Analysis of protein transport through the golgi in a reconstituted cell-free system

The processes which transport membrane proteins between compartments of the Golgi apparatus have been reconstituted in vitro using isolated Golgi fractions. This cell-free system allows a detailed analysis of protein transport not possible in intact cells. Transport of the membrane glycoprotein (G protein) of vesicular stomatitis virus (VSV) is measured from a “donor” to an “acceptor” Golgi fraction. The donor Golgi fraction is prepared from VSV-infected Chinese hamster ovary (CHO) mutant cells deficient in the glycosylation enzyme N-acetylglucosamine transferase I. “Acceptor” is prepared from uninfected wild-type CHO cells. Transport is measured by the addition of N-acetylglucosamine to G protein, which can occur only upon movement of G protein from donor to acceptor. Transport requires physiological pH and osmolarity, is dependent on nucleotide triphosphates, and is mediated by proteins both from cytosol and on the Golgi membranes. Protein movement is inhibited by the non-hydrolyzable GTP analogue, GTPγS. The process of transport proceeds through the budding, pinching off, targeting, and fusion of transport vesicles. In this system these vesicles are initially coated with a non-clathrin coat and are targeted with this coat intact. Several of the proteins which mediate transport have been characterized, and isolated to homogeneity. The successful development of this assay has led to the formulation of cell free assays for protein transport between other compartments. Comparison of these systems indicates that some common mechanisms of vesicular movement are used in transport between a variety of membrane compartments.     

P-糖蛋白基因疫苗的构建与鉴定

目的　制备P 糖蛋白基因疫苗。方法　利用PCR方法扩增编码人类P 糖蛋白多药耐药基因序列胞外区约 1kb片段 ,与真核表达载体pcDNA3进行定向重组 ,限制性内切酶BamHI和XhoI双酶切反应及测序鉴定该重组基因疫苗 ,磷酸钙共沉淀法转染人类K5 62红白血病细胞 ,经G418筛选后 ,免疫组化分析该重组基因疫苗表达产物的抗原特异性。结果　构建了pcDNA3 MDR1重组基因疫苗。限制性内切酶BamHI和XhoI双酶切分别显示 5 .4kb的载体片段及约 1kb的插入序列 ,测序 948个碱基中除 2个碱基突变外均与原序列排列相符。免疫组化方法证实细胞膜表面MDR1约 1kb片段的表达产物可被抗Pgp抗体识别。结论　构建的pcDNA3 MDR1重组基因疫苗可被市售的Pgp抗体特异性识别 ,具有Pgp抗原特异性     

贻贝过氧化氢酶的纯化及部分性质

新鲜贻贝肉匀浆抽提液经硫酸铵盐析、ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＦＦ柱层纯化,得到一种过氧化氢酶。该酶为糖蛋白,亚基分子量约为７６０００,中性糖含量约为１０．５６％,研究结果表明,贻贝过氧化氢酶的最佳ＰＨ７．０左右,对热不稳定,４５℃保温１５ｍｉｎ,酶的残余活力约为７０％;６０℃保温１５ｍｉｎ,则酶的残余活力仅剩１０．６％左右。ＮａＮ３、ＫＣＮ及某些金属离子如Ｐｂ＾２＋、Ｈｇ＾２＋、Ｆｅ＾２＋、Ｃｄ     

Molecular adsorption: early stage surface exploration

In this study, the atomic force microscope has been employed in force spectroscopy mode to gain information on the interaction between long mucin molecules and a positively charged surface during the first few seconds of interaction. Recent studies have revealed that negatively charged mucin molecules introduced to a positively charged surface are kinetically trapped and bind very rapidly, assuming non-equilibrium conformations. This systematic study of surface dwell times has revealed that significant differences exist in mucin adsorption during the first three seconds of introduction to the surface and provides direct evidence of molecular rearrangement for several seconds before trapping occurs. Limited interactions were recorded at dwell times of less than one second, with increased molecular rearrangement observed between 1.5 and 2.25 s. Increasing the surface dwell time beyond this critical limit caused rupture of the tip-tethered mucin molecules during the retract cycle of the cantilever. All subsequent recorded events, at increased dwell times up to 3 s, revealed events at much reduced distances from the point of contact between the mucin functionalised-cantilever and the positively charged surface.     

