HIV－1Protease在大肠杆菌中的高表达

艾滋病的致病因子为人免疫缺陷病毒。该病毒的蛋白酶在病毒复制和成熟中具有决定性的意义。由于目前国内外尚未获得艾滋病病毒蛋白酶高效表达的重组子及简便的活性检测系统，限制了它的研究与应用。本文将用ＰＣＲ技术修饰的ＨＩＶＰｒ基因克隆入原核高效表达载体ｐＴＴＱ１８的ＥｃｏＲⅠ和ＨｉｎｄⅢ酶切位点之间，并用豆芽核酸酶将ＥｃｏＲⅠ的粘端削平，构建了读框正确的表达载体，ＩＰＴＧ诱导表明，该重组子在大肠杆菌中获得了高表达，激光扫描结果表明：重组的ＨＩＶＰｒ占细菌总蛋白８．９％以上。     

肝脏特异性表达载体Kbpala/Alb-ATP7B 的构建及表达

目的：构建小鼠白蛋白启动子引导下携带野 生及常见突变型Arg778Leu、His1069Gln的人ATP7B cDNA的肝脏特异 性表达载体Kbpala/Alb-ATP7B，并检测其表达情况。方法:定点突变法获取人群中常见的Arg778Leu及His1069Gln两种ATP7B突变体，定向克隆将小鼠白蛋白启动子Alb序列及野生和突变的ATP7B cDNA依次亚克隆至转基因载体Kbpala上，得到可在肝脏特异性表达的正常及突变型人 ATP7B的转基因载体Kbpala/Alb-ATP7B。将其 短暂转染BRL细胞及BHK细胞，通过Western blotting检测其蛋白表达情况。结果:经酶切鉴定及定点测序证实，转基因载体Kbpala/Alb-ATP7B 构建成功；Western blotting显示仅在肝脏细胞实现表达。结论:带 有人类ATP7B cDNA的野生及常见致病突变型的转基因载体Kbpala/Alb -ATP7B构建成功并在肝脏细胞特异性表达。     

δ-aminolevulinic acid dehydrase (porphobilinogen synthase) in two families with inherited enzyme deficiency

The inheritance of a deficient delta-aminolevulinic acid dehydrase (ALA-D; synonym: porphobilinogen synthase; EC 4.2.1.24) was studied in blood samples of two families over three generations. The propositus in each family was a young male acute hepatic porphyria patient with an almost complete ALA-D deficiency in the homozygous state (ALA-D activity less than 2% of controls). Heterozygotes are clinically non-affected (mean ALA-D 36% of controls). The mode of transmission could be traced by enzyme activity and electrophoretic polymorphism studies. Heterozygotes are detected by the demonstration of enzyme activity in the gel. The notation D was used for the gene expressing the defective enzyme. The "phenotype" D-1 was observed in six, the "phenotype" D-2 in three of all heterozygotes studied. These results are compatible with a single normal allele in heterozygotes responsible for enzyme activity. Quantitative assays and the segregation pattern in both families suggest a 3-allele-system for the inheritance of ALA-D deficiency.     

Expression and characterization of a soluble rubella virus E1 envelope protein

Individual specific antigenic rubella virus (RV) structural proteins are required for accurate serological diagnosis of acute and congenital rubella infections as well as rubella immune status. The RV envelope glycoprotein E1 is the major target antigen and plays an important role in viral-specific immune responses. The native virion is difficult to produce in large quantities and the protein subunits are also difficult to isolate without loss of antigenicity. The production of a soluble RV E1 (designated E1ΔTm) using the baculovirus-insect cell expression system is described. In contrast to wild-type RV E1, the genetically engineered E1ΔTm protein lacks a transmembrane anchor. It behaved as a secretory protein and was secreted abundantly from insect cells. Pulse-chase studies were used to examine the synthesis, glycosylation, and secretion of E1ΔTm by the insect cells. The secreted E1ΔTm protein was purified from serum-free medium by onestep immunochromatography. The purified E1ΔTm protein retained full antigenicity and may be a convenient source of E1 protein for use in diagnostic assay and rubella vaccine development.     

氯霉素乙酰转移酶检测(CAT assay)

本文介绍一种快速方便的外源基因在哺乳动物细胞中瞬间表达检测系统,即氯霉索乙酰转移酶检测系统。该项检测技术不仅对基因的调控顺序(Cis-acting elements)如启动子、增强子等检测提供了有效的工具,而且为真核基因在哺乳动物细胞表达体系中,质粒的构建及筛选提供了行之有效的手段。并介绍了该技术的全过程及其操作要点。