正交试验优化乙醇回流提取积雪草苷的工艺研究

为了优化积雪草中积雪草苷的提取工艺,本研究采用乙醇回流法,以积雪液草苷含量为研究指标,在单因素试验的基础上采用正交试验设计,考察乙醇浓度、液料比、粉碎粒度以及提取时间等因素对提取结果的影响。结果表明,积雪草苷的最佳提取工艺条件为:乙醇浓度70%、液料比10∶1、粉碎粒度140目、提取时间6h,该工艺条件下积雪草苷的提取含量达到8.32mg·g-1。     

Therapeutic properties and pharmacological activities of asiaticoside and madecassoside: A review

Centella asiatica is an ethnomedicinal herbaceous species that grows abundantly in tropical and sub-tropical regions of China, India, South-Eastern Asia and Africa. It is a popular nutraceutical that is employed in various forms of clinical and cosmetic treatments. C. asiatica extracts are reported widely in Ayurvedic and Chinese traditional medicine to boost memory, prevent cognitive deficits and improve brain functions. The major bioactive constituents of C. asiatica are the pentacyclic triterpenoid glycosides, asiaticoside and madecassoside, and their corresponding aglycones, asiatic acid and madecassic acid. Asiaticoside and madecassoside have been identified as the marker compounds of C. asiatica in the Chinese Pharmacopoeia and these triterpene compounds offer a wide range of pharmacological properties, including neuroprotective, cardioprotective, hepatoprotective, wound healing, anti-inflammatory, anti-oxidant, anti-allergic, anti-depressant, anxiolytic, antifibrotic, antibacterial, anti-arthritic, anti-tumour and immunomodulatory activities. Asiaticoside and madecassoside are also used extensively in treating skin abnormalities, burn injuries, ischaemia, ulcers, asthma, lupus, psoriasis and scleroderma. Besides medicinal applications, these phytocompounds are considered cosmetically beneficial for their role in anti-ageing, skin hydration, collagen synthesis, UV protection and curing scars. Existing reports and experimental studies on these compounds between 2005 and 2022 have been selectively reviewed in this article to provide a comprehensive overview of the numerous therapeutic advantages of asiaticoside and madecassoside and their potential roles in the medical future.     

积雪草活性成分的分析方法及药理作用研究进展

就近年来国内外对积雪草的化学成分、提取纯化、分析检测方法和药理活性的研究进展作了综述,为积雪草的进一步开发利用提供科学依据。     

除草剂2,4-D特异性多克隆抗体的制备及鉴定

目的是制备特异性的2,4-D多克隆抗体.用活性酯和混合酸酐法将半抗原2,4-D分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)共价偶联;以2,4-D-BSA作免疫原免疫两只新西兰白兔;以2,4-D-OVA包被96孔酶标板,用间接酶联免疫吸附分析(ELISA)法检测抗血清效价和竞争性ELISA法检测2,4-D.结果显示2,4-D与BSA和OVA的偶联比率分别为32∶1和18∶1,抗血清效价＞6.4×105,在优化条件下2,4-D的最低检测限达37ng/mL.实验成功地制备了2,4-D特异性多克隆抗体,为其ELISA方法的建立提供了条件.     

Isolation and characterization of squalene synthase cDNA fromcentella asiatica (L) urban

We have cloned and characterized a gene for squalene synthase (SQS) fromCentella asiatica (L) Urban, a species that produces a large quantity of triterpene saponins such as asiaticoside and madecassoside. Its full-length cDNA clone was isolated by RACE PCR. The sequence ofpSQS contains an open reading frame of 1248 nucleotides, which code for 416 amino acids with a molecular mass of 47.3 kDa. Southern analysis revealed that one copy might exist in the C.asiatica genome. We also determined that 0.1 mM methyl jasmonate was sufficient to up-regulate those levels ofCaSQS mRNA.     

