抗冻蛋白结构基因片段的克隆

为了研究和开发利用抗冻蛋白或多肽,木文通过聚合酶链式反应（ＰＣＲ）合成了抗冻蛋白１１０ｂｐ的结构基因片段,然后将该基因片段克隆到大肠杆菌质粒载体Ｐ（Ｂｌｕｅｓｃｒｉｐｔ）Ⅱｋｓ＋／－上,获得了重组质粒,经酶切证明,获得的重组质粒中含有抗冻蛋白结构基因片段,以供该基因在大肠杆菌或酵母中表达抗冻蛋白。     

甲虫抗冻蛋白热滞活性的二维吸附结合模型

甲虫抗冻蛋白是一种具有规则结构的昆虫抗冻蛋白。在相同浓度条件下,甲虫抗冻蛋白比鱼类抗冻蛋白有更高的热滞活性,目前已成为人们重点研究的一类抗冻蛋白。根据甲虫抗冻蛋白的结构特点及其在冰晶表面的吸附模式,应用二维吸附结合模型计算分析了具有6￣11个β-螺旋(β-helix)结构片段的甲虫抗冻蛋白变体分子,得到了它们的热滞活性随溶液浓度变化的规律,特别是热滞活性与甲虫抗冻蛋白的β-螺旋结构片段数的关系。结果显示,抗冻蛋白在冰晶表面的覆盖度是一个影响其热滞活性的重要因素。     

昆虫抗冻蛋白的结构与生物学特性研究

抗冻蛋白(antifreeze proteins AFPs)是一类抑制冰晶生长的蛋白质,它能以非依数性形式降低溶液的冰点而对其熔点影响甚微,因而也被称作热滞蛋白。近几年来对于昆虫抗冻蛋白的研究取得了较快的发展,已有20多种昆虫抗冻蛋白被分离纯化。就昆虫抗冻蛋白的结构特征、生物学特性以及在农业、医学和食品工业等方面的应用进行介绍。     

昆虫抗冻蛋白的结构与生物学特性研究

抗冻蛋白(antifreeze proteins AFPs)是一类抑制冰晶生长的蛋白质,它能以非依数性形式降低溶液的冰点而对其熔点影响甚微,因而也被称作热滞蛋白。近几年来对于昆虫抗冻蛋白的研究取得了较快的发展,已有20多种昆虫抗冻蛋白被分离纯化。就昆虫抗冻蛋白的结构特征、生物学特性以及在农业、医学和食品工业等方面的应用进行介绍。     

昆虫抗冻蛋白: 规则结构适应功能

抗冻蛋白在环境温度低于体液熔点时能够结合到生物体内的冰核表面,通过限制冰核生长和抑制冰晶重结晶而保护有机体免受结冰引起的伤害。与其他生物抗冻蛋白比较,昆虫抗冻蛋白有很强的活性,结构上具有显著特征,如一级结构规律重复,超二级结构为β-螺旋,可与冰晶发生相互作用,具有TXT基序等。该文综述了近年来关于昆虫抗冻蛋白的结构以及分子生物学等方面研究的新进展,讨论了其结构与功能的关系。     

准噶尔小胸鳖甲融合抗冻蛋白活性的比较研究

为了比较准噶尔小胸鳖甲Microdera punctipennis dzhungarica原核表达的不同融合抗冻蛋白活性是否存在差异,分别利用重组表达质粒pGEX-4T-1-Mpafp5和pMAL-p2x-Mpafp5在大肠杆菌Escherichia coli BL21(DE3)中诱导表达融合抗冻蛋白GST-MpAFP5和MBP-MpAFP5,并利用亲和层析纯化蛋白。SDS-PAGE分析结果证明获得了两种高效表达纯化的GST-MpAFP5和MBP-MpAFP5融合蛋白。在浓度为0.5 mg/mL时,两种蛋白的热滞值分别为0.51℃和1.336℃,其溶液冰晶形态均为棱型。抗冻保护实验结果显示： 准噶尔小胸鳖甲两种融合抗冻蛋白均能够显著提高细菌在低温下的存活率,并且MBP-MpAFP5对细菌的保护效果显著高于GST-MpAFP5。研究结果对于准噶尔小胸鳖甲抗冻蛋白的应用具有重要的理论意义。     

低温对转抗冻蛋白基因罗非鱼影响的初步研究

本文阐述了逐渐降低水温对转抗冻蛋白基因罗非鱼生存影响的初步观察结果。用３０尾转抗冻蛋白基因罗非鱼,从２２℃开始,以２℃／ｈ下降至７℃,发现在９℃有６尾死亡,７℃又有１０尾死亡,有１４尾存活,但有不同表现,从存活１４尾鱼和死亡的６尾鱼中提取总ＤＮＡ,作原位杂交,有１０尾为阳性,再从这１０尾呈阳性鱼肝中分离总ＲＮＡ,作ＲＮＡ—ＤＮＡ杂交并未发现阳性结果,表明有外源ＤＮＡ的整合,但未发生转录或弱的转录,不过有部分转基因鱼表现出具有耐低温能力。     

