏等免疫脏器指数、腹腔巨噬细胞的吞噬百分率,和Con A刺激后外周血T淋巴细胞转化率。结果显示:猪胎盘冻干粉高剂量组显著提升小鼠脾脏指数(P0.05),但对胸腺指数影响不明显(P0.05);冻干粉所有剂量组的腹腔巨噬细胞吞噬活性及外周血T淋巴母细胞转化率均显著提高(P0.05),其中雄性组效果更明显,表明猪胎盘冻干粉具有增强小鼠机体免疫功能的作用。     

植物抗体基因工程：II．马铃薯Y病毒中和抗体cDNA基因文库的构建及含抗体轻链和重链基因阳性克隆的筛选

从大量繁殖的Ｄ７３杂交瘤细胞中提取制备其基因组ｍＲＮＡ，并以此为模板，经反转录途径获得基因组ｃＤＮＡ，随后将其插入到λｇｔ１１中，构建了马铃薯Ｙ病毒小鼠单克隆抗体ｃＤＮＡ基因文库。经免疫原位杂交，从该基因文库中筛选出３０个含抗体轻链基因和１０个含抗体重链基因的阳性克隆，进一步将其插入到ｎＧＥＭ－７Ｚ（＋）质粒中，经酶切分析和电泳检测，证实在获得的轻链和重链阳性克隆中各有一个质粒（Ｋ６和Ｇ９）具有较长的外源ＤＮＡ插入（分别为１Ｋｂ和１．６Ｋｂ），它们可能编码完整的抗体轻链和重链蛋白。     

脂质体对牙鲆淋巴囊肿病毒核酸疫苗免疫活性的影响

将淋巴囊肿病毒核酸疫苗pEGFP-N2-LCDV0.6kb用脂质体梭华-SoftastTM包裹后肌肉注射入牙鲆体内,通过测定牙鲆外周血、肠、脾脏和前肾的淋巴细胞增殖反应、呼吸爆发活性以及抗体产生水平,评价脂质体对pEGFP-N2-LCDV0.6kb免疫活性的影响.结果表明,脂质体可显著增强该核酸疫苗的免疫活性和提高保护率,可作为一种有应用前景的核酸疫苗佐剂.     

水产养殖动物核酸疫苗的研究与应用现状

介绍了鱼用核酸疫苗载体的构建、诱导的免疫反应机制、接种途径、佐剂的免疫促进作用及安全性等研究现状。     

Enhancement of Gene Transfer to Cervical Cancer Cells Using Transferrin- Conjugated Liposome

ABSTRACT

Transferrin-conjugated cationic liposome (T_f -DDAB liposome) was developed as a targeted gene delivery system by using heterobifunctional cross-linking agent, N-succimidyl-3-(2-pyridyldithio)propionate (SPDP), and gradient metrizamide ultracentrifugation method. Physico-chemical properties of T_f -liposome were determined by scanning/transmission electron microscopy (SEM/TEM) and dynamic laser-light scattering method (DLS) with a mean diameter of 584±15 nm. Gel retardation assay was performed using various DDAB:DNA ratios, and proved that the 6:1 weight ratio formulation gave the most neutralized complex. In vitro transfection was done in human cervical cancer cells, HeLa, and the transfection efficiency of T_f -liposome was found to be fivefold higher than that of unconjugated (plain) DDAB liposome and twofold higher than that of Lipofectin?. In conclusion, a target-oriented T_f -DDAB liposome was made successfully and proved to be very efficient in DNA delivery into the cervical cancer cells in culture.     

