重组逆转录病毒的制备、浓缩及保存研究

选择含有新霉素磷酸转移酶Ⅱ(Neo^R)的重组逆转录病毒载体,用包装细胞进行包装,得到了含有重组逆转录病毒粒子的病毒上清,该病毒上清经差速离心法浓缩后,－80℃无任何添加物的情况下保存3个月,体外感染靶细胞NIH3T3以测定其滴度,冻存前后的浓缩病毒上清的感染滴度无明显降低,经过冻存的浓缩病毒上清的感染滴度仍可达到10^6cfu/ml,具有理想的感染靶细胞的能力。     

小鼠T-bet逆转录病毒载体的构建与功能验证

为在小鼠细胞中表达并研究T-bet功能,首先构建了含有报告基因Thy1.1的小鼠T-bet逆转录病毒载体,并将构建的载体质粒转染病毒包装细胞系包装成重组病毒,再利用重组病毒分别感染NIH-3T3和D9细胞系检测其感染能力。之后,使用该重组病毒感染T-bet敲除小鼠的CD4+T淋巴细胞,流式细胞术检测T-bet及其下游靶基因Ifng的表达情况。经验证,重组的逆转录病毒感染T-bet敲除小鼠T淋巴细胞后可以在细胞中表达T-bet,并进一步引起下游靶基因Ifng的表达上调,证明本研究中外源表达的转录因子T-bet具有正常功能。综上所述,本实验成功构建了含有报告基因的小鼠T-bet重组逆转录病毒载体,为进一步在小鼠细胞中研究T-bet功能奠定了基础。     

人神经元素3基因重组逆转录病毒表达载体的构建及其包装细胞株的建立

为构建表达人神经元素3基因(neurogenin 3,ngn3)的重组逆转录病毒载体,建立稳定表达ngn3的包装细胞株,本研究以流产人胎儿胰腺组织为材料,通过RT-PCR方法克隆出人ngn3基因,将其连接到pMD18-T载体上并测序,结果表明,测序得到的基因序列与发表的人ngn3基因序列(GenBank Accession No.BC126468)完全一致。将EcoRI和HpaI双酶切后的基因片段构建到pMSCV-neo逆转录病毒载体中,酶切鉴定结果表明,pMSCV-ngn3重组逆转录病毒载体构建成功。脂质体法将pMSCV-ngn3重组载体导入PT67包装细胞,G418筛选后,对得到的细胞株进行RT-PCR和免疫组化检测,结果显示,该细胞株在mRNA水平和蛋白水平均稳定表达Ngn3;收集该细胞株的培养上清液,进行RT-PCR检测及电镜观察,结果表明,该细胞株将导入的重组逆转录病毒载体pMSCV-ngn3包装成了具有感染性的病毒颗粒,并将其释放到了培养上清液中。以上结果表明PT67-ngn3包装细胞株建立成功。该细胞株的成功建立,为下一步将ngn3基因应用于提高人胎儿胰腺祖细胞诱导分化效率方面的研究奠定了基础。     

重组逆转录病毒介导的转蚓激酶基因兔

目的通过重组逆转录病毒介导获得转蚓激酶基因家兔。方法将含有蚓激酶基因的表达盒插入到逆转录病毒载体pLNCL中获得了蚓激酶重组逆转录病毒载体,利用脂质体转染PA317包装细胞,G418进行筛选得到了阳性细胞克隆,克隆扩大培养后病毒上清直接注射雄性兔的睾丸组织转染精原干细胞。结果病毒上清经NIH3T3细胞进行滴度测定,最高滴度为1×104CFU/mL。病毒注射3月龄兔睾丸组织,过1.5个月取其睾丸、肾、脾、肝、肺进行组织切片观察,结果正常。提取交配所得F0、F1代仔兔基因组,经PCR、Southern检测,转基因阳性率F0代为6.3%(2/32)、F1代为15.3%(2/13)。结论通过重组逆转录病毒直接注射3月龄兔睾丸组织可获得转基因兔,病毒对兔的脏器无损害。     

