人α1/158Ⅴ型(α1c型)干扰素基因在大肠杆菌中的表达及其产物的初步纯化

采用DNA聚合酶链反应(PCR)技术,将本实验室从中国人胎肝细胞染色体DNA中发现和分离的IFN-α1/158V基因的原始克隆,改造成适于进行非融合蛋白原核表达的结构形式,并在大肠杆菌中获得高效表达。测得重组IFN-α1/158V的抗病毒活性为1.9×10~7单位/升菌液。随后又采用以单克隆抗体亲和层析为主的纯化流程对表达产物进行初步纯化,获得了在SDS-聚丙烯酰胺凝胶电泳上呈现单一条带的纯化产物。     

几种药物抑制马传染性贫血病毒和人免疫缺陷病毒的实验研究

用马传染性贫血病毒—驴胚肺二倍体细胞(EIAV-DELDC)为实验体系,以细胞中病毒逆转录酶活性及病毒相关抗原的表达为观察指标,检测了叠氮胸苷(AZT)、三氮唑核苷(Ribavirin,病毒唑)、磷羧基甲酸钠(PFA)和苏拉明等4种已知抗人免疫缺陷病毒(HIV-1)药物对马传染性贫血病毒的抑制作用。结果表明,PFA、AZTTP(三磷酸AZT)和苏拉明均能抑制病毒相关抗原的表达,AZT虽无此作用,但能抑制细胞内逆转录酶活性。用~3H-TMP掺入法比较了PFA、AZTTP、苏拉明对体外无细胞系EIAV逆转录酶粗提物和HIV-1基因工程产物逆转录酶活性的抑制作用表明,两种逆转录酶对苏拉明的敏感性相近,而HIV-1逆转录酶对PFA和AZTTP的敏感性较EIAV者高约100倍。又以无细胞系中逆转录酶活性测定法,检测了12种中药提取物的抑制作用,其中小柴胡汤对EIAV和HIV-1逆转录酶活性都有抑制作用,IC_(50)为717μg/ml和700μg/ml(生药浓度)。小柴胡汤对两种病毒感染细胞中抗原的表达和HIV引起细胞病变都有抑制作用,对HIV-1的抑制比EIAV强。这些结果表明,EIAV-DELDC体系可考虑作为抗HIV-1药物筛选模型。     

中华眼镜蛇蛇毒因子(CVF)对补体系统的作用及与人补作C_3和其他蛇毒抗原性关系

中华眼镜蛇蛇毒经DEAE-Sepharose CL-6B。HPLC等多次柱层析分离出有抗补体及溶血活性的眼镜蛇蛇毒因子(Cobra venom factor,CVF),纯化后的CVF在聚丙烯酰胺凝胶电泳图谱上呈单一区带,分子量为225000—230000,等电点为6.20。用二硫苏糖醇还原经SDS-聚丙烯酰胺凝胶电泳得三类亚基,其分子量总和为237,000。 体外抗补体及溶血试验表明,CVF的作用是通过补体旁路途经使总补体活力下降。双向免疫电泳鉴定,发现CVF与人血清作用后,其中补体成分C_3分子的抗原性发生改变,则表明CVF的作用是通过激活补体成分C_3而发挥的。给豚鼠腹腔注射CVF(0.15ug/g体重)后,其血清总补体水平下降到正常值的3％以下,7天后回升,13天后恢复到正常水平。 单相免疫电泳表明,CVF与人补体C_3抗血清间无任何交叉免疫反应,但人血清与CVF抗血清间有微弱的免疫沉淀反应。另外,CVF的氨基酸组成与人补体C_3也较为相似。鉴定还表明眼镜蛇科中四种蛇毒与CVF抗血清有强烈的免疫沉淀反应,蝰蛇毒及海蛇毒也有免疫沉淀反应,但只有眼镜蛇毒具有抗补体活性。     

T3 reverses the changes in met-enkephalin and beta-endorphin contents in the anterior lobe, but not the neuro-intermediate lobe of the pituitary of rats rendered hypothyroid by PTU-treatment

The changes in met-enkephalin and beta-endorphin contents in the pituitary in PTU-induced hypothyroidism were studied in the rat. After 2 weeks of PTU-treatment, both IR-met-enkephalin and IR-beta-endorphin contents in the pituitary were significantly reduced. Gel filtration chromatography followed by radioimmunoassay showed that the immunoactivities in the peaks of precursors, met-enkephalin, beta-lipotropin and beta-endorphin were all lower in the pituitaries from the PTU-treated rats. In another experiment, some of the PTU-treated rats were injected daily with 500 micrograms T3/kg b.w. In the hypothyroid rats, IR-met-enkephalin and IR-beta-endorphin contents were decreased in both the anterior and neurointermediate lobes. Only the changes in the anterior lobe were reversed by T3 treatment. In conclusion, while the effects on the anterior lobe are probably due to a deficiency in thyroid hormones, the mechanism for the decrease of opioid peptide contents in the neurointermediate lobe is still unclear.     

