兔出血症病毒VPg蛋白的原核表达及抗体制备

【目的】构建重组兔出血症病毒(Rabbit hemorrhagic disease virus,RHDV)VPg(Viral Protein Ge-nome-Linked,VPg)蛋白基因的原核表达质粒,在大肠杆菌BL21(DE3)中表达和纯化VPg蛋白,制备多克隆抗体,并检测抗体的基本特性。【方法】用PCR方法扩增RHDVVPg基因,将其克隆至原核表达载体pET-30a(+)中,转化大肠杆菌BL21(DE3)菌株,用IPTG诱导,使之重组表达VPg蛋白,并用镍柱亲和层析法纯化重组蛋白。PCR扩增获得了345bp的VPg基因,用纯化的重组VPg蛋白免疫试验兔,制备抗VPg的多克隆抗体,用Western blot检测其特异性。【结果】构建了pET-30a/VPg原核表达质粒,转化大肠杆菌BL21(DE3)菌株后,成功表达了VPg蛋白。用该蛋白免疫试验兔后,制备的多克隆抗体能与VPg蛋白特异性反应。【结论】成功表达了RHDV VPg蛋白,并制备了特异性良好的多克隆抗体。     

Abundance and community structure of sulfate reducing prokaryotes in a paddy soil of southern China under different fertilization regimes

Flooded rice paddy soils represent a typical anaerobic freshwater habitat of microorganisms. The abundance and community structure of sulfate reducing prokaryotes (SRP) were investigated in order to understand their response to different fertilization practices in rice paddy, including control without fertilizers (CT) and arrangements of different chemical fertilizers of nitrogen (N), phosphorus (P) and potassium (K): N, NP, NK and NPK. The abundance of total bacteria and sulfate reducing prokaryotes of the rice paddy in summer and in winter were quantified by real-time PCR assays based on the 16S rRNA gene and the dissimilatory (bi)sulfite reductase gene (dsrAB) β-subunit. No significant differences in the bacterial and SRP abundance were observed among different fertilization treatments in both winter and summer. The mean copy numbers of bacteria was 7.26 × 10⁹ copies g⁻¹ dry soil in winter and 1.27 × 10¹⁰ copies g⁻¹ dry soil in summer. The average dsrAB gene copy numbers of the SRP was 5.08 × 10⁸ copies g⁻¹ dry soil in winter and 5.92 × 10⁸ copies g⁻¹ dry soil in summer. The dsrAB gene clone libraries of the five fertilization treatments were constructed and their RFLP analysis yielded 22-25 restriction patterns, suggesting a high degree of dsrAB sequence diversity in different fertilization treatments. There was no significant change in the soil SRP community structure among the different fertilization regimes. More than half of the sequences were affiliated with novel branching clusters which were uncultured SRP. Clostridia and Deltaproteobacteria were also found with a high proportion in the clone libraries, while Desulfovibrionaceae was absent. High proportion of novel uncultured SRP implies that they may play important roles in paddy soils and deserve further studies.     

Symbioses of Cyanobacteria in Marine Environments: Ecological Insights and Biotechnological Perspectives

Cyanobacteria are a diversified phylum of nitrogen-fixing, photo-oxygenic bacteria able to colonize a wide array of environments. In addition to their fundamental role as diazotrophs, they produce a plethora of bioactive molecules, often as secondary metabolites, exhibiting various biological and ecological functions to be further investigated. Among all the identified species, cyanobacteria are capable to embrace symbiotic relationships in marine environments with organisms such as protozoans, macroalgae, seagrasses, and sponges, up to ascidians and other invertebrates. These symbioses have been demonstrated to dramatically change the cyanobacteria physiology, inducing the production of usually unexpressed bioactive molecules. Indeed, metabolic changes in cyanobacteria engaged in a symbiotic relationship are triggered by an exchange of infochemicals and activate silenced pathways. Drug discovery studies demonstrated that those molecules have interesting biotechnological perspectives. In this review, we explore the cyanobacterial symbioses in marine environments, considering them not only as diazotrophs but taking into consideration exchanges of infochemicals as well and emphasizing both the chemical ecology of relationship and the candidate biotechnological value for pharmaceutical and nutraceutical applications.     