逆转录病毒载体介导的猪瘟病毒配基因的真核表达

利用DNA重组技术将猪瘟病毒(Classicalswinefevervirus,CSFV)石门株囊膜蛋白E2基因插入逆转录病毒载体pBABE-puro中构建成重组逆转录病毒载体pBABE-puro-E2,该重组逆转录病毒载体与pVSVg质粒经磷酸钙共转染法将其转入293GP细胞中包装逆转录病毒假病毒。用包装的假病毒感染PK-15细胞,经嘌呤霉素筛选阳性细胞后进行流式细胞技术(FACS)分析,结果表明CSFVE2基因在PK-15细胞膜上成功表达。将表达E2蛋白的PK-15细胞腹腔免疫Balb/c小鼠,成功诱导小鼠产生了抗E2蛋白的抗体。     

人IL-12重组逆转录病毒载体在HeLa细胞中的表达

目的:包装携带人白细胞介素12(IL-12)的逆转录病毒,用于宫颈癌的治疗研究.方法:携带IL-12的逆转录病毒重组质粒pL35P40SN经PA317细胞包装,G418筛选.在NIH3T3细胞进行病毒滴度测定.然后用病毒感染人宫颈癌细胞HeLa.PCR、RT-PCR方法检测IL-12基因在HeLa中的整合和表达情况.结果:重组质粒pL35P40SN经PA317细胞包装后收获病毒上清,感染HeLa细胞,检测发现IL-12基因整合到细胞基因组DNA中,并且能有效的转录.结论:成功包装了携带IL-12基因的逆转录病毒,该病毒能有效感染HeLa细胞,并使携带的基因IL-12在细胞中表达,为今后IL-12基因治疗宫颈癌的研究奠定基础.     

逆转录病毒载体介导的猪瘟病毒E2基因的真核表达

利用DNA重组技术将猪瘟病毒(Classical swine fever virus , CSFV)石门株囊膜蛋白E2基因插入逆转录病毒载体pBABE-puro 中构建成重组逆转录病毒载体pBABE-puro-E2,该重组逆转录病毒载体与pVSVg质粒经磷酸钙共转染法将其转入293GP细胞中包装逆转录病毒假病毒.用包装的假病毒感染PK-15细胞,经嘌呤霉素筛选阳性细胞后进行流式细胞技术(FACS)分析,结果表明CSFV E2基因在PK-15细胞膜上成功表达.将表达E2蛋白的PK-15细胞腹腔免疫Balb/c小鼠,成功诱导小鼠产生了抗E2蛋白的抗体.     

骨形成蛋白7基因诱导人肝癌细胞的凋亡

目的 构建表达重组人骨形成蛋白7 （bone morphogenic protein 7, BMP7）基因的重组逆转录病毒,观察其对人肝癌细胞HepG2的凋亡诱导活性,并探讨其作用机制。方法 克隆BMP7基因,以loxP同源重组法构成逆转录病毒载体pLP-LNCX-BMP7（pLLBMP7）,转染包装细胞PT67进行病毒包装并测定病毒滴度;将逆转录病毒感染人成骨细胞,MTT法检测细胞生长变化,琼脂糖凝胶电泳和流式细胞仪检测肿瘤细胞的凋亡;Western blotting检测BMP7,caspase-3和bcl-2蛋白表达。结果 重组逆转录病毒载体pLLBMP7经鉴定连接正确,转染PT67细胞后上清液中可得到病毒,滴度达1×109pfu;MTT检测见pLLBMP7病毒组48和72h细胞抑制率高于对照组（35.1% vs. 5.3%,68.5% vs.18.3%,均p<0.05）,48h可见BMP7蛋白高表达。琼脂糖凝胶电泳出现典型梯形条带;流式细胞仪检测出现凋亡峰,于转染48h后达最高峰,其凋亡百分率高达14.42%;BMP7蛋白高表达时caspase-3蛋白的表达亦有显著升高,但bcl-2蛋白未见表达差异。结论 构建了BMP7逆转录病毒,在体外能够有效地诱导人肝癌细胞HepG2的凋亡,其可能是通过激活caspase-3而发生作用。     