Supplementation of squalene attenuates experimentally induced myocardial infarction in rats

We have investigated the preventive effects of squalene against isoprenaline-induced myocardial infarction in male albino rats. Supplementation with squalene significantly prevented the isoprenaline-induced adverse changes in the levels of protein and glycoprotein components in plasma and heart tissue of experimental groups of rats. It exerted an antioxidant effect by inhibiting the isoprenaline-induced lipid peroxidation and by maintaining the level of non-enzymatic free radical-scavenger, reduced glutathione at near normalcy. Histopathological observations also confirmed the possible cardioprotective action of squalene by maintaining the normal architecture of the heart tissue. The results of the present investigation demonstrate that supplementation with squalene offers cardioprotection in experimental rats by its antioxidant and membrane- stabilizing properties.     

人巨细胞病毒gB基因真核表达载体在小鼠中的免疫效果

目的观察人巨细胞病毒(HCMV)gB基因真核表达载体在小鼠中的免疫效果。方法大量提取目的质粒,分3个剂量组进行多点肌肉注射免疫小鼠,利用MTT法检测免疫小鼠的T细胞增殖活性,ELISA法检测小鼠血清中HCMV特异性抗体,中和试验检测小鼠血清中和抗体。结果ELISA检测结果表明,与空白对照、空载体对照组相比,3个剂量组pcD-NA3.1/gB质粒免疫后小鼠血浆中抗HCMV IgG抗体水平均明显增高(P<0.05),中和抗体水平为1∶20~1∶40,而对照组血清中无中和抗体存在。淋巴细胞增殖指数免疫小鼠与正常小鼠差异无显著意义。结论已构建的HCMV gB基因真核表达载体可以诱导小鼠产生明显的体液免疫,为核酸疫苗研究奠定了基础。     

狂犬病病毒糖蛋白基因重组慢病毒载体的构建及鉴定

目的构建狂犬病病毒SRV9株糖蛋白基因重组慢病毒表达载体,并进行鉴定。方法将狂犬病病毒SRV9株糖蛋白基因克隆至慢病毒载体pLVX-ZsGreen-IRES上,筛选阳性重组克隆。将质粒pLVX-ZsGreen-IRES-SRV9G、包膜质粒pMD2.G和包装质粒psPAX2共转染293T包装细胞系,通过荧光显微镜观察报告基因表达情况。收集上清,经浓缩后接种293T细胞,进行重组慢病毒感染细胞的鉴定;用限制性内切酶切除质粒pLVX-ZsGreen-IRES-SRV9G绿色荧光蛋白基因,采用直接免疫荧光试验检测狂犬病病毒糖蛋白的表达情况。结果重组慢病毒表达载体经酶切和测序证明构建正确。荧光显微镜下可见重组慢病毒感染的293T细胞有大量绿色荧光出现,病毒滴度为3×107 IU/ml;直接免疫荧光检测表明,糖蛋白基因在293T细胞内获得有效表达。结论成功构建了狂犬病病毒SRV9株糖蛋白基因重组慢病毒载体,为狂犬病基因工程疫苗的研制奠定了基础。     

狂犬病毒糖蛋白DNA疫苗的构建及免疫效果的研究

目的构建狂犬病毒糖蛋白基因DNA疫苗,并检测其免疫小鼠的免疫应答。方法用RT-PCR法扩增和分离CTN株狂犬病毒糖蛋白基因,测序后克隆入VR1055载体中,构建重组质粒VR1055-CTN,经碱裂解法提取质粒,并经Sepharose4FF柱层析纯化后免疫NIH小鼠,用ELISA法检测体液抗体,用MTT法检细胞免疫,并进行常规效力试验。结果VR1055-CTNDNA疫苗具有较好的诱导狂犬病毒抗体的能力,在诱导细胞免疫中,刺激脾淋巴细胞转化指数和小鼠血清中的IL-2活性均高于空白载体对照,差异有显著意义(P=0.000)。效力试验的小鼠存活率分别为73%和25%。结论该疫苗既能诱导体液免疫,又能诱导细胞免疫,并具有较好的免疫保护效果。     

狂犬病毒aG株糖蛋白在CHO细胞中的表达

目的克隆狂犬病毒aG株糖蛋白(Glycoprotein)基因,构建重组真核表达载体,在CHO细胞中表达aG株糖蛋白。方法采用RTPCR,从狂犬病毒aG株基因组RNA中扩增GP基因,并将该基因克隆至真核表达载体pCIdhfr上,以脂质体介导法转染CHOdhfr-细胞。用MTX筛选的抗性克隆经ELISA、免疫荧光及Westernblot检测GP蛋白的表达。结果克隆到狂犬病毒aG株糖蛋白全长基因,并在CHO细胞中进行表达。结论成功地在CHO细胞中表达了狂犬病毒aG株的糖蛋白,为进一步开发狂犬病毒基因工程疫苗打下基础。