乙型肝炎病毒基因重组疫苗免疫儿童成人效果及阻断母婴传播的研究

以中试连续生产的6批乙肝基因重组疫苗(R—Hepavac—B)免疫儿童和成人,每剂10μg,3批苗按0、1、6月,另3批苗按0、1、2月方案接种。每批分别接种8～10岁儿童51～66人,6批共363人,18～20岁成人46～52人,6批共287人。同时用两批血源苗作对照,分别接种儿童共85人,成人84人。又用其中89—10批HBsHg含量为20μg/ml的疫苗,按20μg×3,0、1、6月方案接种HBsAg和HBeAg双阳性母亲所生婴儿36名,HBsAg阳性母亲的婴儿19名。结果显示,10μg重组疫苗3针后,无论儿童和成人其抗HBs抗体阳转率均为100％,而对照,儿童成人各有1例未阳转。儿童按0、1、6方案接种者,其抗体GMT为363.8～470.5mIU,按0、1、2方案接种者,为150.8～195.5mIU。前者高于血源苗对照,而后者持平。成人0、1、6方案GMT为189.4～247.2mIU,0、1、2方案为87.9～96.3mIU,均略低于血源苗对照.经复测,此批血源苗含量为15μg/ml,高于基因苗含量,可能是造成以上结果的原因。母婴阻断结果显示,双阳性母亲子女36人,3针免后86.1％(32/36)的婴儿获得保护。单阳性母亲子女19人全部获得保护。     

Mint3/X11gamma is an ADP-ribosylation factor-dependent adaptor that regulates the traffic of the Alzheimer's Precursor protein from the trans-Golgi network

beta-Amyloid peptides (Abeta) are the major component of plaques in brains of Alzheimer's patients, and are they derived from the proteolytic processing of the beta-amyloid precursor protein (APP). The movement of APP between organelles is highly regulated, and it is tightly connected to its processing by secretases. We proposed previously that transport of APP within the cell is mediated in part through its sorting into Mint/X11-containing carriers. To test our hypothesis, we purified APP-containing vesicles from human neuroblastoma SH-SY5Y cells, and we showed that Mint2/3 are specifically enriched and that Mint3 and APP are present in the same vesicles. Increasing cellular APP levels increased the amounts of both APP and Mint3 in purified vesicles. Additional evidence supporting an obligate role for Mint3 in traffic of APP from the trans-Golgi network to the plasma membrane include the observations that depletion of Mint3 by small interference RNA (siRNA) or mutation of the Mint binding domain of APP changes the export route of APP from the basolateral to the endosomal/lysosomal sorting route. Finally, we show that increased expression of Mint3 decreased and siRNA-mediated knockdowns increased the secretion of the neurotoxic beta-amyloid peptide, Abeta(1-40). Together, our data implicate Mint3 activity as a critical determinant of post-Golgi APP traffic.     

The origin of enantioselectivity in aldolase antibodies: crystal structure, site-directed mutagenesis, and computational analysis

Catalytic aldolase antibodies, generated by reactive immunization, catalyze the aldol reaction with the efficiency of natural enzymes, but accept a much broader range of substrates. Two separate groups of aldolase antibodies that catalyze the same aldol reactions with antipodal selectivity were analyzed by comparing their amino acid sequences with their crystal structures, site-directed mutagenesis data, and computational docking of the transition states of the aldol reaction. The crystal structure of aldolase antibody 93F3 Fab' at 2.5A resolution revealed a combining site with two lysine residues, including LysL89 that reacts to form the covalent enamine intermediate. In contrast, antibody 33F12 has one active site lysine, LysH93. The reactive lysine residues in each group of antibodies are differentially located on the heavy and light chain variable regions in pseudo-symmetric opposite orientations, but both within highly hydrophobic environments. Thus, the defining feature for the observed enantioselectivities of these aldolase antibody catalysts is the respective location and relative disposition of the reactive lysine residues within the active sites of these catalysts.     