逆转录病毒载体介导的LacZ基因在NIH-3T3细胞中的稳定表达

精原干细胞(SSCs)介导的转基因技术很可能成为制作转基因动物及治疗雄性不育的一条新途径。为了研究逆转录病毒载体介导法转染体外培养SSCs的可行性,用脂质体介导法将携带LacZ基因的重组逆转录病毒载体pLNCL导入包装细胞PA317,用含G418的培养液筛选得到5株稳定转染的产毒细胞。收集这些克隆的产毒上清,过滤后进行倍比稀释,用NIH-3T3细胞通过X-gal染色测定其浓缩前病毒滴度。结果显示,PA3173培养上清中病毒的浓缩前滴度最高,达1.1×103CFU/mL。再将筛选到的稳定转染的NIH-3T3细胞培养至单层,进行X-gal染色检测β-半乳糖苷酶的表达。结果显示,大多数稳定转染的NIH-3T3细胞均为X-gal ,表明这些细胞成功表达了目的基因LacZ。本研究结果为后期工作中用该载体感染体外培养SSCs奠定了基础。     

水体中病毒浓缩方法及其条件优化

本研究探讨了在自来水和污水两种不同的水体环境中, 三氯化铝沉淀法和正电荷滤膜法浓缩回收病毒的效率, 并比较应用不同的浓缩条件、洗脱物质时的病毒回收率, 从而建立有效的环境样本中病毒的浓缩方法。结果表明, 三氯化铝沉淀法在两种水体中的回收率均较高, 最高回收率分别达到96.0%和92.0%, 但正电荷滤膜法在两种水体中的回收率差别较大。在自来水中, 正电荷滤膜法的最高回收率为93.9%, 在污水中的最高回收率仅为69.9%, 这说明正电荷滤膜法适用于病毒含量较高且悬浮物等杂质较少的样本。在应用于自来水样时, 两种方法均可起到有效的病毒浓缩作用, 但在悬浮物等杂质较多的污水中, 三氯化铝法具有较好的回收效率。     

Poor transduction efficiency of human hematopoietic progenitor cells by a high-titer amphotropic retrovirus producer cell clone.

The transduction efficiency of human bone marrow CD34+ cells with supernatants from the retrovirus producer cell clone PA317/LGSN 16 was only one-fifth of that with supernatants from GP+ envAm12/LGSN 15, even though both producers had similar infection titers on 3T3 cells. PA317/LGSN 16-conditioned medium inhibited the proliferation of the bone marrow CD34+ cells, and this inhibitory effect was partially blocked by anti-transforming growth factor beta antibodies. These studies suggest that cytokine secretion plays a role in the suppression of retrovirus transduction of human CD34+ cells.     

Concentration and purification of two small RNA viruses: mycophage PS-1 and bacteriophage phi6

Two small RNA viruses, mycophage PS-1, and bacteriophage φ6 were concentrated and purified by the sequential use of continuous flow zonal centrifugation, high speed continuous flow sedimentation, and combined rate zonal centrifugation and isopycnic banding. The continuous flow centrifugations were done using the K-II and K-XI rotors while the BXXIX rotor was used for batch rate zonal centrifugation and isopycnic banding. The deseribed procedures are useful for the concentration and purification of virus from large volumes of culture fluid.     

Biochemical and biological analysis of human interleukin 6 expressed in rodent and primate cells.

