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重组人干细胞因子在昆虫细胞中的高效表达 总被引:5,自引:0,他引:5
含信号肽的可溶性人干细胞因子(hSCF)cDNA 基因重组于杆状病毒转移载体pVL941 中,重组转移载体pVL941SCF与野生型苜蓿夜蛾核型多角体病毒(AcNPV)DNA 共转染草地夜蛾细胞Sf9 后,通过体内同源重组构建了重组病毒AcNPVSCF。Southern 杂交表明重组病毒基因组中含有hSCF基因片段。重组病毒感染单层Sf9 细胞后,表达产物分泌到胞外培养液中。用MTT 比色法和TF1 细胞株测定表达产物与IL3 的协同效应,测得感染重组病毒的培养细胞第三天表达量为1970 units/m L培养液。Westernblotting 分析可见分子量为18 ×103 、20 ×103 和22 ×103 三条带。 相似文献
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用逆转录病毒载体表达人粒细胞—巨噬细胞集落刺激因子 总被引:2,自引:1,他引:1
应用基因工程的方法,将含有巨细胞病毒(CMV)启动子的基因片段和人粒细胞-茂噬细胞集落刺激因子(hGM-GSF)的cDNA,克隆进逆转录病毒载体N2A,得到重组质粒N2QA/CMV/hGM-CSF。经脂质体包装并转染包装细胞,通过G418药物筛选,得到抗生克隆。经PCR和Southern blot检测证实,GM-CSF基因已整合到该克隆细胞的染色体上,获得的逆转录病毒滴度达10^4CFU/ml,克 相似文献
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应用基因工程的方法,将含有巨细胞病毒(CMV)启动子的基因片段和人粒细胞-巨噬细胞集落刺激因子(hGM-CSF)的cDNA,克隆进逆转录病毒载体N2A,得到重组质粒N2A/CMV/hGM-CSF.经脂质体包装并转染包装细胞,通过G418药物筛选,得到抗性克隆。经PCR和Southemblot检测证实,GM-CSF基因已整合到该克隆细胞的染色体上,获得的逆转录病毒滴度达10 ̄4CFU/ml,克隆细胞培养上清用TF-1细胞可检测到GM-CSF活性。 相似文献
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人粒-巨噬细胞集落刺激因子(hGM-CSF)是一种重要的造血生长因子.利用基因重组技术构建两个hGM-CSF的E.coli表达菌株,一个为在不改变氨基酸顺序的前提下,对mRNA翻译起始区核苷酸顺序进行优化突变(hGM-CSF(M)),另一个为未突变的对照(hGM-CSF(N)).经酶切电泳、DNA测序、SDS-PAGE和Westernblot等分析鉴定,证明两者均能表达特异性的14.6kDhGM-CSF,但hGM-CSF(M)的表达水平较hGM-CSF(N)提高了1.26倍,占菌体总蛋白的16.9%.mRNA翻译起始区二级结构预测分析表明,优化突变后生成自由能ΔG从原来的-10.2提高至-9.4Kcal,AUG从部分配对状态变为非配对状态. 相似文献
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将大鼠酰胺化酶的信号肽及前导肽编码序列引入昆虫核多角体病毒转移表达载体,构建PABChGRF(Gly)、PABCIGFI融合基因的昆虫细胞分泌表达质粒pBacPAG2、pBacPAI,并与经修饰的银纹夜蛾核多角体病毒BacPAK6线性化DNA共转染秋粘虫细胞Sf21,通过同源重组、筛选和鉴定,得到它们的重组病毒BacPAG、BacPAI。将重组病毒感染Sf21细胞,PABChGRF(Gly)和PABCIGFI均得到有效外泌表达,表达产物通过IgGSepharose柱可获得快速纯化。 相似文献
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优化mRNA翻译起始区提高人粒—巨噬细胞集落刺激因子在E.coli… 总被引:2,自引:0,他引:2
人粒-巨噬细胞集落刺激因子(hGM-CSF)是一种重要的造血生长因子。利用基因重组技术构建两个hGM-CSF的E.coli表达菌株,一个为在不改变氨基酸顺序的前提下,对mRNA翻译起始区核苷酸顺序进行优化突变(hGM-CSF(M)),另一个为未突变的对照(hGM-CSF(N))。经酶切电泳、DNA测序、SDS-PAGE和Western blot等未突变的对照(hGM-CSF(N))。经酶切电泳、D 相似文献
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庚型肝炎病毒包膜糖蛋白E2基因在昆虫细胞中的表达 总被引:2,自引:0,他引:2
用PCR扩增出HGVE2全基因,克隆进杆状病毒表达载体pFASTBACHTa中,构建成重组转座载体pFASTBACE2,转化DH10BAC大肠杆菌感受态细胞,筛选阳性菌落,抽提大分子质粒DNA,获得含HGVE2基因的重组杆状病毒穿梭载体,转染昆虫草地夜蛾Sf9细胞,出现细胞病变后,收集含有重组病毒颗粒的培养上清,重新感染草地夜蛾Sf9单层细胞及甜菜夜蛾幼虫,分别收集Sf9细胞和甜菜夜蛾幼虫体内的血淋巴细胞,进行12%SDS聚丙烯酰胺凝胶电泳,可见表达的融合蛋白带,经亲和层析进行蛋白纯化,用ELISA方法检测各类血清标本,初步研究HGVE2糖蛋白的抗原性 相似文献
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白细胞介素1(IL-1)是一种重要的细胞因子,具有广泛的生物学活性。它通过与细胞表面的白细胞介素 1受体(IL-1R)结合而起作用。以杆状病毒为载体在昆虫细胞中克隆表达了小鼠I型可溶性白细胞介素1受体(sIL-1 RI)基因。以NIH/3T3细胞RNA为模板,采用RT-PCR方法扩增得到小鼠sIL-IRI的cDNA,克隆至杆状病毒转移载体pAcGP67B,将转移重组质粒与野生病毒ACNPV DNA共转染昆虫细胞Sf9,经同源重组得到重组杆状病毒rACNPV。应用经纯化的rAcNPV感染昆虫细胞Sf9,表达获得重组的sIL-1RI。经对亲和层析样品的SDS-PAGE分析和对IL-1β生物活性阻断作用实验证实,表达产物能够与其配基结合,并且能够分泌至细胞培养上清中。 相似文献
