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91.
双裂解法提取血清HBV DNA技术的建立 总被引:1,自引:1,他引:0
为进行PCR实验建立了一种两步裂解血清HBV提取其DNA的方法。该法首先用SDS及蛋白酶消化裂解病毒,随后再进行碱裂解,操作较酚提取法大为简化,而PCR实验进行的两法的灵敏性比较,说明双裂解法至少不低于酚法。 相似文献
92.
用DNA重组法表达丙肝病毒部分NS_4-NS_5基因祁自柏,谷金莲,李河民(中国药品生物制品检定所,北京100050)丙型肝炎病毒(HCV)是一种RNA病毒,为研究其复制机理,制备检测丙肝抗体的诊断试剂,需大量纯化的各种丙肝病毒蛋白,我们在国内首次用D... 相似文献
93.
Takashi Ninomiya Masumi Hirabayashi Junko Sagara Atsushi Yuki 《Molecular reproduction and development》1994,37(3):276-283
Fragments containing 5′ flanking regions of four bovine milk protein genes—alpha lactalbumin (bαLA), alpha S1 casein (bαS1CN), beta casein (bβCN), kappa casein (bkCN)—and mouse whey acidic protein (mWAP) gene were prepared by PCR and ligated to human growth hormone (hGH) gene. These recombinant DNAs were microinjected into rat embryos to produce transgenic rats, and the functions of the 5′ regions to direct secretion of hGH in the milk were tested. Although milk was obtained only in 5 of 19 mWAP/hGH rat lines, more than two-thirds of the rats carrying the other four DNAs produced milk. More than 80% of the lactated rats carrying bαLA/, bβCN/, and mWAP/hGH, and 33% of the laclated bαS1CN/hGH rats secreted detectable amounts of hGH (> 0.05 μg/ml) in the milk. In some rats, the hGH concentrations in the milk were comparable to or more than that of the corresponding milk protein in bovine milk. The ranges of hGH concentrations in the milk of bαLA/, bβCN/, bαS1CN/, and mWAP/hGH rats were 1.13–4,360 μg/ml, 0.11–10,900 μg/ml, 86.8–6,480 μg/ml, and 6.87–151 μg/ml, respectively. HGH was also detected in the sera of these rats, and some abnormalities of growth and reproduction were observed. All but one virgin mWAP/hGH rat secreted up to 0.0722 μg/ml of hGH in the serum, and more than half of them showed abnormal fat accumulations at their abdomen. None of the bαCN/hGH rats secreted detectable amount of hGH into their milk, whereas 8 of the 11 lines secreted hGH into their sera. For the production of hGH in transgenic rat milk, the 5′ region of bαS1CN was shown most suitable, because the bαS1CN/hGH rat secreted > 6,000 μg/ml of hGH into the milk and could be reproduced. © 1994 Wiley-Liss, Inc. 相似文献
94.
锌指基因是一种造血调节基因,编码锌指结构蛋白,主要在髓细胞中表达,促进髓细胞分化,在急性早幼粒白血病维甲酸治疗中,促使病情缓解。本文报道了我们从基因分子上研究锌指基因作用中,探索并建立了单向聚合酶链反应(PCR)扩增特定单链DNA,直接测序的新方法。它能产生质和量均佳的单链DNA,无需纯化即可直接用于测序,使复杂的测序研究简便易行,可在2,3天内完成。这种单向PCR扩增特定单链DNA直接测序的方法,经对锌指基因的cDNA测序,得到验证。此法不仅适用于疾病研究中的DNA测序,还可制各单链DNA探针,更利于基因结构组成的研究。 相似文献
95.
Ralph Rapley 《Molecular biotechnology》1994,2(3):295-298
The polymerase chain reaction (PCR) is a powerful core molecular biology technique, which when coupled to chain termination
sequencing allows gene and DNA sequence information to be derived rapidly. A number of modifications to the basic PCR format
have been developed in an attempt to increase amplification efficiency and the specificity of the reaction. We have applied
the use of DNA-binding protein, gene 32 protein from bacteriophage T4 (T4gp32) to increase amplification efficiency with a
number of diverse templates. In addition, we have found that using single-stranded DNA-binding protein (SSB) or recA protein
in DNA sequencing reactions dramatically increases the resolution of sequencing runs. The use of DNA-binding proteins in amplification
and sequencing may prove to be generally applicable in improving the yield and quality of a number of templates from various
sources. 相似文献
96.