The cDNA for human interleukin 6 (IL 6) was stably expressed at high levels in the three mammalian cell lines COS-7, PA317, and GH3 to yield IL 6 proteins of 25 to 27, 26, 22 to 24, and 23 kDa molecular mass. Both size and relative amounts of the recombinant IL 6 (rIL 6) species produced correspond to those of natural IL 6 secreted by LPS-stimulated monocytes. Oligosaccharide analysis of recombinant IL 6 utilizing tunicamycin and endoglycosidases revealed O- and N-linked glycosylation that is comparable to that of natural IL 6 derived from human monocytes and fibroblasts. IL 6 expressed in each of the three cell lines was phosphorylated similarly to the IL 6 produced in human monocytes and fibroblasts. IL 6 secreted by the three different cell lines have marked differences in specific biological activities. COS-7 IL 6 appeared to be 12-fold more active in its hybridoma growth factor activity than that made in PA317 or GH3 cells. In contrast, PA317 and GH3 IL 6 were 230 and 6.7 times more effective than COS-7 IL 6 in inducing Ig production in CESS cells. Also, PA317 and GH3 IL 6 were more effective than COS-7 IL 6 in inducing the acute-phase protein fibrinogen in human hepatocytes. The rIL 6 species exhibited no antiviral activity.     

Association of a protease (plasminogen activator) with a specific membrane fraction isolated from transformed cells

The intracellular distribution of specific protease, plasminogen activator (PA), has been examined in Rous sarcoma virus-transformed chick embryo fibroblasts (RSV-CEF). Cellular homogenates were fractionated by differential centrifugation followed by sucrose gradient centrifugation. The activities and the percent distribution of a series of marker enzymes, specific for different subcellular organelles, were compared to those of PA. Normal CEF have been similarly fractionated and the relatively low amount of PA activity present in these cells has been analyzed in terms of its subcellular distribution. A membrane fraction was isolated from the RSV-CEF that contained the bulk of the PA activity and less than 8% of the total cellular protein. The specific activity of the PA in this fraction is 40-fold higher than that of a comparable fraction isolated from companion cultures of normal cells. This fraction contains little or no nuclear and cytoplasmic material and is contaminated only to a relatively small degree with mitochondria, lysosomes, endoplasmic reticulum. Significant amounts of a putative Golgi membrane marker are present in this fraction. The relatively high specific activities of Na+,K+-ATPase, 5'-nucleotidase, and [3H]fucose indicate that the fraction is enriched in surface membrane. Further purification of the fraction by equilibrium centrifugation on shallow sucrose gradients reduces further the contaminating activities and results in a PA distribution that closely parallels the distribution of the membrane enzyme, 5'-nucleotidase. PA was not released from its membrane association by hypotonic and hypertonic extraction and ultrasonication, while granule-bound enzymes were released by these treatments. The PA activity from hamster SV40 cells fractionated the same way as that of RSV-CEF. These results suggest that a protease that is dramatically enhanced upon malignant transformation is associated with "plasma membrane-like" elements of the cell and may serve as an intrinsic modifier of cell surface proteins after malignant transformation.     

Release of the phophodiesterase activator by cyclic AMP-dependent ATP:protein phosphotransferase from subcellular fractions of rat brain

The subcellular distribution of the endogenous phosphodiesterase activator and its release from membranes by a cyclic AMP-dependent ATP:protein phosphotransferase was studied in fractions and subfractions of rat brain homogenate. These fractions were obtained by differential centrifugation and sucrose density gradient; their identity was ascertained by electron microscopy and specific enzyme markers.In the subcellular particulate fractions, the concentration of activator is highest in the microsomal fraction, followed by the mictochondrial and nuclear fractions. Gradient centrifugation of the main mitochondrial subfraction revealed that activator was concentrated in those fractions containing mainly synaptic membranes.Activator was released from membranes by a cyclic AMP-dependent phosphorylation of membrane protein. The release of activator occurred mainly from the mitochondrial subfractions containing synaptic membranes and synaptic vesicles.The data support the view that a release of activator from membranes may be important in normalizing the elevated concentration of cyclic AMP following persistent transsynaptic activation of adenylate cyclase.     

Release of the phosphodiesterase activator by cyclic AMP-dependent ATP:protein phosphotransferase from subcellular fractions of rat brain.

