酿酒酵母表达的rHBcAg颗粒的纯化和鉴定及其透射电镜观察

乙肝核心抗原(HBcAg)蛋白基因(C基因)在酿酒酵母中表达,表达产物经过分离和Sepharose CL-4B柱子的初步纯化.产物经SDS-PAGE和Western blotting鉴定为一分子量约21.5kDa的多肽.再经蔗糖密度梯度超离心和CsCl等密度梯度超离心等过程而被纯化.分管收集的超离心纯化产物经ELISA抗原活性检测和密度分析,可知ELISA反应强度较高的收集管中的颗粒密度主要分布在1.27g/mL和1.40 g/mL两个峰值处.将rHBcAg抗原活性最高的收集管合并,再经TEM观察,发现酵母表达的rHBcAg蛋白(核心蛋白)能自主装配成大小不同的两种核心颗粒,大颗粒直径约为30.1±2.4 nm,小颗粒直径约为21.5±3.3 nm.这表明,酿酒酵母表达的rHBcAg颗粒具有大小不同的二态性,其生物学意义还未明了,需进一步研究和探讨.     

HBcAg基因在甲醇型酵母中的表达、产物纯化及其性质的初步研究

为研究乙肝核心抗原蛋白(HBcAg)在甲醇型酵母(PichiaPastoris)中的表达和性质,用PCR方法将HBcAg基因(L)克隆到P.Pastoris胞内表达载体pPIC3k中,并利用电击和同源重组法,将重组质粒pPIC3kL转化感受态的甲醇型GS115酵母菌株。经过筛选得到阳性P.Pastoris重组子。重组菌株经甲醇诱导培养,表达产物的Western印迹结果表明,HBcAg蛋白能在甲醇型酵母(PichiaPastoris)中诱导表达,产物为一215kDa的蛋白。经蔗糖密度梯度超离心和CsCl密度梯度超离心纯化后,ELISA和密度测定结果表明重组HBcAg蛋白主要分布在密度为12576gml和13013gml的2个峰值处。电镜观察表明,该重组HBcAg蛋白能自主装配成大小不同的2种颗粒(即核心颗粒),大颗粒直径约34nm左右,小颗粒直径约30nm左右。同时,我们还观察到,该核心蛋白颗粒在体外可发生集聚现象。     

乙肝病毒preS抗原决定簇与核心抗原的融合表达

将乙肝病毒表面抗原的ｐｒｅＳ抗原决定簇片段与核心抗原进行融合,分别构建了在核心抗原中间对应第７５～８３位氨基酸之间的融合及在核心抗原羧端对应第１５６位氨基酸处的融合,并在ｔａｃ启动子的控制下于大肠杆菌中表达。表达产物经ＥＬＩＳＡ检测和ＷｅｓｔｅｒｎＢｌｏｔｔｉｎｇ分析,表明融合蛋白均被表达,其单体分子量大小与推算值一致．电镜观察和ＣｓＣｌ密度梯度超离心测定都表明融合蛋白能形成颗粒,其密度略小于天然的ＨＢｃ颗粒。初步纯化的融合蛋白免疫Ｂａｌｂ／ｃ小鼠;能产生高滴度的抗－ｐｒｅＳ１抗体,表明ＰｒｅＳｌ（２１～４７）在核心抗原ｅｌｌｏｏｐ区的融合能大大提高其免疫原性．     