抗冻蛋白的生物化学与抗冻作用机制

生物的抗冻作用已引起人们的广泛注意,海洋鱼类血液中含有Nacl和少量的Ca~+、K~+、尿素、糖和游离氨基酸而略微降低体液的冰点,但南北极严寒地区的海洋鱼类则主要依靠血液内所含的抗冻蛋白降低体液的冰点,减少冰晶的生长速度,以防止因体液冻结而致死。     

比较三种防冰晶法对切片保存效果的影响

目的 比较冰冻切片技术中几种防冰晶方法对切片保存效果的影响。方法 分别使用液氮骤冷组织、高渗透压脱水法、单纯OCT胶包埋等几种方法后行冰冻切片、HE染色后,分别于当天、1、3、6个月后比较其染色效果,选出最佳保存方法。结果 单纯OCT包埋法在1个月后染色明显变浅,冰晶数量最多,而在3、6个月后染色基本接近背底色,冰晶面积已占据近半个视野,组织结构破坏极其严重;高渗透压脱水法在1个月后染色深度及冰晶数量上无明显变化,但3、6个月后则明显变浅,冰晶数量明显增加;而低温骤冷法在染色后的几个月,染色深度及冰晶的数量均无明显的变化,结构基本维持染色当天的状态。结论 低温骤冷法是这三种防冰晶法中最利于保存切片的方法。     

昆虫对低温的适应——抗冻蛋白研究进展

昆虫抗冻蛋白的研究主要在几种昆虫中展开,到目前为止已有二十多种昆虫抗冻蛋白被分离纯化。本文综述了关于昆虫抗冻蛋白的结构、组成、生物学活性及功能等方面的研究进展。昆虫抗冻蛋白的二级结构为β折叠和β转角,在其特殊的氨基酸序列结构中,半胱氨酸形成的二硫键对稳定其结构和活性起着很重要的作用。影响昆虫抗冻蛋白的因子,如活化蛋白及低分子量溶质的发现开辟了昆虫抗冻蛋白研究的新领域。     

Antifreeze proteins in higher plants

Overwintering plants produce antifreeze proteins (AFPs) having the ability to adsorb onto the surface of ice crystals and modify their growth. Recently, several AFPs have been isolated and characterized and five full-length AFP cDNAs have been cloned and characterized in higher plants. The derived amino acid sequences have shown low homology for identical residues. Theoretical and experimental models for structure of Lolium perenne AFP have been proposed. In addition, it was found that the hormone ethylene is involved in regulating antifreeze activity in response to cold. In this review, it is seen that the physiological and biochemical roles of AFPs may be important to protect the plant tissues from mechanical stress caused by ice formation.     

The physico-chemical characterization of a boiling stable antifreeze protein from a perennial grass (Lolium perenne)

We have characterized a cold-induced, boiling stable antifreeze protein. This highly active ice recrystallization inhibition protein shows a much lower thermal hysteresis effect and displays binding behavior that is uncharacteristic of any AFP from fish or insects. Ice-binding studies show it binds to the (1 0 1 0) plane of ice and FTIR studies reveal that it has an unusual type of highly beta-sheeted secondary structure. Ice-binding studies of both glycosylated and nonglycosylated expressed forms indicate that it adsorbs to ice through the protein backbone. These results are discussed in light of the currently proposed mechanisms of AFP action.     

Ice restructuring inhibition activities in antifreeze proteins with distinct differences in thermal hysteresis

Antifreeze proteins (AFPs) share two related properties: the ability to depress the freezing temperature below the melting point of ice (thermal hysteresis; TH); and the ability to inhibit the restructuring of ice into larger crystals. Since the ‘hyperactive’ AFPs, which have been more recently discovered, show an order of magnitude more TH than previously characterized AFPs, we have now determined their activities in ice restructuring inhibition (IrI) assays. IrI activities of three TH-hyperactive AFPs and three less TH-active AFPs varied over an 8-fold range. There was no obvious correlation between high TH activity and high IrI activity. However, the use of mutant AFPs demonstrated that severe disruption of ice-binding residues diminished both TH and IrI similarly, revealing that that the same ice-binding residues are crucial for both activities. In addition, bicarbonate ions, which are known to enhance the TH activity of AFPs, also enhanced their IrI activity. We suggest that these seemingly contradictory observations can be partially explained by differences in the coverage of ice by TH-hyperactive and non-hyperactive AFPs, and by differences in the stability of AFP-bound ice under supercooled and recrystallization conditions.     

