利用脊髓灰质炎病毒5‘NCR和RNA聚合酶基因可提高外源基因表达

将含脊髓灰质炎病毒（ＰＶ）ＲＮＡ聚合酶的不同长度基因片段克隆到载体ｐＳＧ５质粒上,分别构建了４个表达ＲＮＡ聚合酶的质粒。体外转录实验证明,ｐＳＧ５－ＰＯＬ１．９９和ｐＳＧ５－ＰＯＬ２．０３质粒转染细胞的提取物促进了特异的ＲＮＡ转录,表明两质闰可表达ＲＮＡ聚合酶。将ＰＶ的５’ＮＣＲ序列插在载体ｐＧＲＥＥＮ ＬＡＮＴＥＲＮ－１的ＣＭＶ启动子下游,构建了ｐＧＲＥＥＮ ＬＡＮＴＥＲＮ－１－５’ＮＣＲ质粒;     

Epstein─Barr病毒膜抗原在中国仓鼠卵巢（CHO）细胞中的表达

将切去３’端穿膜序列的ＥＢ病毒膜抗原（ＭＡ）基因,插入ｐＳＶ２－ｄｈｆｒ质粒的ＳＶ４０早期启动子下游,构建了真核表达载体ｐＳＶ２－ｄｈｆｒＧＰＴＲ,使两个ＳＶ４０早期启动子分别调控ＭＡ和二氢叶酸还原酶（ｄｈｆｒ）基因。将该重组质粒转化ＣＨＯ－ｄｈｆｒ细胞,在选择培养基中筛选阳性克隆,用氨甲喋呤加压扩增,建立了表达ＥＢＶ－ＭＡ的克隆细胞系。ｗｅｓｔｅｒｎｂｌｏｔ分析证明,所表达的蛋白的分子量大约为３４０ｋｄ和２２０ｋｄ。经过细Ｓｅｐｈａｒｏｓｅ２Ｂ琼脂糖凝胶层析初步纯化的抗原与福氏佐剂混合免疫小鼠,２周后小鼠血清中出现明显的ｇｐ３４０／２２０特异性抗体,表明切去嵌膜区结构的ＥＢＶ－ＭＡ基因在ＣＨＯ细胞中的表达产物具有同天然膜抗原相似的分子量大小、糖基化程度、免疫特异性和免疫原性,可望成为ＥＢ病毒人用基因工程亚单位疫苗。     

Epstein-Barr病毒BNLF-1基因的克隆及其在PA317细胞中的表达

本文报道以人Ｅｐｓｔｅｉｎ－Ｂａｒｒ病毒的潜伏膜蛋白基因ＢＮＬＦ－１作为目标基因,插入至逆转录病毒载体ｐＺｉｐＮｅｏＳＶ（ｘ）的克隆位点上,由此获得重组逆转录病毒载体ｐＺｉｐＮｅｏＳＶ（ｘ）－ＬＭＰ及其反义载体ｐＺｉｐＮｅｏＳＶ（ｘ）－ａｎｔｉ－ＬＭＰ,通过质粒ＤＮＡ的电转染和Ｇ４１８培养基筛选,然后对１０个抗性克隆进行基因整合分析,获得６个含ＭＬＶ－ＬＴＲ／ＢＮＬＦ－１融合基因的阳性克隆,采用Ｎｏｒｔｈｅｒｎ杂交及Ｗｅｓｔｅｒｎ印迹证明,在克隆细胞中表达了２．６ｋｂ的ｍＲＮＡ片段及相应的蛋白质分子,这是由ＢＮＬＦ－１基因的ＥＤＬ１启动子表达的产物。     

hBMP—2cDNA在COS细胞和小鼠肌肉中的表达

研究了骨形态发生蛋白ＢＭＰ－２ｃＤＮＡ在ＣＯＳ细胞和小鼠肌肉中的表达的情况,从ｐＳＰＳ６５ＢＭＰ－２质粒中回收ＢＭＰ－２ｃＤＮＡ,删除５端的非翻译序列,插入ｐＳＶＬ载体中,构建了含有ＢＭＰ－２全长编码序列的重组表达质粒ｐＳＶＬＢＭＰ－２将表达质粒导入ＣＯＳ－７细胞中,细胞ＲＮＡ点杂交结果表明,转染ＢＭＰ－２基因的细胞内ＢＭＰ－２的ｍＲＮＡ水平明显升高,细胞培养上清的ＥＬＩＳＡ显示,转染ＢＭＰ－２ｃ     

