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41.
42.
影响大肠杆菌中外源基因表达的因素 总被引:41,自引:1,他引:40
大肠杆菌已经被广泛地应用于表达各种外源基因,但是,不同的外源基因在表达效率上却有很大的差异,文章综述了影响大肠杆菌中外源基因表达的因素,这将有助于认识大肠杆菌中外源基因表达的规律,以便采取有效的方法提高外源基因在大肠杆菌中的表达效率. 相似文献
43.
Christopher M. de Fiebre Ping Wu Dean Notabartolo William J. Millard Edwin M. Meyer 《Neurochemical research》1994,19(6):643-648
The ability of Sendai virosomes or LipofectinTM to introduce an AAV vector into primary rat brain astroglial cultures was characterized. The pJDT95npy vector was constructed by inserting rat NPY cDNA downstream from the indigenous AAV p5, p19 and p40 promoters in pJDT95. LipofectinTM-mediated transfection with pJDT95npy (10 g) resulted in pronounced expression of several NPY mRNA species: p5-driven (3.3 kb), p19-driven (2.7 kb) and p40-driven (0.6, 0.8, 1.1, and 1.8 kb). Exposure to virosomally encapsulated pJDT95npy (50 or 100 ng) resulted in transient expression of some p40-driven mRNA species (0.8 and 1.8 kb). Neither method produced astroglia cells which synthesized mature NPY immunoreactivity. This demonstrates that an AAV-derived vector can drive gene expression in astroglia, that Sendai virosomes can infuse vectors into astroglia, but that the amount of DNA infused in this manner may limit long term expression. 相似文献
44.
M.Luisa Ricci Riccardo Manganelli Cesare Berneri Graziella Orefici Gianni Pozzi 《FEMS microbiology letters》1994,119(1-2):47-52
Abstract A protocol for efficient electrotransformation of Streptococcus agalactiae (group B streptococcus) Lancefield's strain O90R (NTCT 9993) (an unencapsulated derivative of type Ia strain O90) was developed. The Escherichia coli - Streptococcus shuttle vector pDP28 (7.8 kb) carrying the ermB gene for resistance to erythromycin was used as donor DNA. Frozen 'electrocompetent' cells were prepared by repeated washes in 10% glycerol. A 50-μl aliquot containing about 5×109 colony forming units of bacteria was subjected to the electric pulse. Optimal conditions for electrotransformation were determined using different media, harvesting cells at different points of the growth curve, and using different field strengths. The dose-response curve for transformation of S. agalactiae with pDP28 showed one-hit kinetics as donor DNA varied between 0.01 and 3 μg. The efficiency of electrotransformation for this range of amounts of donor DNA was 1.2×104 cfu μg−1 . The transformation frequencies obtained with this electroporation protocol are high enough to allow both subcloning and shotgun cloning of streptococcal DNA in S. agalactiae . 相似文献
45.
46.
Suspension cultures of Sf-9 cells at different stages of growth were infected with a recombinant baculovirus expressing -galactosidase, using a range of multiplicities of infection (MOI) of 0.05 to 50. Following infection, the cells were resuspended either in the medium in which they had been grown or in fresh medium. Specific -galactosidase yields were not markedly affected by either MOI or medium change in cultures infected in early exponential phase (3×106 cells mL–1). In cultures infected at later growth stages, -galactosidase yields could only be maintained by medium replacement. The possibility that this requirement for medium replacement is due either to the accumulation of an inhibitory byproduct or nutrient limitation was examined. Alanine, a major byproduct of cultured insect cell metabolism, did not significantly reduce recombinant protein yield when added to infected cultures in concentrations of up to 40 mM. Following a factorial design, various nutrient concentrates were added alone or in combination to cultures infected in late exponential phase. Additions that included both yeastolate ultrafiltrate and an amino acid mixture restored specific -galactosidase yields to levels observed at earlier growth stages or in late stages with medium replacement; the addition of these concentrates, by permitting production at higher cell density, led to increases in the volumetric yield of recombinant protein. Together or separately, the concentrates when added to uninfected late exponential phase cultures, lead to a doubling of the maximum total cell protein level normally supported by unamended medium. 相似文献
47.
鲑鱼生长激素基因分泌型表达质粒的构建 总被引:1,自引:0,他引:1
生长激素(GH)是动物垂体前叶分泌的一种多肽类激素.应用分子重组及PCR等技术,构建了一种鲑鱼生长激素基因分泌型表达质粒pOsGH153,使编码鲑鱼生长激素成熟肽的序列克隆在大肠杆菌分泌型表达载体PIN-Ⅲ-ompA内,直接位于编码大肠杆菌外膜蛋白A信号肽序列的下游,在Lpp-Lac杂合启动子控制下,经IPTG诱导,分子量约23 000的鲑鱼生长激素在大肠杆菌中获得高效表达,该产物具有天然鲑鱼生长激素的免疫活性,直接分泌到细胞周质,而信号肽被自动剪除. 相似文献
48.
呼和浩特地区蔬菜污染现状综合评价 总被引:1,自引:0,他引:1
呼和浩特地区蔬菜污染现状综合评价杨在中(内蒙古大学,呼和浩特010021)黄雅琴(内蒙古环境监测中心站,呼和浩特010010)ComprehensiveassessmentonpollutionstatusofvegetablesinHuhehot.... 相似文献
49.
生物素标记HBV RNA探针的制备及应用 总被引:1,自引:0,他引:1
本文首次采用SP65特殊质粒与人类的乙型肝炎病毒DNA重组,制备了Bio-HBV RNA探针,能特异地与HBV DNA杂交,将该探针与缺口转移方法标记的Bio-HBV DNA探针进行了比较,结果显示出Bio-HBV RNA探针比Bio-HBV DNA探针的敏感性提高10倍,并分别应用两种探针同时检测70例乙肝病人血清中HBV DNA,阳性率各为31.42%、28.57%(P>0.25)。对Bio- 相似文献
50.
Genetics of lactobacilli: Plasmids and gene expression 总被引:20,自引:0,他引:20