一种可高效转染的青鳉肌肉细胞系的建立与应用

在哺乳动物细胞系中转基因的效率相对较高而在鱼类细胞系中相对较低, 为了获得鱼类高效转基因细胞系, 我们从模式鱼类青鳉(Oryzias latipes)中分离和构建了肌肉细胞系OLM。该细胞系类似于纤维细胞, 在28℃ DMEM和10%的胚胎牛血清中培养了超过88代。通过染色体分析, 它属于两倍体并含有48条染色体。在脂质体介导下, 报告基因的转染效率可以达到40%, 在测试的其他鱼类细胞中转染效率最高。此外还构建了稳定转染转基因或者非编码RNA的细胞系。OLM细胞对常见的病毒SVCV、GCRV、SGIV等不敏感。综上所述, 研究在青鳉中建立了一种能高效转染的肌肉细胞系, 它适用于鱼类瞬时和稳定的转基因研究。     

Immune Responses and Protective Efficacy Induced by 85B Antigen and Early Secreted Antigenic Target-6 kDa Antigen Fusion Protein Secreted by Recombinant Bacille Calmette-Guérin

In an attempt to improve immune responses and protective efficacy, we constructed two recombinant bacille Calmette-Guérin （rBCG） strains expressing an 85B antigen （Ag85B） and early secreted antigenic target-6 kDa antigen （ESAT6） of Mycobacterium tuberculosis （MTB） fusion protein. Both rBCG strains have the same protein insertion but in a different order （Ag85B-ESAT6 and ESAT6-Ag85B）. The cultured supernatant of rBCG strains and the sera from the mice immunized with the fusion protein Ag85B- ESAT6 or ESAT6-Ag85B formed a band with a fraction size of 37 kDa, equalivalent to the sum of Ag85B and ESAT6. Six weeks after BALB/c mice were immunized with BCG or rBCG, spleen lymphocytes showed significant proliferation in response to culture filtrate protein of MTB. Compared with the BCG group, mice vaccinated with rBCG elicited a high level increase of immunoglobulin G antibodies to culture filtrate protein in the serum. The T-interferon levels in the lymphocyte culture medium supernatants increased remarkably in the rBCG1 group, significantly higher than that of the BCG immunized group （P〈0.05）. Four weeks after vaccination, mice were infected with M. tuberculosis H37Rv and a dramatic reduction in the numbers of MTB colony forming units in the spleens and lungs was observed in the two rBCG immunization groups. Although these rBCG strains were more immunogenic, their protective effect was comparable to the classical BCG strain, and there were no significant differences between two rBCG groups （p〉0.05）.     

半夏药材生品及其炮制品指纹图谱的比较研究

目的:该实验旨在用高效液相色谱法对半夏的生品及其炮制品的指纹图谱进行比较研究,通过半夏指纹图谱主峰的变化寻找半夏炮制前后其主要化学成分的变化。方法:采用75％乙醇(含1％的盐酸)对半夏药材生品及其炮制品进行提取, DiamonsilTM C18(150×4．6 mm,5um)色谱柱,0．1％磷酸溶液—乙腈梯度洗脱,洗脱时间定为50 min,检测波长217 nm,以精氨酸为对照品,对半夏的生品及其炮制品的指纹图谱的主峰数目和峰高进行比较分析。结果:该方法精密度和重复性RSD均小于3％,符合有关规定,半夏炮制后主峰数目发生变化。结论:半夏生品清制和姜制后,75％乙醇(含1％的盐酸)提取液中其所含精氨酸与半夏生品相比均有不同程度的下降,而且指纹图谱的主峰数目也有所变化,说明半夏炮制后其主要化学成分发生变化。     

Protein prenylation and human diseases: a balance of protein farnesylation and geranylgeranylation

The protein prenylation is one of the essential post-translational protein modifications, which extensively exists in the eukaryocyte. It includes protein farnesylation and geranylgeranylation, using farnesyl pyrophosphate(FPP) or geranylgeranyl pyrophosphate(GGPP) as the substrate, respectively. The prenylation occurs by covalent addition of these two types of isoprenoids to cysteine residues at or near the carboxyl terminus of the proteins that possess Caa X motif, such as Ras small GTPase family. The attachment of hydrophobic prenyl groups can anchor the proteins to intracellular membranes and trigger downstream cell signaling pathway. Geranylgeranyl biphosphate synthase(GGPPS) catalyzes the synthesis of 20-carbon GGPP from 15-carbon FPP. The abnormal expression of this enzyme will affect the relative content of FPP and GGPP, and thus disrupts the balance between protein farnesylation and geranylgeranylation, which participates into various aspects of cellular physiology and pathology. In this paper, we mainly review the property of this important protein post-translational modification and research progress in its regulation of cigarette smoke induced pulmonary disease, adipocyte insulin sensitivity, the inflammation response of Sertoli cells, the hepatic lipogenesis and the cardiac hypertrophy.     

Effects of cyhalothrin on the transient outward potassium current in central neurons of Helicoverpa armigera

The effects of cyhalothrin on the transient outward potassium current in central neurons of Helicoverpa armigera were studied by using the patch clamp techniques. The results showed that before using cyhalothrin （10.5 mmol/L）, activation potential was approximately -40 mV, after application of the drug, the activation potential shifted roughly 10 mV to the negative potential direction, so channels can be activated more easily. Before and after cyhalothrin application, the change of current amplitude was insignificant. The value of V1/2 and k of activation curves did not change significantly, however, the V1/2 of the inactivation curves changed significantly. Inactivation curves significantly shifted to a negative direction, so that inactivation of the channels was hastened. It is indicated that there may exit a primary way in which cyhalothrin provides neurotoxicity to the nervous system through the regulation of activation potentials and inactivation state of IA channels.     

用RAPD技术检测野生鲫鱼的四个金鱼代表品种的基因组DNA多态性

利用随机扩增多态DNA技术检测了两个不同地区的野生鲫鱼(Carassius a uratus L.)和4个金鱼(Carassius auratus Var.)代表品种的基因组DNA的多态性。用26个随机引物对各品种实验鱼的基因组DNA进行扩增,平均每个品种观察到约134个标记,单个引物获得的标记在1-16个之间。实验结果经统计学分析表明,金鱼和鲫鱼的随机扩增多态DNA共亨度高,进一步证实了金鱼由野生鲫鱼演化而来,聚类结果表明,草金鱼形成后,首先演化为文种鱼,然后由文种再形成龙种和蛋种两个品系。 Abstract:Genomic DNA polymorphisms in the wild crucian from rwo different areas and in four representative varieties of goldfish were detected by using random amplified polymorphic DNA(RAPD)technique.The genomic DNA of each variety of fish was amplified with 26 primers.On average,about 134 RAPD markers were observed by each variety.The markers obtained by a single primer varied from 1 to 16.The statistical analysis of the experimental results indicated that random amplified polymorphic DNA of the goldfish and wild crucian have a high proportion of random amplified polymorphic DNA fragment shared.This further verified that goldfish is evolved from wild crucian.The cluster analysis suggested that after emerging,grass goldfish is evolved to wen goldfish,then through wen goldfish evolved to dragoneye goldfish and oval goldfish.