The functional cell surface glycoprotein CD9 is distinguished by being the major fatty acid acylated and a major iodinated cell-surface component of the human platelet

We showed that a 22 kDa protein (which comigrated with the leukocyte differentiation antigen CD9 as determined by immunoblotting with the platelet-activating mAb 50H.19) is a major iodinated component of the platelet surface. The iodinated protein was identified as CD9 by limited proteolysis analysis. The major acylated protein in platelets incubated with [3H]palmitic acid also had a mobility of 22 kDa. The radiolabelled fatty acid in CD9 appears to be ester bonded, as it is removed by treatment with hydroxylamine. Non-enzymatic ligation of the fatty acid is not involved. Since platelets lack protein synthetic capacity, the palmitolation of a surface protein indicates the existence of a plasma-membrane located transacylase which functions independently of protein synthesis. Limited proteolysis analysis of the palmitylated protein obtained by immunoprecipitation with mAb 50H.19 confirmed its identity as CD9. An additional novel minor component of 27 kDa was detected in platelets by immunoprecipitation of 125I-surface-labelled, or [3H]palmitic acid-labelled protein, and by immunoblotting with mAb 50H.19. The analogous cleavage patterns obtained by the limited proteolysis analysis of the 22, 24 and 27 kDa glycoproteins suggest that they may be differently modified variants of a single polypeptide.     

Establishment and characterization of murine macrophage hybrids

Proteose peptone-induced peritoneal macrophages from CBA/J (H-2k) mice have been fused to a hypoxanthine phosphoribosyltransferase-negative variant of the P388D1 (H-2d) murine macrophage cell line. Six hybrid clones were isolated following HAT selection and further characterized. Five of the six clones express class I antigens of both parental haplotypes by microelisa and by flow cytometric analysis. Class II antigen expression of both haplotypes was apparent following a 72-hr incubation of the hybrids with concanavalin A-stimulated rat spleen cell supernatant. However, I-Ad was expressed in all hybrids to a greater extent than I-Ak. Three clones with the highest level of I-Ak expression, E5, C2, and C4, were capable of antigen presentation to the I-Ak-restricted T-cell line, D10.G4.1. LPS induction of the hybrids resulted in a 2- to 15-fold increase in the amount of IL-1 produced relative to the P388D1 parent. Finally, in distinction to P388D1, all hybrids demonstrated increased Fc-mediated erythrophagocytosis of chromium-labeled antibody-coated erythrocytes. These murine macrophage hybrids appear stable and should serve as useful models in understanding the regulation of macrophage function.     

Modulation of reconstituted cholesterol 7 alpha-hydroxylase by phosphatase and protein kinase

Cholesterol 7 alpha-hydroxylase activity was completely inhibited by incubation with alkaline phosphatase in a reconstituted enzyme system containing a cytochrome P-450, NADPH-cytochrome P-450 reductase and phospholipid. On the other hand, cAMP-dependent protein kinase stimulated cholesterol 7 alpha-hydroxylase activity by 2.5-fold. The modulation of cholesterol 7 alpha-hydroxylase activity was dependent on the amount of phosphatase or kinase added. The phosphatase inhibited enzyme activity was partially reversed by the treatment with protein kinase. These experiments indicate that the reconstituted cholesterol 7 alpha-hydroxylase activity is reversibly regulated by phosphorylation/dephosphorylation mechanism.     

The relative cytotoxicity and mutagenicity of cyclobutane pyrimidine dimers and (6-4) photoproducts in Escherichia coli cells

In order to calculate the relative cytotoxicity and mutagenicity of (5-6) cyclobutane pyrimidine dimers and (6-4) photoproducts, we have measured survival and mutation induction in UV-irradiated excision-deficient E. coli uvrA cells, with or without complete photoreactivation of the (5-6) dimers. Radioimmunoassays with specificity for (5-6) dimers or (6-4) photoproducts have shown that maximum photoreactivation eliminates all of the (5-6) dimers produced up to 10 Jm-2 254-nm light, while it has no effect on (6-4) photoproducts. These results were confirmed by measuring the frequency of T4 endonuclease V-sensitive sites. Based on the best fit equations for survival and mutation induction, we have found that the calculated cytotoxicity of (6-4) photoproducts is similar to that of (5-6) dimers; however, the former is much more mutagenic than the latter.