Ligation of the HCV 5’ NCR,C, E1, E2/NS1 sequences andits cloning into pLNSX

AIM: To construct retrovirus recombinant HCV/pLNSX (pLHC), which is used to investigate new ways to control HCV infection and gene therapy. METHODS and RESULTS: Primers with their sequences homologous to multiple strains of HCV had been designed, synthesized and used in RT-PCR to amplify 5 fragments from 4 regions, 5’ NCR, C, E1 and E2/NS1. The products had been cloned, restrictively cut aside, and overlappingly amplified to ligate a consecutive sequence of 2547 bp in length that contains the whole HCV 5’ NCR and all the structural protein coding regions. The sequence was then inserted into the vector pGEM-3Zf (+) yielding a recombinant pHC2547, and into the retrovirus pLNSX yielding a pLHC. Both plasmid DNA were analyzed by PCR, Southern blot, and enzymatic cut. CONCLUSION: Retrovirus recombinant HCV/pLNSX (pLHC) constructed successfully is useful to study both the regulation of intracellular HCV gene expression and the pathogenesis of HCV infection, as well as the molecular foundation of related transgene animals and gene therapy.     

An investigation into the prevalence of Renibacterium salmoninarum in farmed rainbow trout, Oncorhynchus mykiss (Walbaum), and wild fish populations in selected river catchments in England and Wales between 1998 and 2000

A cross-sectional survey of Renibacterium salmoninarum infection in farmed rainbow trout (RBT) and wild fish populations was carried out in 10 farms and six river catchments, respectively, in England and Wales. The majority of the wild fish were sampled in 1998 and the farmed fish in 2000. Grayling, Thymallus thymallus, and brown trout, Salmo trutta, were the main wild species sampled. Two fish, one grayling and one salmon, Salmo salar, were R. salmoninarum culture-positive, compared with 40 confirmed polymerase chain reaction-positive wild fish. The highest prevalence of R. salmoninarum infection was found in grayling in rivers with RBT farms with a history of R. salmoninarum infection. One hundred and fifty fish were sampled from each RBT farm, but none of the fish was found to be R. salmoninarum-positive. Evidence was found, for the first time, for the presence of R. salmoninarum in an eel, Anguilla anguilla.     

Real time PCR detection of Piscirickettsia salmonis from formalin-fixed paraffin-embedded tissues

Piscirickettsia salmonis is the causative agent of piscirickettsiosis, a transmissible disease of salmonid fish. Diagnosis of piscirickettsiosis has traditionally been based upon identification of typical pathological changes by histological investigation, with confirmation by immunohistochemistry on formalin-fixed, paraffin-embedded tissues. However, implementation of more rapid confirmatory techniques, preferably with higher levels of sensitivity and possibilities for quantification, is desirable. A real-time polymerase chain reaction (PCR) assay was designed for specific detection of P. salmonis and tested on samples extracted from formalin-fixed paraffin-embedded material. Construction of a PCR-target mimic allowed determination of detection limits, linearity of the real-time PCR and quantitative detection of P. salmonis. The present study demonstrates the capability of the described real time PCR assay for detection of P. salmonis from paraffin-embedded material with a high degree of sensitivity and specificity. Implementation of this assay constitutes an important development for a rapid and secure diagnosis of piscirickettsiosis.     

甘肃金鳟RAPD反应体系构建及优化

以甘肃金鳟尾鳍为试验材料,提取基因组DNA。对影响甘肃金鳟RAPD扩增的重要参数进行了优化试验,包括模板DNA浓度、dNTP浓度、引物浓度、Taq酶、变性时间、退火温度等,建立了甘肃金鳟RAPD反应的最适体系。即在25μl反应体系中:模板DNA用量为4.0 ng/μl;Mg2 浓度为2.5mmol/L;dNTP浓度为0.5 mmol/L;引物浓度为0.5μmol/L;Taq酶用量为1 U;扩增程序:94℃预变性4 min,94℃变性45 s,36℃退火50 s,72℃延伸1 min,共35个循环,最后延伸10 min。     

珍珠黄杨DNA的提取方法比较及ISSR反应体系的优化

为从珍珠黄杨幼叶中提取高质量的总DNA,比较了1%CTAB、2%CTAB、4%CTAB和1.5%SDS 4种DNA提取方法.结果表明:2%CTAB提取的DNA A260/A280值最好,ISSR-PCR扩增效果最佳,是有效提取珍珠黄杨基因组DNA的方法.并在此基础上,以(CT)8G为引物,采用单因素实验法,确立了适合珍珠黄杨的ISSR-PCR反应体系:在总体积20 μL的反应体系中,含30 ngDNA模板,2.0 mmol/LMg2 ,0.30μmol/L引物,0.20 mmol/L dNTPs,1 U Taq酶.     

The isolated,perfused bovine ear. A model for pharmacological study of cutaneous vasculature and anaphylaxis

Bovine ears were catheterized and perfused with Krebs-Henseleit (K-H) solution at a constant flow rate. The perfusion pressure (PP) was monitored using a pressure transducer coupled to a carrier preamplifier, amplifier and a pen recorder. Dose-response curves to phenylephrine, histamine and 5-hydroxytryptamine were obtained. The ear preparations responded to these agonists dose-dependently by increases in perfusion pressure. Ear preparations from sensitized calves were challenged with 0.05 ml of horse serum which caused increased perfusion pressure. These preliminary results suggest that the perfused bovine ear represents a promising, simple, inexpensive model for the study of pharmacological mechanisms of the skin.