Establishment of TaqMan Real-time Quantitative PCR Assay for Foreign Gene Copy Numbers in Transgenic Soybean

TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in...     

基于星点设计-响应面法优化藜麦凝集素的提取工艺

为进一步开发利用藜麦的生物活性,利用星点设计-响应面法优选藜麦凝集素的提取工艺。以凝血活性为响应值,在单因素试验的基础上设计响应面试验,优化藜麦凝集素提取条件。结果表明:藜麦凝集素的最佳提取条件为pH 7.2,液料比15∶1(mL∶g),提取温度17℃,提取时间14.3h,该条件下,凝集素血凝活性实为3.612 36。     

Porifera Lectins: Diversity,Physiological Roles and Biotechnological Potential

An overview on the diversity of 39 lectins from the phylum Porifera is presented, including 38 lectins, which were identified from the class of demosponges, and one lectin from the class of hexactinellida. Their purification from crude extracts was mainly performed by using affinity chromatography and gel filtration techniques. Other protocols were also developed in order to collect and study sponge lectins, including screening of sponge genomes and expression in heterologous bacterial systems. The characterization of the lectins was performed by Edman degradation or mass spectrometry. Regarding their physiological roles, sponge lectins showed to be involved in morphogenesis and cell interaction, biomineralization and spiculogenesis, as well as host defense mechanisms and potentially in the association between the sponge and its microorganisms. In addition, these lectins exhibited a broad range of bioactivities, including modulation of inflammatory response, antimicrobial and cytotoxic activities, as well as anticancer and neuromodulatory activity. In view of their potential pharmacological applications, sponge lectins constitute promising molecules of biotechnological interest.     

木豆凝集素提纯

木豆凝集素粗样先经伴刀豆凝集素-琼脂糖凝胶（Con A-Sepharose 4B）亲和层析吸附可去除部分杂质,但凝集素本身并未被亲和吸附,而是随缓冲液排出,再经交联葡聚糖（Sephadex G-100）凝胶过滤层析,得到纯化的木豆凝集素,得率为7．8％,凝血效价提高1280倍。     

棉铃虫C型凝集素抑制多角体病毒体内增殖

为探究C型凝集素（C-type lectin）在病毒传播中的可能作用,采用半定量RT-PCR法检测了棉铃虫Helicoverpa armigera不同C型凝集素在核型多角体病毒（nuclearpolyhedrosis virus,NPV）感染后的表达变化,并通过real-time qPCR和RNA干扰技术研究了NPV在棉铃虫体内的增殖状况及对棉铃虫致死率的影响。结果显示,在感染NPV后,棉铃虫血细胞C型凝集素Ha-lectin1和Ha-lectin5基因表达变化明显,其中Ha-lectin1在血细胞和脂肪体中的表达显著上调;注射dsRNA后,Ha-lectin1基因相对于actin的表达量均值由0.19下降到0.04,下降幅度达79%,显示Ha-lectin1基因被成功干扰。随着Ha-lectin1基因的沉默,病毒滴度由0.0015快速升高到1.95,棉铃虫的死亡率也随之增加到97.7%。表明C型凝集素能够抑制NPV在棉铃虫体内的增殖。     

Food deprivation causes rapid changes in the abundance and glucidic composition of the cutaneous mucous cells of Atlantic salmon Salmo salar L.

Cutaneous mucus is the first physical and chemical barrier of fish. This slime layer is secreted by mucous cells located in the epidermis and is mainly composed of glycoproteins that have their origin in the diet. Therefore, food deprivation can potentially change the abundance and glucidic nature of skin mucous cells, thus changing the mucus properties. To test this hypothesis, we conducted an experiment with Atlantic salmon, Salmo salar L. Changes in the number and glucidic nature of epidermal mucus cells were analysed using standard techniques. The outcome of this study shows that food deprivation caused a rapid decrease in the density of epidermal mucous cells in Atlantic salmon. Lectin histochemistry revealed a change in the presence and stainability of some sugar residues in the mucous cells of unfed fish compared with fed fish. Given that the primary reason for mucus secretion in fish is for protection against infections, we speculate that the changes in the mucus properties caused by nutritional stress may affect their disease resistance. This fact is particularly important for fish that spend a period of time deprived of food, either as a part of their natural life cycle, or as part of farming practices.     