The subcellular distribution of the endogenous phosphodiesterase activator and its release from membranes by a cyclic AMP-dependent ATP:protein phosphotransferase was studied in fractions and subfractions of rat brain homogenate. These fractions were obtained by differential centrifugation and sucrose density gradient; their identity was ascertained by electron microscopy and specific enzyme markers. In the subcellular particulate fractions, the concentration of activator is highest in the microsomal fraction, followed by the mitochondrial and nuclear fractions. Gradient centrifugation of the main mitochondrial subfraction revealed that activator was concentrated in those fractions containing mainly synaptic membranes. Activator was releasted from membranes by a cyclic AMP-dependent phosphorylation of membrane protein. The release of activator occurred mainly from the mitochondrial subfractions containing synaptic membranes and synaptic vesicles. The data support the view that a release of activator from membranes may be important in normalizing the elevated concentration of cyclic AMP following persistent transsynaptic activation of adenylate cyclase.     

Comparison of methods for recovering indigenous viruses from raw wastewater sludge.

Five general methods for recovering indigenous viruses from raw wastewater sludge were compared. Each method included elution, concentration, and disinfection steps. The elution method, found to consistently yield the greatest viral recovery, was a two-phase technique that involved blending sludge with Freon. Other methods, including two being tested as American Society for Testing Materials tentative standard methods, were less effective. Viral recoveries were generally greater (sometimes much greater) if samples were concentrated by high-speed centrifugation rather than by organic flocculation with 3% beef extract. Three cell lines were used to measure viral recoveries by the plaque assay. The efficiency of recovery was greatest on BGM cells, followed by RD and MA-104 cells.     

The isolation of plasma membrane from frog cardiac muscle cells

A vesicular preparation consisting largely of the plasma membrane of frog cardiac cells was isolated and its enzymatic activities and lipid content were investigated.The enriched plasma membrane preparation was obtained by (1) mildly homogenizing washed ventricles, (2) separating away the cellular debris by low speed differential centrifugation, (3) separating the plasma membrane fraction from other membranous components by centrifugation to equilibrium in various sucrose gradients. The frog cardiac plasma membranes were found to be concentrated between specific gravities of 1.07 and 1.11. In the membrane fraction the specific activities of membrane marker enzymes: 5′-nucleotides (EC 3.1.3.5), alklaline phosphatase (EC 3.1.3.1) and Na⁺---:K⁺)-activated ATPase were, respectively, 19, 15, and 14 times greater than in the homogenate. Activities of mitochondrial marker enzymes were either very low or absent. No unusual lipid types were found. Cardiolipin was less than 0.1% (by wt) in the membrane fraction. The molar ratio of phospholipid to cholesterol was approximatley 3 : 2.     

Separation of Non-filamentous Micro-organisms from Soil by Density Gradient Centrifugation in Percoll

Soil suspensions were homogenized, and desorbed non-filamentous micro-organisms were concentrated in a minimum volume of buffer by low speed centrifugation. The cells were separated from inanimate material by flotation at the interface between the buffer and a silica sol/polyvinyl pyrrolidone density gradient medium (Percoll). Cell suspensions were removed from the interface and fractionated according to density by high speed centrifugation on discriminating density gradients in Percoll.
Preliminary experiments indicated that most non-filamentous soil micro-organisms had densities in the range 1.081–1.123 g%sol;ml while Rhizobium isolated from crushed root nodules on Percoll was split into two bands of densities 1.081–1.110 and 1.041–1.073 g/ml. The lighter cells were the more pleomorphic.
The efficiency of extraction of cells from soil was governed by the extent of their desorption from inanimate particles. As rigorous desorption procedures damage cells, extraction efficiencies were low; 10–20% of cells counted microscopically in soil were recovered from density gradients. Electron microscopy of soil micro-organisms isolated by this method showed an unusual range of surface ornamentations on cell-like structures of bacterial dimensions.     

血吸附法浓缩NDV病毒及与其他几种方法的比较

用人O型红细胞吸附－释放病毒的方法纯化被感染的鸡胚尿囊液中的新城疫病毒（NDV）,然后分别对纯化的病毒液及弃去的上清进行血凝检测。结果表明,使用1％的红细胞悬液只能吸附部分病毒,损失较大,只有将红细胞悬液的浓度增加到20％时,才基本将病毒全部吸附住。同时还比较了差速离心,PEG沉淀,PEG包埋等方法。