Insertion of foreign epitopes in HBcAg: how to make the chimeric particle assemble

Summary. Hepatitis B core antigen is one of the most promising protein carriers of foreign epitopes of various human and animal pathogens. Chimeric HBcAg particles can be used as effective artificial immunogenes. Unfortunately, not all chimeric proteins are able to be particulated. The dependence of correct or incorrect folding of chimeric proteins on physical and chemical properties of inserts was studied with the help of ProAnalyst, SALIX and QSARPro computer programs. We have found that insertion of amino acids with high hydrophobicity, large volume, and high β-strand index prevent self-assembling chimeric proteins. These factors are most important for the C-termini of inserts. Recommendations for obtaining correct folding of chimeric HBcAg particles have been given. Received August 8, 1999, Accepted September 26, 1999     

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.     

核糖体结合位点序列对大肠杆菌中HBcAg重组质粒表达的影响

用Bal-31外切酶调整乙肝病毒核心抗原(HBcAg)基因非编码区长度并克隆到质粒pKK 223-3中,获得了不同水平地表达HBcAg的重组质粒pKL系统。该系统所表达的HBcAg蛋白分子量为21000D。DNA序列分析发现高、中、低表达水平的3个重组质粒的SD序列到HBcAg基因的ATG之间的距离分别为12bp、13bp和19bp,高、低表达质粒的mRNA核糖体结合位点序列的二级结构分析显示,二者的自由能相差约3倍,且低表达质粒的mRNA的SD序列中的3个碱基及AUG中的3个碱基都参与配对,而高表达质粒只有SD序列中的2个碱基参与配对,表明SD序列到ATG的距离及mRNA的二级结构均在HBcAg的表达调控中起重要作用。     

广州地区GII.4 Sydney 2012[P31]型诺如病毒GZ2013-L10毒株病毒样颗粒功能和免疫原性鉴定

【背景】诺如病毒(Norovirus,NoV)是全球范围内引起急性胃肠炎暴发的主要病原体之一,其中GII.4型通过不断变异在人群中持续存在并占据诺如病毒感染的主导地位,尤其GII.4 Sydney2012[P31]变异株自2012年出现以来在全球各地持续流行至今。【目的】制备广州地区GII.4 Sydney2012[P31]型诺如病毒毒株GZ2013-L10的病毒样颗粒(virus like particle,VLP),并系统表征其功能及免疫原性特点。【方法】从毒株GZ2013-L10中扩增ORF2基因并克隆构建重组转座载 PFastBac1-L10-ORF2,进一步转化至大肠杆菌DH10Bac构建重组杆状病毒质粒,进而在昆虫细胞sf9中表达病毒样颗粒并通过超速离心纯化,最后经透射电镜、Western blotting和受体结合实验对病毒样颗粒进行表征。此外,将免疫小鼠获得的病毒抗血清通过间接酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)和受体结合阻断试验进行验证。【结果】成功构建了重组杆状病毒质粒Bacmid-L10-ORF2并获得病毒样颗粒,电镜结果表明病毒样颗粒直径约为30 nm,SDS-PAGE和Western blotting显示蛋白大小约为58 kDa。受体结合实验结果显示,病毒样颗粒能与A/B/O等分泌型唾液受体及猪胃黏膜蛋白结合,而与非分泌型唾液受体均不结合。免疫小鼠获得效价为1.3×105的抗血清,但ELISA结果显示其与不同基因型诺如病毒衣壳蛋白无交叉免疫活性。此外,抗血清对同型病毒样颗粒具有受体中和阻断作用,但对不同型别病毒样颗粒(包括GII.8、GII.17和GII.3)无中和效果。【结论】本研究制备并系统表征了广州地区GZ2013-L10毒株的病毒样颗粒及其抗血清,其研究结果可为解析其流行原因以及疫苗研发提供参考。     

