无菌人工养殖海水中副溶血弧菌噬菌体与宿主的消长动态

在3组一定浓度的副溶血弧菌菌液中分别加入3种浓度的副溶血弧菌噬菌体,第12天各实验组再次分别加入一定浓度的副溶血弧菌或副溶血弧菌噬菌体,比较各组水体中副溶血弧菌噬菌体及其宿主的动态关系。结果表明,副溶血弧菌噬菌体可使宿主下降至一定浓度并与之保持动态平衡关系,实验组1、2、3中副溶血弧菌浓度从2.43×106 mL-1分别下降至6.2×104、9.2×104、7.46×105 mL-1,并以此浓度范围维持下去。高浓度副溶血弧菌噬菌体与其宿主之间平衡关系的稳定性较好,再次加入副溶血弧菌,高、中浓度组分别在1 d和4 d后副溶血弧菌下降至平衡时的数量级浓度,并持续下去。低浓度组副溶血弧菌组有所上升,并持续下去。再次加入副溶血弧菌噬菌体对副溶血弧菌浓度影响较小(P>0.05)。     

副溶血弧菌噬菌体微胶囊的制备及在饵料中的应用

采用复相乳液法,以乙基纤维素为包囊材料,明胶溶液为保护液制取微胶囊,对副溶血弧菌噬菌体(Vibrio parahaemolyticus phage)进行固定化,制备噬菌体微胶囊。结果显示,乙基纤维素质量浓度为30 g/L,明胶质量浓度为20 g/L,搅拌速度为600 r/min,时间30 min,副溶血弧菌噬菌体悬液与乙基纤维素溶液的体积比为1∶3时,所得微胶囊有较好的噬菌体缓释性能,综合评价指数最高。对方斑东风螺(Babylonia areolata)饲以混有噬菌体微胶囊的饵料,当饵料中噬菌体微胶囊质量分数为1.0 g/kg时,与对照组比较,东风螺肠道宿主菌平均减少96%,水体中宿主菌减少72%,差异均有统计学意义(P0.05);以黏附剂按1.5 g/kg的剂量将噬菌体微胶囊黏附于对虾饵料,并投喂凡纳滨对虾(Litopenaeus vannamei),对虾肠道的宿主菌比对照组平均减少86%,水体中宿主菌比对照组减少68%,差异均有统计学意义(P0.05)。微胶囊可有效控制宿主弧菌的在养殖体系的浓度,减少养殖动物弧菌病的产生。     

凡纳滨对虾含酪氨酸酶结构域血蓝蛋白的免疫功能

【目的】克隆凡纳滨对虾(Litopenaeus vannamei)一种新的含酪氨酸酶结构域血蓝蛋白(Tyrosinase-domain containing hemocyanin,TDCH),研究其免疫功能。【方法】根据NCBI上公布的tdch基因序列设计克隆引物,通过RT-PCR方法获得tdch的编码区;采用生物信息学方法分析tdch的结构特征;使用荧光定量PCR技术分析tdch在凡纳滨对虾12个组织中的表达,以及在副溶血弧菌(Vibrio parahaemolyticus)、金黄色葡萄球菌(Staphhylococcu saureus)、黑曲霉菌(Aspergillus niger)、白斑综合征病毒、脂多糖及聚肌苷酸-聚胞苷酸[Poly(I:C)]刺激后在鳃和血细胞中的表达变化;利用RNAi技术敲降tdch在对虾中的表达,研究其在对虾抗副溶血弧菌免疫中的作用。【结果与结论】克隆获得tdch的编码区全长,编码551氨基酸的蛋白,理论分子质量为63.37 ku,等电点为7.74;tdch除含有血蓝蛋白结构域以外,还含有酪氨酸酶Tyrosinase结构域;tdch在对虾肌肉、肝胰腺、鳃、...     

Gene cloning and prokaryotic expression of recombinant outer membrane protein from Vibrio parahaemolyticus

Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene (ompW) from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells. The gene coded for a protein that was 42.78 kDa. We purified the prote...     

Cloning, expressing, and hemolysis of tdh, trh and tlh genes of Vibrio parahaemolyticus

Vibrio parahaemolyticus (VP) is one of the pathogenic vibrios endangering net-cage cultured Pseudosciaena crocea,Fennerpenaeus chinensis, and shellfish in coastal areas of China. Several types of hemolysins produced by Vp have been characterized as major virulence factors.They are thermostable direct hemolysin (TDH),TDH-related hemolysin (TRH) and thermolabile hemolysin (TLH). In this study, we cloned tdh, trh, and tlh genes from the genome DNA of VP by polymerase chain reaction (PCR).We ligated the three genes into prokaryotic expression vector pET-28a (+),and transformed the recombinant plasmids into Es-cherichia coli BL21 (DE3). The expression of recombinant proteins was induced by isopropyl-β-D-thiogalacto-pyranoside (IPTG). The recombinant proteins were expressed in a form of inclusion bodies and thus purified with Ni-NTA affinity chromatography. Western blotting results showed that recombinant proteins,TDH, TRH and TLH, could be recognized by rabbit anti-VP serum. The three purified proteins were renatured by gradient dialysis.The renatured proteins exhibited hemolytic activity except for TLH in the presence of phosphatidylcholine. These results not only are helpful for better understanding these genes’ functions under a single factor level, but also provide evidence for VP vaccine engineering.     