Simultaneous Quantification of Tumor Uptake for Targeted and Nontargeted Liposomes and Their Encapsulated Contents by ICPMS

Liposomes are intensively being developed for biomedical applications including drug and gene delivery. However, targeted liposomal delivery in cancer treatment is a very complicated multistep process. Unfavorable liposome biodistribution upon intravenous administration and membrane destabilization in blood circulation could result in only a very small fraction of cargo reaching the tumors. It would therefore be desirable to develop new quantitative strategies to track liposomal delivery systems to improve the therapeutic index and decrease systemic toxicity. Here, we developed a simple and nonradiative method to quantify the tumor uptake of targeted and nontargeted control liposomes as well as their encapsulated contents simultaneously. Specifically, four different chelated lanthanide metals were encapsulated or surface-conjugated onto tumor-targeted and nontargeted liposomes, respectively. The two liposome formulations were then injected into tumor-bearing mice simultaneously, and their tumor delivery was determined quantitatively via inductively coupled plasma mass spectroscopy (ICPMS), allowing for direct comparisons. Tumor uptake of the liposomes themselves and their encapsulated contents was consistent with targeted and nontargeted liposome formulations that were injected individually.     

牙鲆淋巴囊肿病毒主要衣壳蛋白基因片段真核表达载体的构建、表达与免疫效果评价

用淋巴囊肿病毒LCDV-cn感染牙鲆鳃细胞系FG-9307,提取细胞总RNA,用RTPCR法获得了LCDV-cn主要衣壳蛋白(MCP)0.6kb基因片段.将该LCDV-cn-MCP0.6kb片断克隆入真核表达载体pEGFP-N2,得到重组质粒pEGFP-N2-LCDV-cn-MCP 0.6.采用脂质体法将重组质粒转染入牙鲆鳃细胞系FEC,并进行瞬时表达.通过荧光显微镜观察和特异性RT-PCR检测,证实重组质粒pEGFP-N2-LCDV-cn-MCP 0.6kb已成功转染到FEC细胞,并得到了初步表达.将重组质粒肌注入牙鲆体内,检测牙鲆外周血、肠、脾脏、前肾和淋巴细胞的增殖反应、呼吸爆发活性及抗体产生水平.结果表明,构建的核酸疫苗pEGFP-N2-LCDV-cn-MCP 0.6可诱导牙鲆特异性体液免疫和细胞免疫,具有明显的免疫保护作用.     

A novel cationic liposome formulation for efficient gene delivery via a pulmonary route

The clinical success of gene therapy for lung cancer is not only dependent on efficient gene carriers but also on a suitable delivery route. A pulmonary delivery route can directly deliver gene vectors to the lung which is more efficient than a systemic delivery route. For gene carriers, cationic liposomes have recently emerged as leading non-viral vectors in worldwide gene therapy clinical trials. However, cytotoxic effects or apoptosis are often observed which is mostly dependent on the cationic lipid used. Therefore, an efficient and safe cationic lipid, 6-lauroxyhexyl lysinate (LHLN), previously synthesized by our group was first used to prepare cationic liposomes. Physicochemical and biological properties of LHLN-liposomes were investigated. LHLN-liposome/DNA complexes showed positive surface charge, spherical morphology, a relatively narrow particle size distribution and strong DNA binding capability. Compared with Lipofectamine2000, the new cationic liposome formulation using LHLN exhibited not only lower cytotoxicity (P < 0.05) but also similar transfection efficiency in A549 and HepG2 lung cancer cells for in vitro tests. When administered by intratracheal instillation into rat lungs for in vivo evaluation, LHLN-liposome/DNA complexes exhibited higher pulmonary gene transfection efficiency than Lipofectamine2000/DNA complexes (P < 0.05). These results suggested that LHLN-liposomes may have great potential for efficient pulmonary gene delivery.     

Polymeric Materials for Gene Delivery and DNA Vaccination

Gene delivery holds great potential for the treatment of many different diseases. Vaccination with DNA holds particular promise, and may provide a solution to many technical challenges that hinder traditional vaccine systems including rapid development and production and induction of robust cell‐mediated immune responses. However, few candidate DNA vaccines have progressed past preclinical development and none have been approved for human use. This Review focuses on the recent progress and challenges facing materials design for nonviral DNA vaccine drug delivery systems. In particular, we highlight work on new polymeric materials and their effects on protective immune activation, gene delivery, and current efforts to optimize polymeric delivery systems for DNA vaccination.