甘露糖结合凝集素相关的丝氨酸蛋白酶2研究进展

甘露糖结合凝集素相关的丝氨酸蛋白酶(mannan binding lectin-associated serine protease,MASP)和甘露糖结合凝集素(mannan binding lectin,MBL)是补体激活的凝集素途径中的关键因子,发挥着至关重要的作用。MBL或纤维胶凝蛋白(ficolin)分子通过C端糖识别域(carbohydrate recognition domain,CRD)与病原微生物表面的糖类结构结合,其胶原样区(collagen-like region,CLR)与MASP结合,使无活性的MASP转换为活性MASP,从而激活补体凝集素途径。     

秀丽白虾4种C型凝集素基因的克隆、组织表达与进化分析

C型凝集素是一类Ca² 依赖活性的糖蛋白,在虾蟹类的免疫防御中发挥着重要作用。采用RT-PCR及RACE技术首次克隆了秀丽白虾C型凝集素家族4个基因( EmCTL-1、EmCTL-2、EmCTL-3、EmCTL-4)的cDNA全长。这4个C型凝集素基因的cDNA全长分别为1 390、1 119、1 364和1 299 bp,分别含996、969、 1 026和1 044 bp的开放阅读框,各编码332、322、341和347个氨基酸的蛋白。其氨基酸序列与罗氏沼虾相应的C型凝集素的相似性分别为40%、37%、69%和73%, 与其他海水虾类的相似性在30%～41%之间。阳性选择模型分析显示虾类的C型凝集素基因在进化过程中经历了阳性选择。进化树分析表明,秀丽白虾的 C型凝集素基因与罗氏沼虾的亲缘关系最近,并且除EmCTL-4属于十足类C型凝集素亚群A外,其他均属于亚群C。组织表达分析表明,EmCTL-1主要在血液中表 达,EmCTL-2主要在性腺、血液和胸神经节中表达,而EmCTL-3和EmCTL-4则主要在精巢中表达。研究表明,秀丽白虾的C型凝集素基因具有较快的 进化特点,同时又具备凝集素行使免疫功能时必需的保守氨基酸残基及钙离子结合位点,精巢和血液是秀丽白虾C型凝集素基因主要的表达器官。     

三疣梭子蟹C-型凝集素的原核表达和活性检测

通过RT-PCR扩增三疣梭子蟹C-型凝集素（C-type lectin like-domain protein,CTLD）基因序列,将目的基因克隆入质粒pET32a,构建原核表达重组质粒pET32a-CTLD.将重组质粒转化入大肠杆菌BL21中,通过IPTG诱导CTLD的表达,对其表达产物进行SDS-PAGE和Western blot 检测.用His GraviTrap Ni亲和层析柱及超滤离心管纯化目的蛋白,并对其进行活性检验.结果表明,构建得到CTLD基因的原核表达载体,双酶切鉴定得到与预期片段相符的外源基因插入片段,经测序与目的序列完全一致;诱导表达出相对分子质量（M） 39 600的目的蛋白,经SDS-PAGE和Western blot检测,融合蛋白成功表达;重组C-型凝集素对兔的红细胞没有明显的凝集作用,但对小鼠红细胞的凝集效价是22,而对所选5种细菌的凝集作用存在差异,其中对大肠杆菌具有明显的凝集作用,而对溶藻弧菌、哈维氏弧菌、鳗弧菌和金黄色葡萄球菌均无凝集现象.本研究结果为进一步研究C-型凝集素在免疫防御中的作用与地位提供依据.     

益生菌增加IPEC-J2细胞表面半乳糖在抑制RV感染中的作用

轮状病毒通过识别细胞表面糖链附着于肠上皮细胞,一些肠道内常驻菌群可以修改细胞表面糖链。以3种益生菌上清孵育猪小肠上皮细胞IPEC-J2,猪轮状病毒(PRV)感染细胞。采用凝集素荧光技术测定各处理组IPEC-J2细胞表面半乳糖,检测对应轮状病毒滴度和RV含量变化。结果表明,对照组PRV TCID_(50)·0.1 mL~(-1)为10~(-7.15±0.14),干酪乳杆菌组TCID_(50)·0.1mL~(-1)为10~(-3.875±0.125),食淀粉乳杆菌PRV TCID_(50)·0.1 mL~(-1)为10~(-4.125±0.138),枯草芽孢杆菌PRV TCID_(50)·0.1 mL~(-1)为10~(-4.16±0.144)。3组益生菌上清组在RV感染48 h内,RV量显著低于未处理组。凝集素荧光标记表明,3株益生菌上清均可改变细胞表面半乳糖含量。干酪乳杆菌、食淀粉乳杆菌和枯草芽孢杆菌上清可增加IPEC-J2细胞表面半乳糖含量,提高细胞抗RV感染能力,阻断RV黏附,保护肠道上皮细胞。