天胡荽属药用植物的积雪草苷与羟基积雪草苷的含量测定

目的:建立天胡荽属植物的积雪草苷和羟基积雪草苷HPLC含量分析的方法,为开发利用天胡荽属植物药用资源奠定基础。方法:以积雪草苷和羟基积雪草苷为指标成分,采用高效液相色谱法,以Symmetry C18(4.6 mm×250 mm,5μm)为色谱柱;乙腈-水(29∶71,v/v)为流动相;流速0.8 mL/min;柱温30℃;检测波长210 nm,外标法进行测定。结果:积雪草苷的回归方程为:Y=34 958X+154 822 R2=0.999 1(n=8),羟基积雪草苷的回归方程为:Y=114 318X-21 002 R2=0.999 2(n=8),结果表明线性关系与分离度良好,平均加样回收率为99.98%、100.53%,RSD为1.86%、2.31%。结论:该方法稳定性、重复性良好,可做为天胡荽属药用资源的评价方法之一,实验结果为野生与栽培资源的开发与利用奠定了基础。     

人copineV蛋白多克隆抗体的制备

目的：制备兔抗人copineV多克隆抗体。方法：将copineV N端423bp（626-1048bp）构建到原核表达载体pET28a（＋）,转化大肠杆菌BL21（DE3）,在IPTG诱导下进行蛋白表达;以镍柱纯化后的蛋白为抗原,与等体积佐剂混合后免疫家免3次;用ELISA和Western印迹检测抗血清,用（NH4）2SO4沉淀法初步纯化抗体。结果：表达并纯化了copineV N端蛋白,ELISA检测表明抗血清具有高亲和性,Western印迹检测表明抗体能特异性识别内源性和过表达的copineV。结论：制备了具有高亲和性和特异性的抗人copineV多克隆抗体。     

Effect of growth regulators on asiaticoside production in whole plant cultures of<Emphasis Type="Italic">Centella asiatica</Emphasis> (L) urban

We investigated the effects of growth regulators on whole-plant cultures derived from nodes ofCentella asiatica. A B5 liquid medium including 0.01 mg L^-1 2,4-D resulted in decreased growth and asiaticoside production. Among the cytokinins tested (TDZ, BA, zeatin, and kinetin), TDZ was the best supplement for the promotion of asiaticoside biosynthesis. To directly estimate this effect, we measured asiaticoside content in the leaf, the main organ for synthesis. The addition of TDZ did not affect asiaticoside accumulation. Nevertheless, our results suggest that treatment with exogenous TDZ may enhance the production of asiaticoside in cultures simply through an increase in biomass.     

抗谷胱甘肽S转移酶多克隆抗体的制备及特异性鉴定

目的:制备谷胱甘肽S转移酶(GST)的兔多抗,并鉴定该抗体的特异性。方法:用纯化的GST标签蛋白(纯度>98%)免疫新西兰大白兔,获得GST的兔抗血清,并经HiTrap rProtein A柱纯化获得高效价高特异性的抗体;用间接ELISA法检测抗体效价,Western印迹检测抗体的特异性,并与商业化抗体进行对比。结果:通过免疫法得到了GST的兔多克隆抗体血清,抗体效价达1∶1×106,经rProtein A柱纯化后获得了高效价高特异性的抗体,其高效高特异性已达商业化抗体水平。结论:获得了GST的高效价高特异性的兔多克隆抗体。     

An optimized biotechnological system for the production of centellosides based on elicitation and bioconversion of Centella asiatica cell cultures

Centella asiatica is a herbaceous plant of Asian traditional medicine. Besides wound healing, this plant is recommended for the treatment or care of various skin conditions such as dry skin, leprosy, varicose ulcers, eczema, and/or psoriasis. Triterpene saponins, known as centellosides, are the main metabolites associated with these beneficial effects. Considering the interest in these high value active compounds, there is a need to develop biosustainable and economically viable processes to produce them. Previous work using C. asiatica plant cell culture technology demonstrated the efficient conversion of amyrin derivatives into centellosides, opening a new way to access these biomolecules. The current study was aimed at increasing the production of centellosides in C. asiatica plant cell cultures. Herein, we report the application of a new elicitor, coronatine, combined with the addition of amyrin‐enriched resins as potential sustainable precursors in the centelloside pathway, for a positive synergistic effect on centelloside production. Our results show that coronatine is a powerful elicitor for increasing centelloside production and that treatments with sustainable natural sources of amyrins enhance centelloside yields. This process can be scaled up to an orbitally shaken CellBag, thereby increasing the capacity of the system for producing biomass and centellosides.     