应用高效体积排阻色谱偶联多角度激光散射鉴定猪圆环病毒2型灭活疫苗及病毒样颗粒疫苗抗原

本文旨在建立基于高效体积排阻色谱(high-performance size-exclusion chromatography,HPSEC)偶联多角度激光散射仪(multi-angle laser light scattering,MALLS)的猪圆环病毒2型(porcine circovirus type 2,PCV2)疫苗抗原检测方法。以纯化的PCV2灭活病毒及病毒样颗粒(virus-like particles,VLP)为参照,对4家生产企业的2种PCV2灭活病毒疫苗(a、b)及VLP疫苗(c、d)破乳后进行HPSEC-MALLS检测及分子量分析;结合PCV2抗原检测卡、Western blotting和透射电子显微镜(transmission electron microscope,TEM),鉴定了特征色谱峰;考察了方法的重复性和检测线性。结果表明,两家企业生产的PCV2灭活病毒疫苗破乳液水相经HPSEC分离,在保留时间约13.3 min处出现抗原特征峰;MALLS计算该色谱峰分子量分别为2.61×10⁶(±4.34%) Da和2.40×10⁶(±2.51%) Da。两种VLP疫苗也在13.3 min处出现抗原特征峰,分子量分别为2.09×10⁶(±2.94%) Da和2.88×10⁶(±11.85%) Da,接近PCV2的理论分子量;同时在保留时间约11.4 min处也出现色谱峰,经检测分子量为4.37×10⁶(±0.42%) Da,TEM表征显示为VLP二聚体。取疫苗d和PCV2 VLP纯品进行重复检测,抗原色谱峰面积的RSD(n=3)均小于1.5%,重复性好;将PCV2 VLP纯品梯度稀释检测,VLP及其多聚体的色谱峰面积与浓度均呈良好的线性关系,R²分别为0.999及0.997,能够满足定量及多聚体含量分析。该方法有望成为一种准确、高效的PCV2疫苗的体外评价方法,用于质量评价与提升。     

Risk of symptomatic dengue for foreign visitors to the 2014 FIFA World Cup in Brazil

Brazil will host the FIFA World Cup™, the biggest single-event competition in the world, from June 12-July 13 2014 in 12 cities. This event will draw an estimated 600,000 international visitors. Brazil is endemic for dengue. Hence, attendees of the 2014 event are theoretically at risk for dengue. We calculated the risk of dengue acquisition to non-immune international travellers to Brazil, depending on the football match schedules, considering locations and dates of such matches for June and July 2014. We estimated the average per-capita risk and expected number of dengue cases for each host-city and each game schedule chosen based on reported dengue cases to the Brazilian Ministry of Health for the period between 2010-2013. On the average, the expected number of cases among the 600,000 foreigner tourists during the World Cup is 33, varying from 3-59. Such risk estimates will not only benefit individual travellers for adequate pre-travel preparations, but also provide valuable information for public health professionals and policy makers worldwide. Furthermore, estimates of dengue cases in international travellers during the World Cup can help to anticipate the theoretical risk for exportation of dengue into currently non-infected areas.     

我国转基因水稻产业化中的知识产权问题——对“遭遇国外专利陷阱”的回应

摘要：转基因水稻的产业化对缓解我国乃至世界的粮食危机和对保障我国的粮食安全都有重要的意义,而知识产权作为转基因水稻产业化进程中的基础性问题一直得到广泛的关注,尤其是关于我国是否会遭遇专利陷阱的问题。通过对我国知识产权制度以及所进行的转基因水稻专利检索数据的分析来看,转基因水稻的产业化并不会使我国遭遇专利陷阱,专利侵权的指控也并不存;但在整个产业化进程中,为保障转基因水稻知识产权的良性发展,还应当从如何促进技术优势转化为产业优势、如何对国外专利技术加以运用、如何维护稻农利益以及如何加强知识产权行政保护和司法保护等方面加以完善。     