L. Pradier E. Heuillet J. P. Hubert M. Laville S. Le Guern A. Doble 《Journal of neurochemistry》1993,61(5):1850-1858
Abstract— In the human astrocytoma cell line U 373 MG, application of substance P (SP) leads to a transient increase in cytosolic calcium concentration and to a biphasic current response in voltage-clamped cells. Using these two functional assays we have characterized pharmacologically the SP response in U 373 MG cells. SP and [l -Pro9]SP displayed high potencies in both assays with EC50values of 2.5 ± 10?9M and 1 ± 10?9M on calcium responses and 110?9M and 510?9M on ion current responses, respectively. The high potency of SP and [l -Pro9]SP as well as the low potency of [Lys5,MeLeu9,N-Leu10]neurokinin A(4-10) and the inactivity of senktide demonstrate the NK1-type pharmacology of these responses. Furthermore, the NK1 antagonists (±)-CP 96,345, its chloro analogue, (±)-cis-3-(2-chlorobenzylamino)-2-benz-hydrylquinuclidine, and RP 67580 were potent antagonists of both SP responses. For the calcium mobilization induced by SP (1 (10?7M), the IC50 values for the three antagonists were 4 ± 10?10M, 4 ± 10?9M, and 9 ± 10?9M, respectively, whereas on the current response evoked by SP 10?8M), the IC50 values were 8 ± 10?9M, 2.4 ± 10?8M, and 1.2 10?7M, respectively. Despite differences in the absolute IC50 values obtained with both techniques, the relative potencies of the three antagonists correlate fairly well. The U 373 MG cell line provides a useful model system for studies of the pharmacology of the human NK1receptor and its transduction mechanisms at the level of second messengers and modulation of ion currents. 相似文献
97.
Christopher M. Elvin Vicki Whan Peter W. Riddles 《Molecular & general genetics : MGG》1993,240(1):132-139
Gene fragments encoding serine proteases expressed in adult buffalo fly (Haematobia irritans exigua) were amplified from cDNA using generic oligonucleotide PCR primers, based on conserved residues surrounding the active-site His and Ser amino acids found in all serine proteases. The PCR product consisted of a broad band extending from about 450 by to 520 bp, which suggested that the PCR product actually consisted of numerous DNA fragments of slightly variable sizes. Seventeen independent clones of these fragments, each with an insert of approximately 480 bp, were digested with HaeIII. Comparison of restriction fragment patterns indicated that 13 of these clones harboured different PCR products. This was confirmed by DNA sequence analysis of 9 clones. Each of the sequenced clones contained an open reading frame which included structurally conserved regions characteristic of the serine protease superfamily. This study reveals the expression of a large and highly variable repertoire of serine proteases in adult buffalo fly. Importantly, these data also demonstrate the utility of such an approach in obtaining DNA probes for use in further investigations of gene family organization and expression, as well as providing recombinant antigens in the form of fusion proteins which may be used as candidates for vaccine production. 相似文献
98.
A panel of bovine, ovine and caprine polymorphic microsatellites 总被引:1,自引:0,他引:1
We report a set of six new bovine microsatellite polymorphisms based on (CA)n repeats. They are highly polymorphic and thus represent valuable markers for genome mapping. Four of the six are polymorphic in sheep and two are polymorphic in goats. One, which is polymorphic in cattle and sheep and apparently monomorphic in goats, is X-chromosome specific and has potential value in, for example, sex determination and detection of chimaerism. 相似文献
99.
《Microbes and infection / Institut Pasteur》2023,25(3):105046
Although arbovirus transmission and identifying target vectors may provide a baseline for planning disease control strategies, there are many gaps in knowledge regarding these mosquitoes and viral species in urban, rural, or sylvatic habitats in the Brazilian Amazon. Our goal was to screen for dengue, chikungunya, and Zika viruses in synanthropic mosquitoes and with Flinders Technology Associates (FTA) cards using insect saliva. Mosquitoes were caught using ovitraps and aspirators in the city of Porto Velho, Rondônia, Brazil. Honey-baited FTA cards were placed in mosquito cages for 15 days; whole mosquitoes and FTA cards were analysed for viral RNA using RT-qPCR assays. One pool of Aedes aegypti females was found to be infected with the Zika virus and one male mosquito was infected with dengue-4, suggesting natural vertical/venereal transmission. Our study also reported evidence of vertical/venereal transmission of ZIKV in Culex quinquefasciatus males for the first time in the Brazilian Amazon, and the feasibility of using FTA cards to detect arboviruses in the saliva of field-collected mosquitoes. Vertical/venereal transmission of viruses by atypical mosquito species reinforces the need for combined viral and entomological screening in arbovirus surveillance programs. 相似文献
100.
在地衣芽孢杆菌NCIB 6816菌株碱性蛋白酶基因已知序列的基础上,通过设计合适的引物,利用PCR(Polymerase Chain Reaction)技术从地衣芽孢杆菌2709菌株的柒色体DNA中扩增了2709碱性蛋白酶的编码序列。对两个克隆的PCR片段的全序列分析结果显示,2709碱性蛋白酶的编码序列同相应的NCIB 6816序列相比有3%左右的碱基组成差异。由此推定的2709碱性蛋白酶的氨基酸序列肯定了2709碱性蛋白酶属典型的subtilisin Carlsberg类,同时还表明来源于不同地衣芽孢杆菌菌株的subtilisin Carlsberg存在着若干氨基酸组成上的差异。 相似文献