安徽省猪伪狂犬病毒的分离鉴定及其主要毒力基因分子特征

猪伪狂犬病是由猪伪狂犬病毒(porcine pseudorabies virus,PRV)引起的一种急性、热性传染病。自2011年底以来,PRV发生了变异,传统的PRV弱毒疫苗已不能对PRV变异株提供完全保护,这给我国PR防控带来了巨大挑战。为了解安徽省PRV流行特征及其主要毒力基因遗传变异情况。利用PCR技术、细胞接种试验、电子显微镜观察、间接免疫荧光试验及兔体接种试验等方法,对安徽省临诊病例中疑似PRV感染的病猪进行病原检测及PRV分离鉴定,并通过设计6对特异性引物对PRV分离株主要毒力基因(gE、gI、TK、gB、gC、gD)进行克隆及测序分析。2016—2018年安徽省临诊病例中共分离鉴定15株PRV;PRV分离株主要毒力基因序列均与2011年后国内PRV变异株同源性较高;与2011年前国内PRV经典株序列比对,PRV分离株gE、gB、gC及gD基因存在多个位点的一致性替换、插入或缺失,且gE、gC基因多位点突变位于其重要的抗原表位区。本研究分离的15株PRV均为变异株,变异株已成为安徽省主要的流行毒株。15株PRV分离株的gI、TK基因序列较为保守,而gE、gC蛋白抗原表位区域氨基酸的突变可能导致其毒力及抗原性发生改变。部分PRV分离株与邻近地区PRV序列同源性均为100%,可能与频繁跨省调运生猪、跨区引种等原因有关。     

Ligand-binding characteristics of feline insulin-binding immunoglobulin G

Polyclonal immunoglobulin (Ig) G autoantibodies against insulin have been identified in sera of healthy cats. We purified and fractionated insulin-binding IgGs from cat sera by affinity chromatography and analyzed affinity of insulin-binding IgGs for insulin and their epitopes. Following the passing of fraction A, which did not bind to insulin, insulin-binding IgGs were eluted into two fractions, B and C, by affinity chromatography using a column fixed with bovine insulin. Dissociation constant (KD) values between insulin-binding IgGs and insulin, determined by surface plasmon resonance analysis (Biacore™system), were 1.64e⁻⁴ M for fraction B (low affinity IgGs) and 2e⁻⁵ M for fraction C (high affinity IgGs). Epitope analysis was conducted using 16 peptide fragments synthesized in concord with the amino acid sequence of feline insulin by an enzyme-linked immunosorbent assay. Fractions B and C showed higher absorbance (affinity) of the peptide fragment of 10 amino acid residues at the carboxyl-terminal of the B chain (peptide No. 19), followed by peptide fragments of 6 to 15 amino acid residues of the B chain (peptide No. 8). Fraction C showed a higher absorbance to 7 to 16 amino acid residues of the B chain (peptide No. 5) compared with the absorbance of fraction B. Polyclonal insulin-binding IgGs may form a macromolecule complex with insulin through the multiple affinity sites of IgG molecules. Feline insulin-binding IgGs are multifocal and may be composed of multiple IgG components and insulin.     

Isolation and identification of a viral haemorrhagic septicaemia virus (VHSV) isolate from wild largemouth bass Micropterus salmoides in China

Fish rhabdoviruses are a family of viruses responsible for large‐scale fish die‐offs worldwide. Here, we reported the isolation and identification of a member of rhabdoviruses from wild largemouth bass (Micropterus salmoides) in the coastal area of the Pearl River Estuary, China. This virus isolate was identified as viral haemorrhagic septicaemia virus (VHSV) by specific RT‐PCR. Furthermore, the virus (VHSVLB2018) was isolated by cell culture using fathead minnow cells and confirmed by RT‐PCR. Electron microscopy showed the presence of bullet‐shaped viral particles in the cytoplasm of infected cells. The complete sequencing of VHSVLB2018 confirmed that it was genome configuration typical of rhabdoviruses. Phylogenetic analysis based on whole‐genome sequences and G gene nucleotides sequences revealed that VHSVLB2018 was assigned to VHSV genogroup Ⅳa. The pathogenicity of VHSVLB2018 was determined in infection experiments using specific pathogen‐free largemouth bass juveniles. VHSVLB2018‐infected fish showed typical clinical signs of VHSV disease, including darkened skin, petechial haemorrhages and pale enlarged livers, with the cumulative mortalities reached 63.3%–93.3% by 7 days post‐infection. VHSVLB2018 was re‐isolated from dead fish and confirmed by RT‐PCR. Together, this is the first report of isolation and identification of a VHSV isolate from wild largemouth bass in China.     