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人粒细胞-巨噬细胞集落刺激因子基因表达受到转录调控与转录后调控。5'非转录区的一些顺式调控成分,如CATT(A/T)重复序列,富含GC序列,CK-1、CK-2、kB特异序列与可诱导的CsA敏感增强子成分等在转录水平上调控hGM-CSF的表达。3'非翻译区有一62bp富含AU序列,这与mRNA的稳定性相关,在翻译水平调控hGM-CSF的表达,细胞因子与一些刺激因子通过不同的机制作用于hGM-CSF基 相似文献
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将含有鸡传染性支气管炎病毒 S1 基因c D N A 的重组转移质粒p S X I V V I+ X3 S1 . Holte 和p S X I V V I+ X3/4 S1 . Holte 分别与粉纹夜蛾核型多角体病毒 Tn N P V S V I- G D N A( O C C- ,gal+ ) 共转染草地夜蛾( Sf9) 细胞,经空斑纯化得到重组病毒 Tn N P V( X3) S1 . Holte O C C+ 和 Tn N P V( X3/4) S1 . Holte O C C+ 。将重组毒株分别感染 Tn5 B1 细胞,并进行 S D S P A G E 与 Westernblot 检测。结果表明, Tn N P V( X3/4) S1 . Holte O C C+ 在感染的细胞中高效表达了 S1 蛋白, S D S P A G E 凝胶薄层色谱分析结果显示,感染病毒后72 h S1 蛋白的表达量占细胞内总蛋白量的35 .8 % ,而 Tn N P V( X3) S1 . Holte O C C+ 感染的细胞内检测不出 S1 蛋白。经分析认为这一差异主要来自 S1 基因翻译起始位点及其附近的周围环境。 相似文献
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正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases 相似文献
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Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme
responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare
the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show
that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by
distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of
demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least
one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of
the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable
potent antiproliferative synthetic drugs. 相似文献
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RICHARD E. NORRIS 《Botanical journal of the Linnean Society. Linnean Society of London》1991,106(1):1-40
Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera. 相似文献
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JOAN VALUÈS MONTSERRAT TORRELL NÚRIA GARCIA JACAS 《Botanical journal of the Linnean Society. Linnean Society of London》2001,137(4):399-407
Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted. 相似文献
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The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle. 相似文献
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肝癌中HBV和HCV基因和抗原的分布及意义 总被引:1,自引:0,他引:1
采用原位分子杂交方法检测HCV
RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV
RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV
RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV
X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV
C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细 相似文献