Objective To develop directly molecular evolution of nitrite oxido-reductase using DNA-shuffling technique because nitrobacteria grow extremely slow and are unable to nitrify effectively inorganic nitrogen in wastewat...Objective To develop directly molecular evolution of nitrite oxido-reductase using DNA-shuffling technique because nitrobacteria grow extremely slow and are unable to nitrify effectively inorganic nitrogen in wastewater treatment. Methods The norB gene coding the nitrite oxido-reductase in nitrobacteria was cloned and sequenced. Then,directed molecular evolution of nitrite oxido-reductase was developed by DNA-shuffling of 15 norB genes from different nitrobacteria. Results After DNA-shuffling with sexual PCR and staggered extension process PCR,the sequence was different from its parental DNA fragments and the homology ranged from 98% to 99%. The maximum nitrification rate of the modified bacterium of X16 by DNA-shuffling was up to 42.9 mg/L·d,which was almost 10 times higher than that of its parental bacteria. Furthermore,the modified bacterium had the same characteristics of its parental bacteria of E. coli and could grow rapidly in normal cultures. Conclusion DNA-shuffling was successfully used to engineer E. coli,which had norB gene and could degrade inorganic nitrogen effectively.展开更多
目的探讨中药复方提取物湘A-1（Hu Nan A-1,HNA-1）对猴免疫缺陷病毒（Simian immunodeficiency virus,SIV）慢性感染中国恒河猴胸腺输出功能的影响。方法采用SIVmac239病毒液对8只中国恒河猴进行感染16~21个月,随机分为对照组和治疗组...目的探讨中药复方提取物湘A-1（Hu Nan A-1,HNA-1）对猴免疫缺陷病毒（Simian immunodeficiency virus,SIV）慢性感染中国恒河猴胸腺输出功能的影响。方法采用SIVmac239病毒液对8只中国恒河猴进行感染16~21个月,随机分为对照组和治疗组,每组4只。治疗组给予HNA-1灌胃20 m L治疗,每日1次;对照组给予等体积生理盐水灌胃。两组给药2个月。观察两组一般情况、体重,采用实时荧光定量PCR方法检测两组血浆病毒载量,采用流式细胞法检测CD4比例及数量、CD4的初始亚群,实时荧光定量PCR法检测T细胞受体DNA重排删除环（TREC）,常规HE染色后观察胸腺组织的病理情况。分析胸腺组织病变情况与CD4细胞数、初始型CD4细胞数及TREC的关联性。结果治疗后两组体重、病毒载量、CD4比例绝对数比较,差异无统计学意义（P〉0.05）。对照组TREC改变倍数明显呈下降趋势,治疗组总体呈明显的上升趋势,两组间差异有统计学意义（P〈0.05）。两组动物的胸腺组织均可见结构破坏、粉红色无结构样物质填充、结缔组织增多、细胞密度降低、排列紊乱等病理改变,未见明显区别。TREC含量与初始型CD4去除极值后,呈正相关（r=0.926,P=0.001）;而初始型CD4数量与CD4数量呈正相关（r=0.961,P=0.005）。结论检测TREC含量来测定新胸腺迁出细胞数量可作为评价胸腺输出功能的检测方法,且HNA-1可增加胸腺的输出功能,而TREC、初始型CD4数量及胸腺组织病理情况之间存在一定相关关系,在感染晚期尤为明显。展开更多
基金This study was supported by the National High Technology Research and Development Program of China (863 Program) (No. 2001AA214191).
文摘Objective To develop directly molecular evolution of nitrite oxido-reductase using DNA-shuffling technique because nitrobacteria grow extremely slow and are unable to nitrify effectively inorganic nitrogen in wastewater treatment. Methods The norB gene coding the nitrite oxido-reductase in nitrobacteria was cloned and sequenced. Then,directed molecular evolution of nitrite oxido-reductase was developed by DNA-shuffling of 15 norB genes from different nitrobacteria. Results After DNA-shuffling with sexual PCR and staggered extension process PCR,the sequence was different from its parental DNA fragments and the homology ranged from 98% to 99%. The maximum nitrification rate of the modified bacterium of X16 by DNA-shuffling was up to 42.9 mg/L·d,which was almost 10 times higher than that of its parental bacteria. Furthermore,the modified bacterium had the same characteristics of its parental bacteria of E. coli and could grow rapidly in normal cultures. Conclusion DNA-shuffling was successfully used to engineer E. coli,which had norB gene and could degrade inorganic nitrogen effectively.
文摘目的探讨中药复方提取物湘A-1（Hu Nan A-1,HNA-1）对猴免疫缺陷病毒（Simian immunodeficiency virus,SIV）慢性感染中国恒河猴胸腺输出功能的影响。方法采用SIVmac239病毒液对8只中国恒河猴进行感染16~21个月,随机分为对照组和治疗组,每组4只。治疗组给予HNA-1灌胃20 m L治疗,每日1次;对照组给予等体积生理盐水灌胃。两组给药2个月。观察两组一般情况、体重,采用实时荧光定量PCR方法检测两组血浆病毒载量,采用流式细胞法检测CD4比例及数量、CD4的初始亚群,实时荧光定量PCR法检测T细胞受体DNA重排删除环（TREC）,常规HE染色后观察胸腺组织的病理情况。分析胸腺组织病变情况与CD4细胞数、初始型CD4细胞数及TREC的关联性。结果治疗后两组体重、病毒载量、CD4比例绝对数比较,差异无统计学意义（P〉0.05）。对照组TREC改变倍数明显呈下降趋势,治疗组总体呈明显的上升趋势,两组间差异有统计学意义（P〈0.05）。两组动物的胸腺组织均可见结构破坏、粉红色无结构样物质填充、结缔组织增多、细胞密度降低、排列紊乱等病理改变,未见明显区别。TREC含量与初始型CD4去除极值后,呈正相关（r=0.926,P=0.001）;而初始型CD4数量与CD4数量呈正相关（r=0.961,P=0.005）。结论检测TREC含量来测定新胸腺迁出细胞数量可作为评价胸腺输出功能的检测方法,且HNA-1可增加胸腺的输出功能,而TREC、初始型CD4数量及胸腺组织病理情况之间存在一定相关关系,在感染晚期尤为明显。