Design strategies to address the effect of hydrophobic epitope on stability and in vitro assembly of modular virus‐like particle

Virus‐like particles (VLPs) and capsomere subunits have shown promising potential as safe and effective vaccine candidates. They can serve as platforms for the display of foreign epitopes on their surfaces in a modular architecture. Depending on the physicochemical properties of the antigenic modules, modularization may affect the expression, solubility and stability of capsomeres, and VLP assembly. In this study, three module designs of a rotavirus hydrophobic peptide (RV10) were synthesized using synthetic biology. Among the three synthetic modules, modularization of the murine polyomavirus VP1 with a single copy of RV10 flanked by long linkers and charged residues resulted in the expression of stable modular capsomeres. Further employing the approach of module titration of RV10 modules on each capsomere via Escherichia coli co‐expression of unmodified VP1 and modular VP1‐RV10 successfully translated purified modular capomeres into modular VLPs when assembled in vitro. Our results demonstrate that tailoring the physicochemical properties of modules to enhance modular capsomeres stability is achievable through synthetic biology designs. Combined with module titration strategy to avoid steric hindrance to intercapsomere interactions, this allows bioprocessing of bacterially produced in vitro assembled modular VLPs.     

Species-specific aggregation factor in sponges. XIII. Entire and core structure of the large circular proteid particle from Geodia cydonium

High-molecular weight particles have been isolated from the sponge Geodica cydonium. In the "native" from these particles consist of a spherical center and have 25-30 filaments attached to it. The core structure of the particles is assembled of a central circle and 25 radially-arranged filaments. The core structure is obtained from the entire structure by incubation in a medium, containing a non-ionic detergent and EDTA. The molecular weight of the enitre structure was in the range of 1.4 X 10(9) daltons or more and of the core structure 6.1 x 10(8) daltons. Two functional proteins are released from the "native" particles: the aggregation factor and the sialytransferase.     

Sex in the wild: How and why field‐based studies contribute to solving the problem of sex*

Why and how sexual reproduction is maintained in natural populations, the so‐called “queen of problems,” is a key unanswered question in evolutionary biology. Recent efforts to solve the problem of sex have often emphasized results generated from laboratory settings. Here, we use a survey of representative “sex in the wild” literature to review and synthesize the outcomes of empirical studies focused on natural populations. Especially notable results included relatively strong support for mechanisms involving niche differentiation and a near absence of attention to adaptive evolution. Support for a major role of parasites is largely confined to a single study system, and only three systems contribute most of the support for mutation accumulation hypotheses. This evidence for taxon specificity suggests that outcomes of particular studies should not be more broadly extrapolated without extreme caution. We conclude by suggesting steps forward, highlighting tests of niche differentiation mechanisms in both laboratory and nature, and empirical evaluation of adaptive evolution‐focused hypotheses in the wild. We also emphasize the value of leveraging the growing body of genomic resources for nonmodel taxa to address whether the clearance of harmful mutations and spread of beneficial variants in natural populations proceeds as expected under various hypotheses for sex.     

农林院校《遗传学》与相关课程间教学内容的重复及解决途径

遗传学是农林院校的主干课程之一。通过对遗传学与相关课程教学内容的分析,它们之间存在着严重的重复现象,课程间的衔接也存在一些问题。文章探讨了教学内容重复和不完善衔接所导致的不良后果,提出了解决教学内容重复问题的途径及改善相关课程间衔接的建议,旨在缓解遗传学本科教学内容大幅度扩增与教学时数缩减的矛盾,并为今后有关课程的教材编写提供参考。     

Analysis of Different Methodological Approaches to Measuring Microtubule Length in the Cytoplasm of Cultured Cells