含RGD修饰的病毒样颗粒递送ICG靶向肿瘤的研究

目的:获取含RGD靶向肽的乙肝核心病毒样颗粒,为药物靶向纳米递送系统提供一种新型载体。方法:将实验室前期构建测序正确的含RGD修饰的乙肝核心病毒重组质粒转化入大肠杆菌BL21(DE3)中,单因素分析及正交试验探究重组蛋白最适表达条件。在最适表达条件下扩培,收集菌体超声破碎后离心,采用凝胶过滤层析、离子交换和蔗糖密度梯度离心进行纯化,利用透射电镜对形成的RGD-HBc VLPs的形态及稳定性进行鉴定。纯化的RGD-HBc VLPs利用其体外自组装的特性,将光敏剂ICG装载到颗粒的内部,通过静脉注射到4T1乳腺癌荷瘤小鼠,探究重组RGD-HBc VLPs作为纳米递送系统的靶向性。结果:RGD-HBc VLPs在温度32℃、IPTG0.5mmol/L、诱导4h时以可溶性蛋白的形式得到高效表达。经蔗糖密度梯度离心纯化后纯度到达95%以上。透射电镜下观察纯化的RGD-HBc VLPs形态、大小均一,直径约为32nm,通过近红外荧光活体成像证实了RGD-HBc作为纳米载体的靶向性。结论:经表达和纯化后,RGD-HBc VLPs具有较高的表达量和大小均一的形态外貌,近红外荧光活体成像证实具有较好的靶向性,这不仅为肿瘤的可视化诊断提供一种快速、精准、方便的方法,而且为今后靶向免疫治疗提供一种新型载体。     

丙肝病毒核心蛋白与乙肝病毒核心抗原的融合表达及其性质研究

构建了丙肝病毒核心蛋白 ( 1～ 191)及其N端 ( 1～ 69)和 ( 1～ 40 )与乙肝病毒核心抗原 ( 1～ 14 4 )羧端的融合克隆 ,在大肠杆菌中进行了表达 .其表达产物B14 4C191,B14 4C69和B14 4C40同时具有乙肝核心抗原 (HBc)和丙肝核心蛋白 (HCc)的双重抗原性和免疫原性 .CsCl密度梯度超离心和电镜观察表明 ,融合蛋白能组装成颗粒 .比较等量的融合蛋白的抗原性和免疫原性后发现 ,融合的HCc长度对HBc的抗原性和免疫原性影响不大 .而B14 4C69和B14 4C40比B14 4C191免疫小鼠能产生更高的抗HCc抗体 .利用表达的融合蛋白建立了ELISA法 ,对人血清中抗HBc抗体和抗HCc抗体进行了检测 .     

呈现新型冠状病毒RBD抗原病毒样颗粒的表达与鉴定*

目的:以乙型肝炎病毒核心抗原HBcAg为载体,构建呈现新冠病毒刺突蛋白受体结合域的病毒样颗粒,并鉴定其免疫原性,为新冠病毒疫苗的开发提供新思路。方法:在乙型肝炎病毒核心蛋白氨基酸编码序列第78和81位插入新冠病毒刺突蛋白受体结合域(RBD),并通过柔性linker(G4S)3进行连接,序列优化后将融合基因克隆到原核表达载体pET-28a(+),转化表达菌Rosetta,在自诱导培养基中诱导表达,菌体破碎后经蔗糖密度梯度离心,透析浓缩的方法纯化病毒样颗粒。SDS-PAGE、Western blot、透射电子显微镜检测和鉴定VLPs。将制备的VLPs与佐剂等比例混合经皮下免疫BALB/c小鼠,ELISA检测小鼠血清中特异性抗体,分析该HBc-RBD VLPs的免疫原性。结果:在自诱导培养基中,大肠埃希菌可表达部分可溶的VLPs,经蔗糖密度梯度离心纯化后在透射电子显微镜下可以观察到病毒样颗粒的存在。动物实验表明HBc-RBD VLPs刺激小鼠产生了特异性抗体。结论:在原核表达系统中成功表达了展示RBD抗原的VLPs,并通过小鼠实验初步验证了免疫原性,为新冠病毒疫苗的研发提供了新方向。     