汉滩病毒S基因及其5‘端真核表达载体的构建及在细胞中的表达

体外研究汉滩病毒（ＨＴＮＶ）Ｓ基因及其５'端表达的意义,为核蛋　白Ｔ细胞表位的研究奠定基础。设计２套引物,用ＰＣＲ方法从ＰＢＶ２２０－Ｓ２２原核质粒中扩增出Ｓ　基因全读码框（３７－１３２６ｂｐ）及Ｓ基因５'端（３７－５０１ｂｐ）,用ＴＡ克隆将其克隆入ｐｃＤＮＡ３．１／Ｖ５－Ｈｉｓ－ＴＯＰＯ载体中,成功构建ｐｃＤＮＡ３．１－Ｓ及ｐｃＤＮＡ３．１－Ｓ－Ｎ真核表达载体,并通过脂质体转　染至Ｖｅｒｏ细胞中,进行了瞬时表达。间接免疫荧光成功检测到ｐｃＤＮＡ３．１－Ｓ及ｐｃＤＮＡ３．１－Ｓ－Ｎ在Ｖｅｒｏ细胞中的表达。ｐｃＤＮＡ３．１－Ｓ及ｐｃＤＮＡ３．１－Ｓ－Ｎ真核表达载体有较高的转染效率,目　的基因能在宿主细胞中表达,有利于研究ＨＴＮＶ－Ｓ基因在Ｔ细胞表位研究中的意义。     

hBMP－2cDNA在COS细胞和小鼠肌肉中的表达

研究了骨形态发生蛋白ＢＭＰ－２ｃＤＮＡ在ＣＯＳ细胞和小鼠肌肉中的表达的情况,从ｐＳＰＳ６５ＢＭＰ－２质粒中回收ＢＭＰ－２ｃＤＮＡ,删除５＇端的非翻译序列,插入ｐＳＶＬ载体中,构建了含有ＢＭＰ－２全长编码序列的重组表达质粒ｐＳＶＬＢＭＰ－２。将表达质粒导入ＣＯＳ－７细胞中,细胞ＲＮＡ点杂交结果表明,转染ＢＭＰ－２基因的细胞内ＢＭＰ－２的ｍＲＮＡ水平明显升高;细胞培养上清的ＥＬＩＳＡ显示,转染ＢＭＰ－２ｃＤＮＡ后,细胞分泌产生的ＢＭＰ－２显著增加。小鼠实验发现,在肌肉内用注射法导入ＢＭＰ－２重组质粒后,局部组织内ＢＭＰ－２的ｍＲＮＡ转录水平也明显提高。     

小鼠4.5S RNA逆转座子对Luc基因表达的影响

利用ＲＴ－ＰＣＲ方法,从小鼠肝脏组织总ＲＮＡ中扩增出４．５ＳＲＮＡ的ｃＤＮＡ。该ｃＤＮＡ被克隆到ｐＧＥＭ３Ｚｆ（＋）质粒上,酶切鉴定并测序。然后将该序列插入以Ｌｕｃ基因作为报道基因的表达载体ｐＳＶｌｕｃ２０的ＰｖｕⅡ位点,构建了含４．２ＳＲＮＡ逆转座子的表达载体ｐＳＶｌｕｃ２０－４．５Ｓ。脂质转染法将表达载体导入小鼠骨髓瘤细胞ＮＳ－１、ＳＰ２／０和人乳腺癌细胞Ｂｃａ６１。结果表明,小鼠４．５ＳＲＮ     