It is generally assumed that microtubules in tissue culture cells extend from the centrosome to cell periphery, and the length of individual microtubules averages several dozens of microns. However, direct electron-microscopic measurements have cast some doubt on this assumption. In this study, the average length of microtubules in cultured Vero cells was estimated using a combined approach. The length of free cytoplasmic and centrosomal microtubules was determined by means of electron microscopy in serial sections; concurrently, the length of free microtubules in the lamella was measured in preparations stained with tubulin antibodies (an indirect immunofluorescent method), by tracing saltatory particle movements along the microtubules in living cells. According to the data of immunofluorescent microscopy, microtubule length in the lamella averaged 4.57 ± 3.69 m. However, since two or more microtubules can overlap, their length may be slightly overestimated by this method. On the other hand, saltatory movements are easy to monitor and measure fairly accurately, but their range may be shorter than the actual microtubule length because of a limited processiveness of motors (kinesin and dynein). On average, the trajectories of saltatory movements in living cells were 3.85 ± 0.72 m long. At the electron-microscopic level, microtubule length was analyzed using pseudo-three-dimensional reconstructions of the microtubule systems around the centrosome and in the lamella. The length of free microtubules in the lamella reached 18 m, averaging 3.33 ± 2.43 m; the average length of centrosomal microtubules was 1.49 ± 0.82 m. Good correspondence between the data on microtubule length and arrangement obtained by different methods allows the conclusion that most of the free microtubules in Vero cells actually have a length of 2–5 m; i.e., they are much shorter than the cell radius (about 25 m). Microtubules extending from the centrosome are shorter still and do not reach the cell periphery. Thus, most microtubules in the lamella of Vero cells are free and their ordered arrangement is not associated with their attachment to the centrosome.     

A solution to the problem of monotone likelihood in Cox regression

The phenomenon of monotone likelihood is observed in the fitting process of a Cox model if the likelihood converges to a finite value while at least one parameter estimate diverges to +/- infinity. Monotone likelihood primarily occurs in small samples with substantial censoring of survival times and several highly predictive covariates. Previous options to deal with monotone likelihood have been unsatisfactory. The solution we suggest is an adaptation of a procedure by Firth (1993, Biometrika 80, 27-38) originally developed to reduce the bias of maximum likelihood estimates. This procedure produces finite parameter estimates by means of penalized maximum likelihood estimation. Corresponding Wald-type tests and confidence intervals are available, but it is shown that penalized likelihood ratio tests and profile penalized likelihood confidence intervals are often preferable. An empirical study of the suggested procedures confirms satisfactory performance of both estimation and inference. The advantage of the procedure over previous options of analysis is finally exemplified in the analysis of a breast cancer study.     

Approaches to reconstruction of early Celtic land-use in the cent ral Neckar region in southwestern Germany

In four early Celtic settlements in the central Neckar region of Baden-Württemberg (Germany), 250 archaeobotanical samples were collected and studied for their plant macrofossils.Hordeum vulgare L. (hulled barley) andTrilicum spella L. (spelt wheat) could be recognized as the main crops, as well asPanicum miliaceum L. (millet) in some places. Legumes andLinum usitatissimum L. (flax) were important crops. Typical field weeds dominated the wild plant remains, but some present-day grassland taxa were abundantly and frequently found. The analysis of the data set of finds of the cereal remains, field weeds and grassland plants was done by Correspondence Analysis, but no significant differences between the various groups were found. Samples dominated by remains of grassland taxa, which could indicate that there were hay meadows, were absent. Consideration of plant macrofossil results as well as archaeological, osteological and pollen analytical data shows that there is likely to have been a land management system of arable fields and grassland. The densely settled fertile central Neckar region could therefore have been intensively utilised, using all available resources. Permanent grasslands in the form of pasture on river floodplains and on dry soils were certainly present. Hay meadows, on the other hand, were not identified. Cultivated plants such asFicus carica L. (fig) andVicia ervilia (L.) Willd. (bitter vetch) as well as some field weeds suggest connections with the Mediterranean lands.     

Inducible expression of the alpha2-macroglobulin signaling receptor in response to antigenic stimulation: a study of second messenger generation.