基于HPV16E7蛋白CTLs抗原肽的病毒样颗粒治疗性疫苗的构建

目的:构建呈递HPV16 E7 CTLs抗原表位的病毒样颗粒,并初步评价病毒样颗粒作为治疗性疫苗载体的潜能。方法:根据文献选择有效的HPV16 E7 CTLs表位,合成其正负链寡核苷酸序列,并通过退火形成双链DNA片段。将片段克隆于表达乙肝核心抗原的重组质粒p Thio His AHBc Ag,使抗原肽得以呈现于病毒样颗粒。重组菌经IPTG诱导后经SDS-PAGE鉴定目的蛋白表达。菌体破碎后经硫酸铵盐析法和蔗糖密度梯度离心进行纯化,并经高效液相凝胶过滤色谱和电镜鉴定病毒样颗粒的存在。制备的病毒样颗粒免疫接种了TC-1细胞的肿瘤模型小鼠,检测小鼠肿瘤大小。此外,在体外以抗原肽刺激脾细胞,以ELISA检测IFN-γ表达水平。结果:构建的三个重组表达质粒经酶切鉴定及测序分析证实构建正确。表达的重组蛋白大小与预期相符,并形成了病毒样颗粒。免疫小鼠后显示了抑制肿瘤增长的一定作用趋势。此外,抗原肽体外刺激促进了疫苗免疫小鼠脾细胞IFN-γ的表达,显示疫苗引起机体产生E7特异性的细胞免疫应答。结论:HBc Ag VLPs是有潜能的治疗性疫苗载体。     

密度梯度离心技术在分子生物学研究中的应用

密度梯度离心技术是随着分子生物学的飞速发展而建立起来的一种重要的实验技术,广泛应用于核酸,蛋白、酶等生物大分子和生物膜、细胞大颗粒,亚细胞颗粒的分离、纯化及分析。可以说七十年代分子生物学研究中许多重大的成果都和这种技术的应用分不开。密度梯度离心大致可分二大类,一类是速率区带密度梯度离心,这类离心主要是利用生物大分子或亚细胞颗粒不同的沉降常数,在离心后分布在不同的预先铺制好的介质密度区,从而达到分离、纯化的目的。离心密度介质主要用蔗糖也可用甘露糖、甘油,多聚糖类、中性硅胶油等。这类离心技术广     

金黄色葡萄球菌肠毒素C的原核表达、纯化及活性鉴定

目的：在大肠杆菌中表达金黄色葡萄球菌肠毒素C（SEC）,并对其活性进行检测。方法：以金黄色葡萄球菌基因组为模板扩增SEC基因,经测序正确后插入原核表达载体pBV220中,转化大肠杆菌DH5α后温度热诱导重组SEC表达;表达产物经阳离子柱纯化后,用ELISA鉴定其抗原性;通过观测表达产物对鼠脾淋巴细胞的刺激增殖情况,检测其超抗原活性。结果：构建了SEC-pBV220原核表达载体,在大肠杆菌中可快速、高效表达SEC蛋白,表达产物具有超抗原活性。结论：实现了SEC的原核表达。     

共表达构建呈现人白细胞介素-13抗原肽的Qβ噬菌体病毒样颗粒

目的:构建呈现人白细胞介素-13(interleukin-13,IL-13)抗原肽的Qβ噬菌体病毒样颗粒(virus-like particles,VLPs)疫苗。方法:将人IL-13抗原肽经基因重组插入Qβ噬菌体衣壳蛋白(CP)的C端。在BL21细菌中,经IPTG诱导CP及C端接有IL-13抗原肽的CP(CP/IL-13)同时表达。以硫酸铵沉淀及蔗糖密度梯度离心进行VLPs纯化及分析嵌合VLPs的存在,以HPLC分析VLPs纯度,以电镜观察颗粒形态。小鼠经皮下免疫VLPs后采集血清,以ELISA检测人IL-13特异性Ig G抗体水平。结果:重组蛋白CP与CP/IL-13获得成功表达,两者在密度梯度离心中有一致的、与QβVLPs相同的沉降行为,而CP/IL-13单独无Qβ颗粒行为。经纯化获得了高纯度颗粒,嵌合颗粒与Qβ颗粒形态相似。此外,该VLPs疫苗诱导小鼠产生了IL-13特异的抗体应答。结论:利用共表达策略可成功构建呈现人IL-13抗原表位的嵌合VLPs,为以主动免疫方式调控IL-13在疾病中的病理作用,提供了具有临床应用潜能的疫苗形式。     

Expression of HBcAg mutant with long internal deletion in Saccharomyces cerevisiae and observation of its self-assembly particles by atomic force microscopy (AFM)

