重组腺相关病毒(rAAV)介导的人凝血因子Ⅸ高活性突变衍生物在血友病B基因治疗研究中的应用

采用定点诱变技术, 将R338A点突变引入人凝血因子Ⅸ基因, 并构建于AAV载体上, 以rHSV/AAV杂合辅助病毒系统介导制备rAAV-hFIX重组病毒, 然后, 经肌肉注射对血友病B小鼠进行治疗实验, 观察该突变基因在小鼠体内的表达、活性以及机体对该突变衍生物的免疫反应与治疗效果. 结果显示: (i)治疗小鼠体内可检测到hFIX-R338A突变衍生物的存在, 并持续15周以上; (ii)突变衍生物hFIX-R338A在小鼠血浆中的凝血活性达(34.2±5.23)%, 显著高于野生型Ⅸ因子凝血活性((14.27±3.4)%); (iii)治疗小鼠体内未检测到抗Ⅸ因子突变衍生物抗体的存在; (iv)未发现与治疗相关的局部及全身性毒副作用. 提示: 以AAV介导人凝血因子Ⅸ高活性突变衍生物hFIX-R338A基因治疗可能成为替代野生型Ⅸ因子进行血友病B基因治疗的一个更为有效的途径.     

重组腺相关病毒(rAAV)介导的人凝血因子IX高活性突变衍生物在血友病B基因治疗研究中的应用

采用定点诱变技术,将R338A点突变引入人凝血因子Ⅸ基因,并构建于AAV载体上,以rHSV/AAV杂合辅助病毒系统介导制备rAAV-hFIX重组病毒,然后,经肌肉注射对血友病B小鼠进行治疗实验,观察该突变基因在小鼠体内的表达、活性以及机体对该突变衍生物的免疫反应与治疗效果.结果显示:(i)治疗小鼠体内可检测到hFIX-R338A突变衍生物的存在,并持续15周以上;(ii)突变衍生物hFIX-R338A在小鼠血浆中的凝血活性达(34.2±5.23)%,显著高于野生型Ⅸ因子凝血活性((14.27±3.4)%);(iii)治疗小鼠体内未检测到抗Ⅸ因子突变衍生物抗体的存在;(iv)未发现与治疗相关的局部及全身性毒副作用.提示:以AAV介导人凝血因子Ⅸ高活性突变衍生物hFLX-R338A基因治疗可能成为替代野生型Ⅸ因子进行血友病B基因治疗的一个更为有效的途径.     

Gene therapy for hemophilia B mediated by recombinant adeno-associated viral vector with hFIXR338A, a high catalytic activity mutation of human coagulation factor IX

A mutant human factor IX with arginine at 338 residual changed to alanine (hFIXR338A) by site-directed mutagenesis was introduced into AAV vectors, and a recombinant adeno-associ-ated viral vector containing hFIXR338A, prepared by rHSV/AAV hybrid helper virus system, was directly introduced to the hind leg muscle of factor IX knock out mice. The expression and the biological activity of human factor IX mutant, hFIXR338A, and the immune response against it in the treated mice were assayed and detected. The results showed that (i) the high-level expression of human factor IX mutant protein, hFIXR338A, has been detected in rAAV-hFIXR338A treated hemophilia B mice and lasted more than 15 weeks; (ii) the clotting activity of hFIXR338A in plasma is 34.2%± 5.23%, which is remarkably higher than that of (14.27%±3.4%) of wild type hFIX treated mice in the activated partial thromboplastin assay; (iii) immune response against factor IX R338A was absent, with no factor IX mutant protein (hFIXR338A) inhibitors deve     

Gene therapy for hemophilia B mediated by recombinant adeno-associated viral vector with hFIXR338A, a high catalytic activity mutation of human coagulation factor IX

A mutant human factor IX with arginine at 338 residual changed to alanine (hFIXR338A) by site-directed mutagenesis was introduced into AAV vectors, and a recombinant adeno-associ- ated viral vector containing hFIXR338A, prepared by rHSV/AAV hybrid helper virus system, was directly introduced to the hind leg muscle of factor IX knock out mice. The expression and the biological activity of human factor IX mutant, hFIXR338A, and the immune response against it in the treated mice were assayed and detected. The results showed that (i) the high-level expression of human factor IX mutant protein, hFIXR338A, has been detected in rAAV-hFIXR338A treated hemophilia B mice and lasted more than 15 weeks; (ii) the clotting activity of hFIXR338A in plasma is 34.2%± 5.23%, which is remarkably higher than that of (14.27% ± 3.4%) of wild type hFIX treated mice in the activated partial thromboplastin assay; (iii) immune response against factor IX R338A was absent, with no factor IX mutant protein (hFIXR338A) inhibitors development in the treated mice; and (iv) no local or systemic side-effects and toxicity associated with the gene transfer were found. It demonstrated the potential use of treating hemophilia B by recombinant adeno-associated viral vectors with mutant hFIXR338A gene, an alternative strategy for hemophilia B gene therapy to wild-type human factor IX.     

双线嗜粘液蛞蝓危害平菇的生态观察及防治的初步研究

双线嗜粘液蛞蝓在合肥地区一年只发生一代。在它们的生命周期中,从孵出到再产卵需299—325天,寿命约为340—375天。双线嗜粘液蛞蝓对平菇的危害程度随气温的变化而增减。气温在12.2—15℃时,每克蛞蝓一昼夜能取食0.2—0.35克平菇。双线嗜粘液蛞蝓的幼体对2%的甲酚皂或芳香灭害灵较敏感,根据该蛞蝓的生长发育规律,5月下旬至6月上旬为采用药物杀灭幼蛞蝓的最佳时期。     

蒙古沙冬青AmRD22基因的克隆及耐逆功能研究

脱水应答蛋白22(RD22)属于植物特有的BURP蛋白家族中的一个亚族,与耐逆性关系密切。该研究从中国西北荒漠区特有的强耐逆植物蒙古沙冬青克隆到一个RD22基因(AmRD22)的全长cDNA,并对其编码蛋白、表达模式和耐逆功能进行了研究。结果表明：(1)AmRD22蛋白(360 aa)的初级结构中含有RD22亚族共有的4个结构域,预测其定位于细胞壁;在功能已知的RD22蛋白中,AmRD22与大豆GmRD22的进化关系最近。(2)在室内培养的蒙古沙冬青幼苗中,AmRD22的表达受失水、高盐、低温和ABA胁迫的诱导显著上调,其中失水和低温胁迫诱导其上调幅度较大;在野外生长的蒙古沙冬青植株嫩叶中,其表达量从中秋至隆冬远高于其他季节。(3)转AmRD22基因拟南芥的耐盐性显著提高且Na⁺含量降低,其耐旱性也有较明显的改善且在种子萌发早期对外源ABA的敏感性降低,但耐冷性和耐冻性无明显变化。