The structure of microbial communities in redoximorphic microsites of Gleysol

Microbial communities were studied in redoximorphic microsites of highly heterogeneous Gleysol at a mm scale using 16S and 18S amplicon sequencing to demonstrate if the composition of soil microbes reflects the differences in ferric and ferrous micro-sites. In both explored gley horizons with redoximorphic features (Bg2 and Cg), ferric mottles were significantly enriched with total P and Fe and depleted of O, Si, Al, K and Ca compared with the adjacent ferrous groundmass (SEM–EDS). Ferric mottles were determined as Fe oxide coatings and hypocoatings. In Bg2, both prokaryotic and micro-eukaryotic communities differed significantly between mottles and groundmass in composition of operational taxonomic units (OTUs) and in proportions of phyla, reflecting heterogeneities in the soil properties there. Mottles in Bg2 were characterized by increased proportion of Proteobacteria, decreased proportion of Acidobacteriota among prokaryotes and by dominance of a single proteobacterial OTU from Anaplasmataceae compared to all other samples. The composition of micro-eukaryotes showed an opposite trend, as micro-eukaryotes of Bg2 groundmass were unique among the other horizons, while micro-eukaryotes of Bg2 mottles had similar composition to neighbouring horizons. Microbial communities of adjacent samples were not more similar to each other than communities of randomly selected ones in Bg2 horizon. That suggests that at mm scale, the sample distance does not represent the driving factor of microbial community composition and that the adjacent samples differ rather due to physicochemical factors. The spatial organization of microbial communities revealed in Bg2 has not reappeared in similarly organized Cg horizon, probably due to other overriding factors. The differences revealed between Bg2 and Cg horizons, including granulometric composition, content of crystalline Fe, exchangeable Al, and organic carbon, as well as exposition to groundwater, were discussed as possible reasons of the distinct organization in Cg. The similarity of pro−/eukaryotic communities of adjacent and non-adjacent couples suggests no distance decay pattern at a mm scale. The agreement between patchiness in soil properties and microbial communities was revealed for the first time and confirms the importance of microscale patterns in soil.     

兔出血症病毒VP60蛋白的原核表达及检测方法初步应用

VP60是免出血症病毒（RHDV）的主要结构蛋白，也是免疫原性蛋白，与诱导抗病毒免疫反应直接相关，因而也是检测用首选抗原。本实验在原核系统pPRoEX^TMHTa中表达了VP60基因，经SDS—PAGE电泳中可见表达带。Western blot鉴定结果表明，重组蛋白可被RHD阳性血清特异性识别，具有相应的抗原性。将表达蛋白经SDS—PAGE电泳切胶纯化后免疫BALB／c小鼠，免疫血清经全病毒ELISA检测，效价可达1：3200-1：6400，经琼脂扩散试验检测效价为1：16。重组蛋白纯化、复性后，作为包被抗原初步建立了间接ELISA方法，并检测了48份免血清样品，与全病毒间接ELSIA试剂盒检测结果相同。实验结果表明．用原核系统表达的VP60蛋白．可以作为RHD新型诊断试剂的候选抗原。     

The effects of soil properties,cropping systems and geographic location on soil prokaryotic communities in four maize production regions across China

The diversity of prokaryotic communities in soil is shaped by both biotic and abiotic factors. However, little is known about the major factors shaping soil prokaryotic communities at a large scale in agroecosystems. To this end, we undertook a study to investigate the impact of maize production cropping systems, soil properties and geographic location(latitude and longitude) on soil prokaryotic communities using metagenomic techniques, across four distinct maize production regions in China. Acr...     

鲤脂蛋白脂肪酶基因特性、表达分布、重组蛋白获得及酶活性比较

为研究鲤脂蛋白脂肪酶(Cc LPLs)的基因特征、时空表达分布及酶活性，实验利用基因组同源搜索获取鲤CcLPLs同源基因并分析其序列特征；通过荧光定量PCR(qPCR)方法对CcLPLs在不同组织的表达进行分析；采用原核表达系统获取CcLPLs重组蛋白，并使用对硝基苯酚法测定各重组蛋白的酶活性。结果显示，鲤基因组中挖掘到5个CcLPLs基因(CcLPLA1a、CcLPLA1b、CcLPLA2a、CcLPLA2b*和CcLPLBa)，经验证，CcLPLA2b*是假基因，共线性分析显示，鱼类特有基因组加倍过程中出现基因丢失的现象，而鲤特有的基因组加倍致使鲤存在5个CcLPLs。CcLPLA1a和CcLPLA1b核苷酸序列和氨基酸序列相同，同源性分析显示，CcLPLBa与CcLPLA1s的同源性为64%，与CcLPLA2a同源性为50.8%。qPCR结果显示，CcLPLs的表达量在肝脏、心脏、脂肪、肌肉、脑、肠道和脾脏中依次降低，在各个组织中，各基因表达量从高到底依次为CcLPLA1s、CcLPLA2a和CcLPLBa。鲤正常投喂、饥饿及再投喂状态下CcLPLA1s和CcLPLA2a在肝脏、...