Physicochemical and Structural Properties of Recovered Elastin from Jumbo Squid (Dosidicus gigas) By-Products

Physicochemical and structural properties of soluble jumbo squid (Dosidicus gigas) elastin recovered from skin by-products were evaluated. The molecular weight of isolated elastin was ~40 kDa with an isoelectric point (pI) between 9 and 10. Aspartic, glutamic, arginine, proline, glycine, and lysine amino acids were the most abundant in squid elastin, whereas the hydroxyproline absence, ~0.7% cysteine content, and the calculated 0.35 isoleucine/leucine ratio were used as purity index. Total and reactive sulfhydryl contents were similar (247.0 ± 5.1 vs 242.0 ± 7.5 μmol mg^?1 of protein, P ≥ 0.05) in purified squid elastin but surprisingly higher than previously reported in other elastins. On the other hand, the secondary structures of squid elastin analyzed by Fourier transform infrared spectroscopy (FTIR) were ~45% β-sheets, ~15% α-helices, ~10% β-turns, and ~30% undefined structures. In addition, squid elastin experienced glass transition at 82.01 ± 0.01ºC, denaturation temperature at 110.45 ± 0.64ºC, and aggregation at 197.5 ± 0.23ºC. In conclusion, the prevalence of charged amino acids and pI of squid elastin can facilitate its solubilization in hydrophilic systems, whereas the secondary structure profile and thermostability are desirable features in proteins used for biopolymer designs such as food biofilms or barrier systems.     

黑蜣(Bess beetle)纤维素酶的组分及其特性分析

以30日龄的黑蜣幼虫为材料,对其整体匀浆后组织液中的纤维素酶组成及活性进行了分析。结果表明:黑蜣体内存在内切β-1,4-葡聚糖酶(Cx酶)、β-葡萄糖苷酶;β-葡萄糖苷酶和Cx酶的最适宜温度为分别为40℃和50℃;Cx酶和β-葡萄糖苷酶分别在pH值为6.4和5.2时具有最大的活力;β-葡萄糖苷酶和Cx酶的最适宜反应时间为80 min。Cx酶具有比β-葡萄糖苷酶更强的温度适应性,在30～50℃温度范围内可维持最大酶活力的66.20%。     

基于溶解无机碳中碳氧同位素及化学组分的瓶装饮用水产地真实性鉴别

为了鉴别我国市场上销售的瓶装饮用水的产地和产品类型,本试验分析了瓶装饮用水溶解无机碳(DIC)中的δ¹⁸O_DIC和δ¹³C_DIC、水中的δ¹⁸O和δ²H值、矿物元素(K、Ca、Na、Mg、Sr)含量、阴离子(Cl^-、SO₄^2-、HCO₃^-)含量、pH值与电导率等水化学指标,结合费希尔判别分析(Fisher-DA)建立模型,对瓶装饮用水的类别和产地进行鉴别。结果表明,不同产地瓶装饮用水的δ¹⁸O_DIC、δ¹³C_DIC、δ¹⁸O、δ²H值均具有显著差异,而且矿物元素与阴离子的含量变化范围广,具有不同的地域性特征。产地溯源判别模型对不同产地瓶装饮用水的判别准确率高达100%,且采用水化学指标还能区分瓶装饮用水的类型。正态化重要性分析显示,对产地具有指示效应的前五项重要指标分别为K、Cl^-和δ¹³C_DIC、δ²H和pH值。综上所述,通过稳定同位素指纹信息和水化学指标的差异进行融合分析,结合多元统计模型可有效鉴别市场上瓶装饮用水的真实性,保护消费者的合法权益,维护市场公平。     