融合蛋白IL6-OmpW的质粒构建及表达纯化

以重组质粒pMD18-T/IL6和pMD18-T/OmpW为模板,分别扩增红笛鲷IL-6基因和哈维氏弧菌外膜蛋白OmpW基因,运用PCR重叠延伸剪切技术,将IL-6和OmpW基因融合,将融合基因定向克隆到原核表达载体pET-32a(+),转化大肠杆菌BL21(DE3)感受态,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导融合蛋白高效表达,融合蛋白分子质量约为66.6 ku。优化后表达条件为温度37℃,IPTG浓度0.2 mmol·L-1,诱导时间5 h。用HisTrap HP亲和柱纯化重组蛋白,最佳咪唑洗脱浓度为400 mmol·L-1,纯化蛋白的质量浓度为480μg·mL-1。Western-blot分析显示,该融合蛋白可与鼠抗His-tag单克隆抗体发生特异反应,表明目的蛋白得以正确表达。     

Identification of two major histocompatibility (MH) class II A genes and their association to Vibrio anguillarum infection in half-smooth tongue sole (Cynoglossus semilaevis)

Major histocompatibility complex class II antigens are important in vertebrate immune system.In the present study,the full cDNA sequence of class II A gene was synthesized by RACE-PCR from half-smooth tongue sole(Cynoglossus semilaevis),and its open reading frame(ORF) polymorphism was studied.The whole cDNA sequence was 992 bp in length,including the ORF with 717 bp.Twenty-five alleles were identified and clustered into two distinct groups according to the specific nucleotides/amino acids in specific positions.Eleven alleles belonged to Cyse-DAA while fourteen alleles belonged to Cyse-DBA.Four Cyse-DAA alleles were observed in one individual,and three to five Cyse-DBA alleles were observed in each of the three detected individuals,which indicated that at least two loci existed in each gene.Moreover,in order to study the function of the alleles in resistance to infection,200 individuals were intraperitoneally injected with Vibrio anguillarum and the first 20 dead individuals and 20 surviving ones were selected for genotype analysis.Fifty-six alleles were identified among the 40 individuals.Twenty-nine alleles belonged to Cyse-DAA and the other 27 alleles belonged to Cyse-DBA.Eighteen alleles were selected for studying their function in resistance to infection.Alleles Cyse-DAA*0201,Cyse-DAA*1101,Cyse-DBA*0401,Cyse-DBA*1102,Cyse-DBA*1801 and Cyse-DBA*2201 were identi-fied only in surviving individuals,while alleles Cyse-DAA*0901,Cyse-DBA*1101 and Cyse-DBA*1401 occurred more frequently in dead individuals.This study confirmed the existence and polymorphism of two class II A genes as well as the relationship between alleles of class II A genes and disease susceptibility/resistance in half-smooth tongue sole.     

溶藻弧菌双组分调控系统PhoR/PhoB的基因克隆及生物信息学分析

双组分调控系统（Two-component Regulatory System）在致病菌的生长及毒力调控中起重要作用.克隆溶藻弧菌（Vibrio alginolyticus）HY9901 株组氨酸激酶PhoR 和反应调控因子PhoB 的全长基因,并对其进行生物信息学分析.序列分析结果显示,phoR（GenBank 登录号：KJ958404）全长1 299 bp,共编码432 个氨基酸;phoB（GenBank 登录号：KJ863646）全长690 bp,编码229 个氨基酸.构建PhoR/PhoB 的系统进化树,结果显示,溶藻弧菌PhoR/PhoB 与副溶血弧菌（Vibrio parahaemolyticus）、哈氏弧菌（Vibrio harveyi）有较近亲缘关系.利用SWISS-MODEL 软件,对PhoR/PhoB 中两个相对保守的功能域HATPase_c 和REC 进行同源建模,发现HATPase_c具有1 个ATP 结合位点,REC 具有4 个天冬氨酸活性位点.     

近20年烟台主城区土地利用变化特征与驱动分析

以TM影像为基本数据源,结合土地利用现状图和土地详查数据,分析了烟台主城区1992年以来土地利用空间扩张特征与机制,以能够更好地对城市进行科学规划控制,促进城市经济协调发展。通过对1992年及2013年TM遥感影像进行处理分析,结合烟台市的社会经济统计数据,得出结论：烟台市1992年到2013年城市空间扩张向西面及沿海方向发展;城市建筑用地面积增加较多,耕地和水域面积则呈减小趋势。影响城市土地利用空间扩张的驱动因子主要是经济发展、人口增长、产业结构、交通发展及国家政策等,其中经济发展和人口增长是促进烟台城市土地利用空间扩张的主要因素。     

川滇地区次级地块及其主要断裂带现今活动研究

以位于川滇区域的陆态网和四川省CORS站速度场为约束,结合研究区已知的断裂分布情况,通过使用欧拉矢量计算方法和反欧拉矢量计算方法求出每个次级块体的欧拉矢量,进而对川滇区域次级块体作进一步的调整。同时对该区域进行1°×1°的网格插值,并利用各次级地块的欧拉矢量反演出每个次级块体的长期运动方向及其速度;对相邻块体的边界处,结合各自块体的欧拉矢量求出同一点在2个块体的速度并作差,进而研究分析各主要断裂带的现今活动速率。