果蝇心脏发育标记基因Kruppel的多克隆抗体制备

Kruppel在果蝇发育过程中起着重要的调控作用。为了进一步研究Kruppel的功能,需要制备Kruppel蛋白及其抗体.对已有的Kruppel序列进行分析,选取适当区域进行引物设计,从果蝇心脏cDNA文库中PCR扩增得到Kruppel部分编码区序列,并其连接到pET-28a载体上。将重组质粒（pET-28a-Kruppel）转化rosetta受体菌,通过IPTG（Isopropylβ-D-thiogalactoside）诱导表达融合蛋白,用镍柱进行亲和纯化。将纯化得到的融合蛋白免疫新西兰大白兔制备多克隆抗体,并用Western blot检测抗体的效价。     

果蝇心脏发育标记基因lbe的多克隆抗体制备

lbe是已经证明的心脏标记基因。为了深入研究lbe在心脏发育中的功能,需要获得lbe蛋白并制备其抗体。首先提取野生型成体果蝇的总RNA,反转录获得其cDNA文库,通过PCR克隆出lbe编码区序列,将其连接到pET-28a原核表达载体上。经酶切及测序鉴定后,质粒构建成功。将重组质粒（pET-28a-lbe）转化大肠杆菌菌株Rosseta,用IPTG诱导表达出融合蛋白,经Ni-IDA凝胶柱纯化后,最后将纯化的融合蛋白免疫新西兰大白兔制备lbe多克隆抗体,并用Western blotting检测抗体的效价和特异性。结果显示获得了lbe原核表达重组融合蛋白及高效价的特异性兔抗lbe多克隆抗体,为lbe功能的进一步研究奠定了基础。     

抗人Elp3 的N末端抗体制备及在染色质免疫沉淀中的应用

人 Elp3（human elongator protein 3, hElp3）具有组蛋白乙酰转移酶活性,是与延伸中的 RNA 聚合酶Ⅱ结合的 elongator 复合物的催化亚基,可参与组蛋白的乙酰化修饰与基因的转录延伸. Elp3 及其复合物功能异常与人类多种疾病相关. 为运用染色质免疫沉淀等手段深入研究 Elp3 功能,PCR 法克隆 pYES2-hElp3 质粒中编码 hElp3的N端亲水区段（1～69 氨基酸残基）,构建原核表达载体 pMXB10-hElp3-210,经 IPTG 诱导和几丁质柱纯化后,免疫兔制备多克隆抗体. ELISA 检测显示,该抗体有较高的效价(不低于1∶2　500).免疫印迹实验结果表明,该抗体可与纯化的及 HeLa 细胞中的 hElp3 蛋白特异性结合.运用该抗体对转入 elp3Δ菌株的人 Elp3 的染色质免疫沉淀实验结果表明,人 Elp3 可参与酵母 SSA3 基因的转录调控,这可能是人Elp3 能够部分补偿酵母 SSA3 基因延迟表达缺陷的原因.     

转录因子XBP1的融合表达、纯化及多克隆抗体的制备

人X盒结合蛋白 1(XBP 1)为一种转录因子 ,与多种肿瘤的发生、发展有密切关系 .XBP 1有2种剪切形式 ,即XBP 1S和XBP 1U .将这 2种剪切形式中的一段相同编码序列 (编码 82～ 14 7位氨基酸 )重组于谷胱甘肽S转移酶 (GST)融合蛋白表达载体pGEX KG中 ,构建成重组质粒pGST XBP 1(82～ 14 7位氨基酸 ) .将该重组质粒转化E .coliDH5α后 ,表达GST XBP 1(82～ 14 7位氨基酸 )融合蛋白 ,经谷胱甘肽 Sepharose 4B亲和层析获得纯化的融合蛋白 .用此融合蛋白免疫家兔制备多克隆抗体 .利用制备的抗体分别用Western印迹和免疫细胞化学检测XBP 1的 2种剪切形式在哺乳动物细胞中的表达 .结果表明 ,该抗体对XBP 1的 2种剪切形式均具有反应原性 ,效价高 ,特异性好 ,可以用于进一步研究XBP 1的功能     

柑橘衰退病毒多克隆和单克隆抗体的制备及检测效果分析

通过改进提纯方法获得了柑橘衰退病毒(Citrustristezavirus,CTV)的提纯液,其产量为1mg/100g植物组织。用CTV免疫大耳白兔,获得多克隆抗体,间接ELISA效价为1∶25600。用CTV免疫小鼠,经细胞融合、ELISA筛选和克隆化培养,获得18株能稳定分泌抗CTV单克隆抗体的杂交瘤单细胞株。对其中4株单克隆腹水抗体进行分析的结果表明,这些抗体的ELISA效价为1∶51200~1∶204800,其中2G和3H的抗体类型及亚类为IgG2a,1E和4H为IgG2b。用所制备抗体对不同来源柑橘样品的CTV检测结果显示,单克隆和多克隆抗体结合使用,采用三抗体夹心ELISA(TAS-ELISA)可以获得理想的检测效果,其特异性强、灵敏度高。同时发现所分析4株单克隆抗体对不同的CTV分离物鉴别能力存在差异,但有关这些CTV分离物的特性及其血清学关系还需进一步研究。     