Anomalous high activity of a subfraction of polyvinyl alcohol ice blocker

Low molecular weight copolymers of polyvinyl alcohol (PVA) are known to be potent inhibitors of ice formation in solutions used for cryopreservation by vitrification, even at concentrations as low as one part per million. Concentrated aqueous solutions of these polymers tend to become turbid after preparation. Condensed particles causing turbidity were isolated from a commercially available PVA-based ice blocker (X-1000) and found to consist of a polymer subfraction that is especially effective at ice blocking. Fifty seven percentage (w/w) of ethylene glycol (EG) in distilled water and 0.025% of the condensate polymer showed similar stability against devitrification as 57% EG+0.1% ordinary X-1000. At higher concentrations, 56.9% EG+0.1% condensate polymer was as effective as 56% EG+1% ordinary X-1000. All solutions containing ice blocker showed much less devitrification during warming than a 57% EG control solution. The condensate polymer was found to be strongly self-associating and less water soluble than ordinary X-1000. The mean molecular weight of the condensate polymer was approximately 1400 compared to 2100 for ordinary X-1000. Proton NMR revealed no large chemical differences. Subtle differences in composition or stereochemistry, perhaps in local regions of molecules, must be responsible for the dramatic differences in physical behavior and ice blocking effectiveness of the condensate polymer.     

The presence and quantification of splenic ice in the McMurdo Sound Notothenioid fish, Pagothenia borchgrevinki (Boulenger, 1902)

Survival of some polar fishes is associated with high levels of circulating antifreeze glycoproteins (AFGPs). AFGP prevent ice growth giving rise to thermal hysteresis. The inhibiting action of AFGPs implies that polar fish contain ice to which AFGPs adsorb. Cryopelagic Pagothenia borchgrevinki, inhabiting the ice-laden waters of McMurdo Sound, Antarctica, were assayed for ice and ice was found on skin, gills, in the intestine, and in the spleen. Two methods used to assess the number of ice crystals in spleens gave comparable results (12.1 +/− 1.9 and 22 +/− 3.8 per spleen). Attempts were made to measure the rate of uptake of ice by P. borchgrevinki held in cages immediately beneath the sub-ice platelet layer in McMurdo Sound; uptake was sporadic. Introduction of ice into fish by spray freezing a small patch of the integument resulted in detection of splenic ice after 1 h, illustrating that a mechanism exists for ice transport from the periphery to the spleen. Splenic ice did not seem to be eliminated from fish held in ice-free water at − 1.6 °C for approximately two months. The relatively small number of splenic ice crystals and the slow rate of ice uptake suggest efficient ice barriers exist in P. borchgrevinki.     

A facile method for determining ice recrystallization inhibition by antifreeze proteins

Ice recrystallization, the growth of large ice crystals at the expense of small ones, stresses freeze tolerant organisms and causes spoilage of frozen foods. This process is inhibited by antifreeze proteins (AFPs). Here, we present a simple method for determining the ice recrystallization inhibition (RI) activity of an AFP under physiological conditions using 10microl glass capillaries. Serial dilutions were prepared to determine the concentration below which RI activity was no longer detected, termed the RI endpoint. For type III AFP this was 200nM. The capillary method allows samples to be aligned and viewed simultaneously, which facilitates RI endpoint determination. Once prepared, the samples can be used reproducibly in subsequent RI assays and can be archived in a freezer for future reference. This method was used to detect the elution of type III AFP from a Sephadex G-75 size-exclusion column. RI activity was found at the expected V(e) for a 7kDa protein and also unexpectedly in the void volume.     

Expression and localization of an ice nucleating protein from a soil bacterium, Pseudomonas borealis

An ice nucleating protein (INP) coding region with 66% sequence identity to the INP of Pseudomonas syringae was previously cloned from P. borealis, a plant beneficial soil bacterium. Ice nucleating activity (INA) in the P. borealis DL7 strain was highest after transfer of cultures to temperatures just above freezing. The corresponding INP coding sequence (inaPb or ina) was used to construct recombinant plasmids, with recombinant expression visualized using a green fluorescent protein marker (gfp encoding GFP). Although the P. borealis strain was originally isolated by ice-affinity, bacterial cultures with membrane-associated INP-GFP did not adsorb to pre-formed ice. Employment of a shuttle vector allowed expression of ina-gfp in both Escherichia coli and Pseudomonas cells. At 27 °C, diffuse fluorescence appeared throughout the cells and was associated with low INA. However, after transfer of cultures to 4 °C, the protein localized to the poles coincident with high INA. Transformants with truncated INP sequences ligated to either gfp, or an antifreeze protein-gfp fusion showed that the repetitive ice-nucleation domain was not necessary for localization. Such localization is consistent with the flanking residues of the INP associating with a temperature-dependent secretion apparatus. A polar location would facilitate INP–INP interactions resulting in the formation of larger aggregates, serving to increase INA. Expression of INPs by P. borealis could function as an efficient atmospheric dispersal mechanism for these soil bacteria, which are less likely to use these proteins for nutrient procurement, as has been suggested for P. syringae.