Thioglycollate (TG)-elicited murine, peritoneal macrophages express two receptors for activated forms of the proteinase inhibitor alpha2-macroglobulin (alpha2M*)--namely, the low density lipoprotein receptor-related protein (LRP) and the alpha2M signaling receptor (alpha2MSR). We now report that resident peritoneal macrophages express only 400+/-50 alpha2MSR receptors/cell compared to 5000+/-500 receptor/TG-elicited macrophage. By contrast, LRP expression is only 2-2.5-fold greater on elicited cells. The low level of alpha2MSR expression by resident cells is insufficient to trigger signal transduction in contrast to TG-elicited cells which when exposed to alpha2M* demonstrate a rapid rise in inositol 1,4,5-trisphosphate and a concomitant increase in cytosolic free Ca2+. We then studied a variety of preparations injected subcutaneously for their ability to upregulate alpha2MSR. Macroaggregated bovine serum albumin (macroBSA) injection upregulated alpha2MSR and triggered signaling responses by splenic macrophages. Nonaggregated BSA injection alone or in the presence of alum, by contrast, did not alter alpha2MSR expression. Recombivax (hepatitis B antigen adsorbed to alum) injection also upregulated alpha2MSR on splenic macrophages while the alum carrier had no effect. We conclude that macrophage alpha2M* receptors are inducible and their expression may be regulated, in part, by potential antigens.     

Specific cyanylation and cleavage at cysteine-104 human hemoglobin alpha-chain. A novel approach to the problem of the alpha-chain tryptic core in the study of haemoglobin variants by "fingerprinting" methods.

1. A new approach to the analysis, by "fingerprinting", of the tryptic core region of human haemoglobin alpha-chain is described. 2. The alpha-chain is cyanylated at its single cysteine residue (alpha104) and then split, by exposure to mild alkali, at the N-peptide bond of the resulting beta-thiocyanoalanine residue. 3. The two cleavage fragments, alpha1-103 and alpha104-141, are separated by gel filtration, and the fragment alpha104-141, which contains all the residues of the alpha-chain tryptic core, is digested with pepsin. 4. Preparative "fingerprints" of these peptic peptides yield eight major peptides, which provide complete sequence information for the whole region alpha104-141. 5. The utility of the method is demonstrated by repeating the determination of the substitution in haemoglobin Hopkins-2, a known alpha-chain core variant in which histidine-alpha112 (G19) is replaced by an aspartic acid residue.     

Allometry and prediction in hominoids: a solution to the problem of intervening variables.

To avoid misinterpretation of allometric exponents determined from interspecific allometric comparisons, specific conditions must be met with respect to the common reference variable. Body weight is considered to be the best general indication of overall size and is hence widely acknowledged to be the most suitable reference variable. However, because of the paucity of recorded body weights for museum specimens, various comparative studies have used other size indicators as intervening variables, although the allometric relationships to body size/weight were often unknown and possibly differed between species. Because of differences in the scaling properties of alternative intervening variables across the species investigated, conflicting conclusions may be drawn if different variables are chosen as substitutes for overall size. This is illustrated with two examples. In this study, series of skeletons with associated body weights of Gorilla, Pan, Pongo, and Homo were investigated. Both ontogenetic and static adult allometric relationships between several widely used reference variables and body weight were determined. Neither these variables nor additional estimators investigated in this study displayed allometric exponents and coefficients similar enough across species to justify direct interspecific comparison. To generate an alternative size estimator for both ontogenetic and static interspecific investigations, equations for combined sexes were derived to predict body weight from various long bone dimensions for individual hominoid species. From a total of 25 predictors, 12 prediction equations per species (six for nonadults and six for adults) were selected according to their relative suitability for reliable prediction of body weight. It is shown that the derived reference variable "predicted body weight" avoids problems of intervening variables, is valid for any interspecific ontogenetic and static allometric comparison, and displays less fluctuation in comparison to actual body weight.