Biosafety of DNA vaccines: New generation of DNA vectors and current knowledge on the fate of plasmids after injection

DNA vaccination has been widely studied to develop new, alternative, efficient and safe vaccines for humans and animals. Many efforts have been made to increase the immunising potential of these vaccines and three veterinary vaccines are now available on the market. Much work is also being dedicated to develop effective DNA vaccines for humans. However, this new vaccination technique raises issues concerning biosafety due to the nature of the vector, i.e. a DNA molecule that contains sequences of prokaryotic origin (e.g. genes for antibiotic resistance). This review describes the development of the new generation of DNA vectors that are partially or completely devoid of elements of prokaryotic origin and outlines the results of studies on the fate of plasmids after their injection in vivo.     

携带SKP2基因的shRNA表达质粒的构建与鉴定

目的:构建含人SKP2基因不同位点的一系列shRNA真核表达质粒，并进行酶切鉴定、测序。方法:设计合成3对SKP2基因干扰寡核苷酸序列，形成双链后将其依次连入带有U6启动子的pSIREN-RetroQ载体，构建成能产生SKP2短发卡RNA的质粒。结果:构建的核苷酸序列经鉴定构建成功，能成功的克隆入带有pSIKEN RetroQ的载体中。结论:成功构建并鉴定了针对人SKP2基因3个不同位点的shRNA的真核表达质粒，为SKP2为靶点的肿瘤基因治疗研究奠定了基础。     

系统性硬化病成纤维细胞单克隆Ⅰ型前胶原基因转录特性的研究

维细胞克隆所占的比例(50.0%)高于正常对照组(12.5%),SS组成纤维细胞克隆的平均COL1A1mRNA水平显著高于正常对照组(t=3.690,P<0.01).5个COL1A1上游转录调控序列中-174～+42 bp在SS组和正常对照组不同成纤维细胞克隆均具有最强启动活性.COL1A1转录调控序列的活性与成纤维细胞克隆的COL1A1mRNA水平呈正相关.结论 SS高胶原合成的成纤维细胞克隆的COL1A1基因在转录水平即被激活.     

生姜中一种新化合物的抗氧化活性

目的和方法:采用溶血实验,小鼠肝组织脂质过氧化物(MDA)测定,采用受辐射质粒构象改变等方法测定从生姜中分离得到的一新的环状二苯基庚烷类化合物(化合物1)的抗氧自由基损伤活性。结果:化合物1能显著抑制H₂O₂致人红细胞溶血作用(P＜0.01);明显抑制小鼠肝组织脂质过氧化物(MDA)的产生(P＜0.01);显著抑制受辐射质粒超螺旋构象百分率的下降(P＜0.05或P＜0.01)。结论:化合物1具有明显抗氧自由基损伤的作用。     

小鼠骨髓树突状细胞pcDNA3-hCEA转染疫苗的抗瘤作用

目的:将pcDNA3-hCEA转染小鼠骨髓树突状细胞 (DCs),观察其诱导BALB/c小鼠对CT2 6(hCEA⁺)的抗肿瘤免疫效应。方法:采用rmGM-CSF和rmIL-4体外诱导培养小鼠骨髓DCs;构建真核表达质粒pcDNA3-hCEA;并用lipofectamine将其转染DCs,制备DCs(pCDNA3-hCEA)疫苗;同时制备CT2 6(hCEA⁺);采用RT-PCR检测DCs(pCDNA3-hCEA)中CEAmRNA的表达,采用放射免疫法检测DCs(pCDNA3-hCEA)培养上清中CEA的含量;使用DCs(pCDNA3-hCEA)疫苗体外诱导小鼠同种异体T淋巴细胞靶向性杀伤。采用DCs(pcDNA3-hCEA)疫苗预防免疫BALB/c小鼠,观察其对于荷临界致瘤量CT2 6(hCEA⁺)小鼠的抗肿瘤效应。结果:经G₄₁₈筛选,DCs(pCDNA3-hCEA)有14 %的获得率;RT-PCR检测表明DCs(pCDNA3-hCEA)内CEAmRNA呈阳性表达;放射免疫法检测表明DCs(pCDNA3-hCEA)能微量表达人CEA;DCs(pCDNA3-hCEA)能够有效诱导CEA靶向免疫杀伤。DCs(pcDNA3-hCEA)疫苗延长荷CT2 6(hCEA⁺)瘤小鼠生存期1周至 4周。结论:编码肿瘤抗原基因的真核表达质粒转染的树突状细胞,能够诱导较强的特异性抗肿瘤免疫效应.     