An internally truncated C gene of adr hepatitis B virus core antigen with long internal deletion (aa81–aa116) (ΔHBcAg with 36aa truncation) was expressed in Saccharomyces cerevisiae and the products (ΔrHBcAg) were purified from a crude lysate of the yeast by three steps: Sephrose CL-4B chromatography, sucrose step-gradient ultracentrifugation and CsCl-isopycnic ultracentrifugation. Results of ELISA test and density analysis of CsCl-isopycnic ultracentrifugation indicated that the purified products (ΔrHBcAg protein) with HBeAg antigenicity mainly located at the densities of 1.23 g ml⁻¹. Observation and analysis of the purified ΔrHBcAg products by AFM indicated that the ΔrHBcAg (core) protein produced in S. cerevisiae could self-assemble into three or more size classes of core particles which exhibited a polymorphous distribution of ΔrHBcAg (core) particles. These different size classes of core particles mainly centred on the range whose mean diameter was from 10 nm to 48 nm, especially on the position of 11 nm, 15.6 nm and the range from 27 nm to 41 nm, respectively. Furthermore, the most number of core particles mainly centred on the range whose mean diameter was from 27 nm to 41 nm. These results above indicated that the truncated internal long fragment (aa81–aa116) probably had no effect on self-assembly of the HBcAg core particles which implied the internal length fragment (aa81–aa116) was not the sole domain for self-assembly of HBcAg dimer or the truncated HBcAg protein subunit formed the fresh interactive domain with each other. These initial results above by AFM analysis were very important for further research on the self-assembly, ultrastructure, subunit interaction and core internal deletion mutant (CIDM) function of HBcAg core particles.     

Ectogenesis and the case against the right to the death of the foetus

Ectogenesis, or the use of an artificial womb to allow a foetus to develop, will likely become a reality within a few decades, and could significantly affect the abortion debate. We first examine the implications for Judith Jarvis Thomson’s violinist analogy, which argues for a woman’s right to withdraw life support from the foetus and so terminate her pregnancy, even if the foetus is granted full moral status. We show that on Thomson’s reasoning, there is no right to the death of the foetus, and abortion is not permissible if ectogenesis is available, provided it is safe and inexpensive. This raises the question of whether there are persuasive reasons for the right to the death of the foetus that could be exercised in the context of ectogenesis. Eric Mathison and Jeremy Davis have examined several arguments for this right, doubting that it exists, while Joona Räsänen has recently criticized their reasoning. We respond to Räsänen’s analysis, concluding that his arguments are unsuccessful, and that there is no right to the death of the foetus in these circumstances.     

On the diversity of the Cladocera in the tropics

The mythical concept of an impoverished tropical cladoceran fauna is refuted. On a planetary scale, around half of the cladoceran species presently known occur exclusively in the tropics-subtropics, often with considerable restriction to particular geographical subzones. On a regional (political) scale, the situation is often unclear because of the continued fragmentary nature of studies, and because political units are not a good basis for biogeographical comparisons. At the finest level of resolution (lake-perlake comparisons), there appears to be an upper limit of c. 50 cladoceran species per individual lake. No significant difference between lakes in the temperate zone and in the tropics could be established here. Daphnia is largely absent from the tropics, but is replaced by more Sidids, Moinids, and Bosminids, such that the average cladoceran community in the limnetic zone of a tropical lake is not characterized by less species but rather by lower population densities. This, in turn, is considered a consequence of higher prevalent predation levels in the tropics.     

Tubular networks in the terminal endings of the visual receptor cells in the human,the monkey,the cat and the dog

Summary A tubular network was found in the terminal endings of the visual receptor cells in the human, the monkey (Macaca mulatta), the cat and the dog. These tubules are arranged in close groups in the vicinity of the synaptic lamellae and the invaginated dendrites. According to the form, diameter, density of the tubules and to the consistence of the network formed by them one can distinguish at these places an initial type (type I), a transitory (type II) and a vesicular one (type III). In the the type III branching, bizarre forms are frequent. The diameter of all the tubules reaches 500–600 Å, their density and walls being the same as in the synaptic vesicles.Similar networks also occur in the axons of the visual receptor cells of the monkey.