含有Epstein-Barr病毒膜抗原的重组表达质粒及其基因免疫

将Ｅｐｓｔｅｉｎ－Ｂａｒ（ＥＢ）病毒主要的膜抗原（ＭＡ）ＢＬＬＦ１基因片段插入ｐＨＤ１０１－３质粒的ＣＭＶ启动子下游,构建了真核表达质粒ｐＨＤ－ｇｐ３５０,并转染２９３细胞进行瞬间表达。用免疫荧光法从细胞膜检测到表达的抗原能与其单克隆抗体发生特异性结合,Ｗｅｓｔｅｒｎ－ｂｌｏｔ法证实,表达的抗原分子量为３５０ｋＤ．用能在真核细胞表达的重组质粒ｐＨＤ－ｇｐ３５０的ＤＮＡ,经Ｓｅｐｈａｒｏｓｅ２Ｂ柱纯化后,注射经普鲁卡因预处理的Ｂａｌｂ／Ｃ小鼠的四头肌,观察到ＥＢＶ－ＩｇＡ／ＭＡ抗体水平比ＥＢＶ－ＩｇＧ／ＭＡ低,而ＥＢＶ－ＩｇＡ／ＭＡ的持续时间比ＥＢＶ－ＩｇＧ／ＭＡ长。采用表达ＥＢＶＭＡ的质粒ＤＮＡ与ＣＨＯ细胞表达的ＭＡ蛋白免疫小鼠,均获得抗ＥＢＶＭＡ的抗体。     

牛疱疹病毒Ⅰ型前早期基因BICPO的表达对牛免疫缺陷病毒LTR的反式激活作用

由含有ＢＨＢＶ－１（ＢｏｖｉｎｅＨｅｒｐｅｓＶｉｒｕｓ－１）前早期基因的基因组片段亚克隆ＢＩＣＰＯ（ＢＨＶ－１ＩｎｆｅｃｔｅｄＣｅｌｌＰｒｏｔｅｉｎＯ）的ＤＮＡ序列至表达载体ｐＳＶＫ３,构建质粒ｐＳＶ２．９。将该质粒与ｐＢＬＴＲ－Ｌｕｃ共转染小牛肺细胞,检测转染细胞裂解物的荧光素酶活性,ＢＩＣＰＯ的表达产物可以显著地激活ＢＩＶＬＴＲ启动子控制下的荧光素酶基因的表达。根据ｐＳＶ２．９与含有ＢＩＶＬＴＲ不同区段缺失的质粒ｐＤ－３１９－Ｌｕｃ、ｐＤ－１１５－Ｌｕｃ、ｐＤ－５２－Ｌｕｃ共转染小牛肺细胞的实验结果,推测ＢＩＶＬＴＲ－３１９位上游区的ＤＮＡ序列影响ＢＩＣＰＯ基因产物对ＢＩＶＬＴＲ表达的激活作用。     

Epstein－Barr病毒潜伏膜蛋白（LMP）基因在哺乳动物传代细胞中的表达

ＥＢ病毒潜伏膜蛋白（ＬＭＰ）是由病毒编码的主要的与病毒致宿主细胞潜伏感染有关的蛋白之一。我们用基因重组技术,把含有ＬＭＰ基因（ＢＮＬＦ１）３个外显子（ｅｘｏｎ）开放阅读框架（ＯＲＦ）的长１．８０ｋｂｐ的ＤＮＡ片段,和能分解ＨｙｇｒｏｍｙｃｉｎＢ的含有ＳＶ４０早期启动子和ＨｇｒｙｏｍｙｃｉｎＢ磷酸转移酶全基因（长１０２５ｂｐ）的ＤＮＡ片段（长１．６０ｂｐ）,同时重组于亚克隆载体ｐＢｌｕｅｓｃｒｉｐｔＳＫ（ｐＢＳ）中,并使该重组质粒ｐＢＳ－ＬＭＰ－Ｈｙｇ（长５７６７ｂｐ）在乳地鼠肾传代细胞（ＢＨＫ）中获得表达。ＢＨＫ细胞在经此重组质粒转染后,ＬＭＰ阳性细胞是２％,在ＨｙｇｒｏｍｙｃｉｎＢ的持续压力下,ＬＭＰ表达细胞率可达２０％。３个月后,ＬＭＰ表达细胞逐渐减少。５个月后,不能测到ＬＭＰ表达细胞。经免疫荧光和蛋白印迹（Ｗｅｓｔｅｍｂｌｏｔ）实验证实,人鼻咽癌、风湿性关节炎和正常人血清中不含有抗ＬＭＰ抗体。     