Secretion of heterologous proteins by genetically engineered Streptococcus gordonii

The secretion of heterologous proteins by Streptococcus gordonii from genes present on plasmids or on the chromosome has been achieved with a novel recombination system. We have constructed an integration-mediated transformation system in oral streptococci to clone foreign DNA into either resident plasmids or the host chromosome. In this system, Escherichia coli plasmid pACYC184 was extensively modified and utilized to construct anchor, integration, and heterodimer plasmids for introduction of the foreign genes. In order to evaluate this system, we cloned the gene for cycloisomaltooligosaccharide glucanotransferase (CITase) isolated from Bacillus circulans T3040 into S. gordonii. A portion of the CITase gene devoid of its signal sequence was fused inframe to the signal sequence of the Streptococcus sobrinus gtfI gene. The CITase fused gene was then introduced into either a resident plasmid or the S. gordonii chromosome. The presence of the enzymatically active CITase in the culture fluids from plasmid-borne transformants was confirmed. These results indicate that the integration-mediated transformation system described is an effective means of engineering recombinant streptococci capable of secreting heterologous proteins.     

Nurrl基因shRNA表达载体的构建及其鉴定

目的构建针对小鼠核受体相关因子1(Nurr1)的短发夹RNA(shRNA)表达载体,为进一步体内外研究Nurr1基因的功能奠定基础。方法选择两段RNA干扰靶序列,在体外分别合成两段互补的寡核苷酸,退火后与线性化的pSilenCirele质粒连接、转化感受态细胞DH5α,扩增、纯化得到所需质粒,通过酶切后琼脂糖电泳以及基因测序鉴定其分子量及插人片段的序列。结果纯化质粒的大小为4．6kb,插入的寡核苷酸序列与设计的序列完全相符。结论成功构建了针对:Nurr1基因的shRNA的表达载体。     

Plasmid profiles demonstrate that an upsurge inSalmonella berta in humans in England and Wales is associated with imported poultry meat

Sixteen plasmid profile types have been identified in drug-sensitive isolates ofSalmonella berta isolated from humans and human food in England and Wales in the ten-year period 1981–1990. Since 1988 six profile types of epidemiological importance have caused infections in widelyseparated geographical areas and of these, four types have been identified inS. berta isolated from chicken carcasses imported from Denmark. The findings suggest that imported Danish poultry has substantially contributed to a recent upsurge ofS. berta in humans in England and Wales.     

质粒DNA对大鼠骨骼肌肌质网Ca2+转运的影响

目的：观察ＤＮＡ与骨骼肌肌质网蛋白结合后对肌质网功能的影响。方法：应用差速离心分离大鼠骨骼肌肌质网,并用４５ＣａＣｌ２作为示踪剂,观察质粒ＤＮＡｐｃＤＮＡ３和ｐｃＤＮＡ３／ＡＮＦ对肌质网钙转运功能的影响。结果：两种质粒均可明显增加肌质网Ｃａ２＋摄取,呈明显的量效关系,并且也可促进肌质网钙释放。结论：外源质粒ＤＮＡ可影响肌质网钙转运功能。     

小RNA干扰对高表达RegIα基因胃癌细胞株增殖的影响

目的:探讨小RNA干扰(small interfering RNA,RNAi)对胃癌细胞株RegIα基因表达的抑制作用及对细胞增殖和凋亡的影响。方法:RT-PCR检测6株胃癌细胞的RegIα基因mRNA表达水平;体外构建RegIα基因小RNA干扰的重组质粒,分别稳定转染RegIα高表达细胞株MKN45和AGS,并设空载体作为对照组;应用RT-PCR和Western blot测定转染后细胞RegIα基因及其蛋白的表达水平;应用MTT法和流式细胞仪检测RegIα小RNA干扰对细胞增殖抑制率和凋亡的影响。结果:RT-PCR显示:与空载体组相比,转染RegIα小RNA干扰质粒后,MKN45和AGS细胞的RegIαmRNA明显被抑制;Western blot结果显示:MKN45和AGS细胞的RegIα蛋白表达水平分别降低至空载体组的(44±4)%和(25±4)%;MTT结果显示:RegIα小RNA干扰后MKN45和AGS细胞增殖均受到抑制,凋亡率分别为(12.96±0.50)%和(11.59±1.10)%,与空载体组比差异有统计学意义(P<0.05)。结论:小RNA干扰可有效抑制胃癌细胞株中RegIα基因的表达,具有抑...