水稻半外卷叶突变体sol1的表型分析与基因定位

适度卷曲有利于提高水稻叶片的光合效率,增加植株光合产物的有效积累量。我们利用甲基磺酸乙酯(EMS)处理籼型水稻保持系西农1B,获得一个稳定遗传的水稻半外卷叶突变体。该突变体从十叶期开始各叶片逐渐向外卷曲直至半卷状,并伴随茎秆半矮化和叶片披垂,暂被命名为semi-outcurved leaf 1(sol1)。与野生型(WT)相比,sol1的叶片卷曲指数均达到30%以上(P<0.01);倒一、倒二、倒三、倒四节节间长度和穗长极显著缩短,倒一、倒二、倒三叶的叶夹角显著或极显著增加;有效穗数、千粒重、每穗实粒数、结实率显著或极显著下降,一次枝梗数则增加11.3%(P<0.05)。sol1的蒸腾速率、胞间CO2浓度、气孔导度显著高于野生型。石蜡切片显示,sol1倒一叶的泡状细胞体积变小,数量显著增多,表皮细胞体积略微增大。遗传分析表明,sol1的半外卷叶性状受1对隐性核基因调控,定位于6号染色体标记JY6-3和JY6-10之间165 kb的物理范围内,共含15个注释基因。qRT-PCR结果表明,与泡状细胞相关的内卷基因和外卷叶基因RL14、Roc5、REL1在突变体sol1中呈不同程度的上调,NRL、BRD1、OsHox32、ADL1、LC2则呈不同程度的下调。研究结果为SOL1基因的克隆和功能研究奠定了基础。     

Occurrence of multiple antibiotic resistance and genotypic characterization in Edwardsiella tarda isolated from cage‐cultured hybrid red tilapia (Oreochromis sp.) in the Ping River,Northern Thailand

Edwardsiella tarda is a pathogen that causes edwardsiellosis in aquatic animals. The emergence of multiple antibiotic‐resistant strains makes antibiotic treatment difficult. This study aimed to investigate the antibiotic susceptibility patterns and the genotypic characterization of E. tarda isolated from cage‐cultured red tilapia in Thailand. A total of 30 isolates were identified as E. tarda using biochemical and molecular analysis. The disc diffusion method for testing antibiotic susceptibility showed all the isolates were resistant to colistin sulphate and oxolinic acid. High levels of resistance to amoxicillin, ampicillin, ceftazidime, oxytetracycline and sulphamethoxazole/trimethoprim were observed as well. The multiple antibiotic resistance index ranged from 0.25 to 0.92, indicating that these isolates had been exposed to high risk sources of contamination where antibiotics were commonly used. All the isolates carried the bla_TEM gene based on polymerase chain reaction (PCR). The tetA and sul3 genes were detected in 90% (27/30) and 26.7% (8/30) of the isolates respectively. Nine different genetic groups of isolates were obtained using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC‐PCR). A correlation between genetic types and multiple antibiotic‐resistant patterns was found. These results highlight the potential risks of multiple antibiotic‐resistant isolates for humans and the environment.     

Steam explosion of crop straws improves the characteristics of biochar as a soil amendment

Five crop straws(wheat, rice, maize, oil-rape, and cotton) were first steam-exploded for 2 min at 210°C, 2.5 MPa and then pyrolyzed at 500°C for 2 h. Steam explosion(SE) induced 47–95% and 5–16% reduction of hemicellulose and cellulose, respectively, in the crop straws. The biochars derived from SE-treated feedstocks had a lower specific surface area(SSA) and pore volume, compared to those from pristine feedstocks, with one exception that SE enhanced SSA of oil-rape straw biochar by approximately 16 times. After SE, biochars had significant higher anion exchange capacity(AEC)(6.88–11.44 cmol kg~(–1)) and point of zero net charges(PZNC)(pH 3.61–5.32) values. It can thus be speculated that these biochars may have higher potential for anions adsorption. In addition, oil-rape straw might be suitable to SE pretreatment for preparing biochar as a soil amendment and sorbent as well. Further work is required for testing its application in soil.     

建鲤组织蛋白酶L 的原核表达、纯化鉴定及多克隆抗体的制备

对原核表达的重组建鲤组织蛋白酶L(Cathepsin L,CAT L)蛋白进行尿素洗涤和Ni-NTA亲和层析纯化,该目的蛋白经300 mmol/L咪唑洗脱为单一峰,SDS-PAGE结合TSK-GEL G2000SWxl凝胶过滤高效液相色谱分析表明重组CAT L获得了高度纯化,分子量约28 k D,纯度超过95%。Z-Phe-Arg-MCA底物测活法显示该重组CAT L表现为半胱氨酸蛋白酶活性,能与其内源抑制因子Cystatin以1︰1的摩尔比结合,具有生物学活性。以纯化的重组CAT L蛋白免疫Balb/C小鼠获得抗血清,经ELISA法检测获得的CAT L抗血清效价高于1︰512000;Western blotting鉴定结果表明该抗体具有良好的特异性,能够识别原核表达的重组CAT L蛋白。免疫组织化学分析结果表明,该抗体还能识别建鲤小肠、肝胰脏、脾、背肌和心肌组织表达的内源性CAT L蛋白。因此可利用该抗体从蛋白水平检测CAT L在鱼类不同组织中的表达和分布情况。