斑马鱼基因ATF4多克隆抗体的制备

ATF4是含有bZIP结构域的ATF／CREB转录因子家族成员,对胚胎的发育以及细胞的增殖、分化有重要的调节作用。制备ATF4的多克隆抗体对于研究其在斑马鱼心脏发育过程中的作用有重要的意义。研究首先通过生物信息学方法,选择ATF4基因中特异性强、具亲水性的一段核苷酸序列（1017bp）,通过PCR扩增,将片段重组到原核表达载体pET-28a,然后转化入Rosetta菌株中。经测序鉴定正确后,用IPTG诱导表达融合蛋白,以该融合蛋白免疫小鼠,获得ATF4多克隆抗鼠血清。对该多抗血清抗体进行验证,具有很好特异性和较高效价,可以用作Western—blotting、免疫印迹等试验分析。     

人类新基因Bzw2的克隆、抗体制备及表达分析

Bzw2( basic leucine zipper and W2 domains 2)基因是人类心脏发育候选基因,它由bzip结构域和W2结构域构成.为了研究该基因在心脏发育过程中的作用,利用生物信息学信息克隆了人类Bzw2基因全长,将所得的片段插入到原核表达载体pGEXT-4T-1载体中,利用BL21菌株表达该重组...     

非洲爪蟾PAPC多克隆抗体的制备及其特异性鉴定

非洲爪蟾ParaxialProtocadherin(PAPC)是一个在爪蟾Spemann组织者特异表达的膜蛋白.它在爪蟾原肠运动阶段的汇聚延伸运动和体节发生阶段的体节边界形成,以及早期听泡的形态发生和细胞特化过程中都有重要的作用.为了研究PAPC基因在早期胚胎发育过程中的表达及其生物学功能,需要制备PAPC抗体.应用谷胱甘肽S-转移酶(glutathioneStransferase,GST)表达系统表达GST-PAPC融合蛋白,亲和纯化后用以免疫新西兰大白兔,获得PAPC多克隆抗体.免疫印迹分析发现,以1∶3000稀释的该多克隆抗体为一抗时,能够在转染了全长PAPC质粒的HEK293T细胞的蛋白质抽提物中,特异地识别出150ku的印迹条带.同时,GST-PAPC融合蛋白可以竞争性抑制该抗体对全长PAPC质粒转染细胞的蛋白质抽提物的特异性条带.用1∶500稀释的该抗体为一抗进行免疫荧光分析时,发现,PAPC多克隆抗体能够识别在HEK293T细胞中过表达以及爪蟾动物极细胞中过表达的PAPC蛋白,荧光信号定位在细胞膜上.免疫印迹分析证明,PAPC抗体能够识别爪蟾胚胎中内源表达的PAPC蛋白.     

GST/MKRN1融合蛋白的表达、纯化及多克隆抗体制备

目的:利用大肠杆菌BL21(DE3)表达GST/MKRN1融合蛋白,亲和层析分离纯化目的蛋白,进行动物免疫制备多克隆抗体。方法:MKRN1cDNA全长1449bp,编码482个氨基酸残基。将其基因片段克隆到pGEX-4T-1载体上,获得pGEX-4T-1-MKRN1原核表达重组质粒,转化大肠杆菌BL21(DE3),经IPTG诱导表达,在大肠杆菌表达系统中获得可溶性表达,经谷胱甘肽Sepharose4B介质填充的层析柱分离纯化蛋白,制备抗原免疫动物,得到pGEX-4T-1-MKRN1多克隆抗体。结果:ELISA结果显示血清抗体效价可达到1∶256000。通过Western免疫印迹及免疫细胞荧光分析,自制的多克隆抗体能特异地与MKRN1蛋白相互作用,可用于免疫细胞化学分析。结论:制备了效价和特异性良好的抗MKRN1多克隆抗体,经实验验证获得的免疫血清能够满足针对MKRN1的Western免疫印迹和细胞免疫组化检测的实验要求,为今后深入研究MKRN1表达的组织分布、细胞内定位及与端粒酶催化亚基(hTERT)之间相互作用的生物学意义提供了有用的实验工具。