Neuroprotective effects of purslane herb aquenous extracts against D-galactose induced neurotoxicity

In order to evaluate mechanisms of natural plant purslane herb aquenous extracts (PHAS) for neuroprotective, we assessed neuroprotective effects of PHAS at doses of 2.5, 5 and 10 mg/(kg day) on SD mice injected daily with D-gal (50 mg/(kg day)) by behavioral tests. PHAS-fed mice showed higher activity upon induction by new environmental stimuli, lower anxiety and higher novelty-seeking behavior in the open field tasks, and significantly improved learning and memory ability in step-through compared with D-gal-treated mice. We further examined the mechanisms involved in neuroprotective effects of PHAS on mouse brain. PHAS significantly increased superoxide dismutase (SOD) activity and decreased the malondialdehyde (MDA) level. Meanwhile, PHAS also could up-regulate telomere lengths and telomerase activity in PHAS-fed groups. Furthermore, we examined the expression of p21(waf1) and p53 mRNA and protein in mouse brain by western blot analysis and real-time RT-PCR. We found that p21(waf1)was down-regulated by PHAS without changing the expression of p53. The results of this study suggested that the PHAS might be a primary target of p21(waf1)and the neuroprotective effect of PHAS might be carried out through a p21(waf1)-dependent and p53-independent pathway.     

基于引物扩增受阻突变技术开发水稻耐冷基因CTB4a特异性分子标记

水稻(Oryza sativa)作为热带与亚热带起源的作物对低温敏感.对水稻种质进行耐冷性鉴定,能筛选出耐冷性强的种质,发展耐冷基因分子标记,能够有效鉴别种质中耐冷基因的基因型.本研究使用芽期4℃低温处理10d对41份水稻材料进行芽期耐冷鉴定,对品种的芽期耐冷能力进行评价,获得了参试材料中除了'昆明小白谷'之外的芽期耐冷性最强的品种'南特号'.对已克隆的耐冷基因CTB4a开发分子标记,能够辅助选择水稻的耐冷育种.水稻孕穗期耐冷基因CTB4a来源于'昆明小白谷',能够影响水稻抵抗低温的能力.参照公布的CTB4a序列信息,从中挑选出序列中的作用位点SNP(单核苷酸多态性,single nucleotide polymorphism),结合引物扩增受阻突变技术(Penta-primer amplification refractory mutation system,PARMS),用Primer 6.0设计引物,建立CTB4a基因荧光分子标记GM-CTB4a,使用荧光分子标记GM-CTB4a对41份水稻品种进行鉴定,使用酶标仪在'昆明小白谷'中检测到利用标记扩增产物中包含'昆明小白谷'特异SNP、T碱基引物携带的FAM荧光信号,在另外40份品种的扩增产物中检测到包含作用位点的C碱基引物携带的HEX荧光信号.本研究利用设计的分子标记,鉴定了 41份水稻品种的耐冷性和基因型.比对分析耐冷性和基因型鉴定结果,说明我们开发的分子标记GM-CTB4a特异性较强,具有实际应用价值.研究结果为利用水稻孕穗期耐冷基因CTB4a培育强耐冷水稻品种奠定坚实基础.     

蜜环菌遗传测定的单孢分离和培养方法

从平板中直接分离担子菌萌发孢子的单孢分离方法,主要利用自制的毛细管和显徽玻璃针等简单工具操作。该法简便快速,抗污染,获得单孢率高。同时报道一种改进型的担子菌互交不育性测定用培养基MEJA,与国内外目前常用的SR和MEA培养基相比,增加一项判断标准,使互交不育性和互交可育性的反应更加清晰可靠。     

痢疾菌苗研究进展

痢疾是全世界范围内流行的肠道传染病。随着分子生物学技术的发展,迄今已构建了新一代预防痢疾的疫苗,其中包括:具有侵袭力和不具侵袭力的单价口服活菌苗候选株;它们与大肠杆菌、伤寒沙门氏菌和血清型不同的痢疾杆菌组成的双价和三价菌苗候选株;以及以脂多糖和核糖体为基础的不经胃肠的组分菌。猴体和人体试验证明它们安全有效。预计在未来的10年中,将会有一个或几个安全有效的痢疾疫苗投放市场。     

志贺氏菌属弗氏2a染色体基因文库的构建

以λ噬菌体EMBL3作载体,用体外包装技术构建了志贺氏菌属弗氏2a染色体基因文库。该体外包装系统的包装效率达1.6×10~2pfu/μg DNA,重组噬菌体的产量达2×10~6pfu/μg DNA。对重组噬菌体进行了遗传分析,即分别对7个标记(leu,pro,his,arg,thr,purIaroA)作了测定。测得当噬菌体母液的效价为8.7×10~8pfu/ml时,带有以上标记的重组噬菌体的效价为5×10~4-8.2×10~5pfu/ml。这个令人满意的结果为尔后克隆志贺氏菌属弗氏2a染色体上的与群抗原3,4和型抗原Ⅱ有关的基因打下了基础。