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Zusammenfassung In den Endigungen der Photorezeptorzellen von Mensch, Affe (Macaca mulatta), Katze und Hund kommen aus Tubuli bestehende Komplexe vor. Organellenartig in geschlossenen Gruppen angeordnet, liegen sie in Nähe der synaptischen Lamellen und der invaginierten Dendriten. An diesen Stellen kann man nach Form, Durchmesser, Dichte und Konsistenz der von den Tubuli gebildeten Komplexe drei Typen unterscheiden: 1. einen initialen (Typus I), 2. einen Übergangstypus (Typus II) und 3. einen vesiculären Typus (Typus III). In letzterem kommen häufig verzweigte, bizarre Formen vor. Der Durchmesser sämtlicher Tubuli erreicht 500–600 Å. Ihre Dichte und ihre Wand gleicht denen der synaptischen Vesikel.Ähnliche Komplexe fanden wir auch in den Axonen der Photorezeptorzellen vom Affen.

     

Assessing the Physiological State of the Fucoids from the Barents Sea Littoral by the End of the Polar Night

Methods of amperometry and potentiometric titration were used to follow dark respiration (DR) and apparent photosynthesis (AP) in the fucoids Ascophyllum nodosum (L.) Le Jol, Fucus vesiculosus L., and F. serratus L. from the Barents Sea littoral by the end of the 40-day-long polar night. The macroalgae were shown to manifest species-specific low rates of photosynthesis and respiration. However, in spite of their low photosynthetic status due to the effects of subzero temperature and prolonged low or zero illumination, the macroalgae have been able to restore DR and AP to the initial level already by the day 9; the ability to restore AP depended on the level of illumination. The study of the changes in the carbonate–bicarbonate system in the light and darkness demonstrated that the macroalgae grown in darkness, in contrast to those grown in twilight, could absorb bicarbonate in darkness; however, they lost this capacity after two-day-long illumination at an irradiance of 7 mol/(m² s). Bicarbonate uptake in darkness and the capacity to restore the systems of photosynthesis and respiration in fucoid cells are discussed in the context of algal energy metabolism under the polar night conditions.     

Arranging the elements of the potassium channel: the T1 domain occludes the cytoplasmic face of the channel

The voltage-gated potassium channel is currently one of the few membrane proteins where functional roles have been mapped onto specific segments of sequence. Although high-resolution structures of the transmembrane portions of three bacterial potassium channels, the tetramerization domain and the cytoplasmic ball are available, their relative spatial arrangement in mammalian channels remains a matter of ongoing debate. Cryo-electron microscopic images of the six transmembrane voltage-gated Kv channel have been reconstructed at up to 18 Å resolution, revealing that the T1 domain tetramerizes and is suspended below the transmembrane segments. However, the resolution of these images is insufficient to reveal the location of the third piece of the puzzle, the inactivating ball domain. We have used the aberrant interactions observed in a series of chimæric channels to establish that an assembled T1 domain restricts access to the cytoplasmic face of the channel, suggesting that the N-terminal ball and chain may be confined in the space between the T1 domain and the transmembrane portion of the channel.     

Feeding the vertebrate retina from the Cambrian to the Tertiary

The retina of the vertebrate eye is metabolically active and requires nutritive support. During the last 540 million years it has evolved into forms as complicated and nutritionally demanding as those found in avian or primate eyes. Diffusion from the choroid is generally able to supply the metabolic needs of thin retinae. However, when the thickness exceeds the limits of diffusion, structures are needed to supplement the vascular supply from the choroid. These supplemental nutritive devices include the choroidal gland, the falciform process and preretinal vascular plexus of fish, the conus papillaris of lizards, the pecten oculi of birds, the intraretinal vessels of mammals and a few novel systems that remain difficult to classify. These vascular systems are among the most variable features of the vertebrate eye. Here, we review classical and recent findings regarding such retinal nutrition systems, propose a three category classification for them based on histologic origins and speculate on the evolutionary forces which drove their development.