大熊猫及其近种活化素基因β_A亚基成熟肽序列的克隆分析及其在分类地位上的应用

借鉴互联网中已经克隆到的Activin基因 βA 亚基成熟肽序列 ,设计并合成 1对兼并引物 ,利用PCR技术从大熊猫、小熊猫、马来熊的基因组DNA中直接扩增目的基因片段 ,并分别克隆到大肠杆菌载体pBlueScript+ 当中 ,然后对培养产物进行序列测定。DNA序列分析表明 ,三种物种的Activin基因 βA 亚基成熟肽序列长度均为 35 9bp ,无内含子。基因片段编码一个含有 119个氨基酸残基的肽段。大熊猫、小熊猫、马来熊在该成熟肽核苷酸和氨基酸序列上表现出高度的同源性 ,其中核苷酸同源性为 93.9% ,氨基酸同源性高达 99%以上。此外 ,3种动物的核酸限制性酶切图谱也高度相似。与GenBank中收录的其他物种Activin基因 βA 亚基成熟肽序列相比较 ,显示此片段在处于不同进化程度的物种之间仍具有高度保守性。运用系统发育与进化树软件包PHILIP ,并结合克隆序列对大熊猫、小熊猫、马来熊进化与分类地位进行了探讨。采用不同的统计学分析方法 ,所得到的 3个物种系统发育进化树的拓扑结构完全一致。相比较而言 ,大熊猫与马来熊有着较近的亲缘关系 ,而小熊猫与上述两个物种的亲缘关系相对疏远。结果支持将大熊猫与马来熊归为熊科、而将小熊猫单列成科的学术观点。这是首次以生殖相关的核基因作为研究对象 。

Comparative investigation on spindle behavior and MPF activity changes during oocyte maturation between gynogenetic and amphimictic crucian carp

The spindle behavior and MPF activity changes in the progression of oocyte maturation were investigated and compared with cytological observation and kinase assay between gynogenetic silver crucian carp and amphimictic colored crucian carp. MPF activity was measured by using histone H1 as phosphorylation substrate. There were two similar oscillatory MPF kinase activity changes during oocyte maturation in two kinds of fishes with different reproductive modes, but there existed some subtle difference between them. The subtle difference was that the first peak of MPF kinase activity was kept to a longerlasting time in the gynogenetic silver crucian carp than in the amphimictic colored crucian carp. It was suggested that the difference may be related to the spindle behavior changes, such as tripolar spindle formation and spindle rearrangement in the gynogenetic crucian carp.

天然产物对树突状细胞免疫调节作用的研究进展

树突状细胞(dendritic cells,DCs)是连接先天免疫和获得性免疫的关键。在感染和过敏反应中,DCs作为专职抗原递呈细胞引起T细胞免疫应答,并维持自身抗原免疫耐受。DCs介导的T细胞免疫应答有益于感染性疾病以及癌症等的治疗,同时DCs诱导的免疫耐受在控制自身免疫疾病、过敏或炎性疾病中发挥作用。研究表明多种天然产物能够调控DCs的表型与功能,并在免疫疗法的临床应用中被证明有效。因此,利用天然产物调控DCs免疫功能将为开发有效且低成本的细胞治疗方法提供新的思路。对近年来关于天然产物调控DCs功能的研究进行综述,总结天然产物对DCs表型变化的影响及其对免疫应答和免疫抑制的调控作用。

小鼠Flt3配体的原核表达及其在不同亚型树突状细胞制备中的应用

目的原核表达小鼠Flt3配体,分析其在不同亚型DCs体外诱导中的应用潜能,为不同亚型DCs的深入研究提供实验资料。方法 PCR扩增flt3l基因,构建pCold-flt3l质粒,转化大肠埃希菌BL21（DE3）诱导表达并纯化。重组蛋白进行SDS-PAGE和Western blot分析,并用于小鼠骨髓源DCs的诱导制备,流式细胞术分析不同DC亚型相关分子标志以及CD40、CD80、CD86和MHCⅡ的表达,同时ELISA测定IL-12和IFN-α的表达。结果 SDS-PAGE结果表明目的蛋白成功表达,纯化后纯度达93.3%,Western blot结果表明其具备良好的免疫反应性。同时,该蛋白能诱导小鼠骨髓细胞分化为较高比例（约60%）的DCs,且包含cDCs（ CD11c＋CD45RA-）和pDCs（ CD11c＋CD45RA＋）亚型；LPS刺激后2种DC亚型均上调CD40、CD80、CD86和MHCⅡ的表达,且分泌较高水平的 IL-12和IFN-α。结论本研究成功获得重组小鼠Flt3配体蛋白,可用于制备具有良好生物学功能的cDCs和pDCs亚型,为下一步研究提供基础材料。

Enhancing polyphenol extraction from unripe apples by carbohydrate-hydrolyzing enzymes

The effects of process variables such as enzyme types, enzyme ratio, reaction temperature, pH, time, and ethanol concentration on the extraction of unripe apple polyphenol were investigated. The results indicated that Viscozyme L had the strongest effect on polyphenols extraction and was selected to study the polyphenol composition. The ratio of enzyme （Viscozyme L） to sobstrate （2 fungal beta-glucanase units （FBG）） at 0.02, reaction at pH 3.7, 50℃ for 12 h, and ethanol concentration of 70% were chosen as the most favorable extraction condition. Total phenolic content （TPC）, reducing sugar content （RSC）, and extraction yield increased by about 3, 1.5, and 2 times, respectively, compared with control. The contents ofp-coumaric acid, ferulic acid, and caffeic acid increased to 8, 4, and 32 times, respectively. The enzyme-aided polyphenol extraction process from unripe apples might be applied to food industry for enhancing bioactive compound production.