PCR检定OSM cDNA转染细胞中基因组整合与转录

用PCR和RT-PCR方法对人OSM cDNA转染的小鼠黑色素瘤细胞进行基因组整合和mRNA转录的检定.基因组整合检定时,采用与调控序列和cDNA序列相对应的上、下游引物,以连续的转录单位进行扩增,能够更准确地反映整合与表达的关系;mRNA检定时,采用与cDNA序列和质粒克隆位点与加polyA信号之间序列相对应的上、下游引物,可以区分宿主细胞中内源性与外源性基因的转录.     

IL-6, leukemia inhibitory factor,and oncostatin M stimulate bone resorption and regulate the expression of receptor activator of NF-kappa B ligand,osteoprotegerin, and receptor activator of NF-kappa B in mouse calvariae

IL-6, leukemia inhibitory factor (LIF), and oncostatin M (OSM) are IL-6-type cytokines that stimulate osteoclast formation and function. In the present study, the resorptive effects of these agents and their regulation of receptor activator of NF-kappaB ligand (RANKL), RANK, and osteoprotegerin (OPG) were studied in neonatal mouse calvaria. When tested separately, neither human (h) IL-6 nor the human soluble IL-6R (shIL-6R) stimulated bone resorption, but when hIL-6 and the shIL-6R were combined, significant stimulation of both mineral and matrix release from bone explants was noted. Semiquantitative RT-PCR showed that hIL-6 plus shIL-6R enhanced the expression of RANKL and OPG in calvarial bones, but decreased RANK expression. Human LIF, hOSM, and mouse OSM (mOSM) also stimulated 45Ca release and enhanced the mRNA expression of RANKL and OPG in mouse calvaria, but had no effect on the expression of RANK. In agreement with the RT-PCR analyses, ELISA measurements showed that both hIL-6 plus shIL-6R and mOSM increased RANKL and OPG proteins. 1,25-Dihydroxyvitamin D3 (D3) also increased the RANKL protein level, but decreased the protein level of OPG. OPG inhibited 45Ca release stimulated by RANKL, hIL-6 plus shIL-6R, hLIF, hOSM, mOSM, and D3. An Ab neutralizing mouse gp130 inhibited 45Ca release induced by hIL-6 plus shIL-6R. These experiments demonstrated stimulation of calvarial bone resorption and regulation of mRNA and protein expression of RANKL and OPG by D3 and IL-6 family cytokines as well as regulation of RANK expression in preosteoclasts/osteoclasts of mouse calvaria by D3 and hIL-6 plus shIL-6R.     

Secretion of oncostatin M by infiltrating neutrophils: regulation of IL-6 and chemokine expression in human mesothelial cells

Recently, we identified that regulation of leukocyte recruitment by IL-6 requires shedding of the IL-6R from infiltrating neutrophils. In this study, experiments have examined whether other IL-6-related cytokines possess similar properties. Levels of oncostatin M (OSM) and leukemia inhibitory factor were analyzed in patients with overt bacterial peritonitis during the first 5 days of infection. Although no change in leukemia inhibitory factor was observed throughout the duration of infection, OSM was significantly elevated on day 1 and rapidly returned to baseline by days 2-3. The source of OSM was identified as the infiltrating neutrophils, and OSM levels correlated both with leukocyte numbers and i.p. soluble IL-6R (sIL-6R) levels. FACS analysis revealed that OSM receptor beta expression was restricted to human peritoneal mesothelial cells. Stimulation of human peritoneal mesothelial cells with OSM induced phosphorylation of gp130 and OSM receptor beta, which was accompanied by activation of STAT3 and secretion of CC chemokine ligand 2/monocyte chemoattractant protein-1 and IL-6. Although OSM itself did not modulate CXC chemokine ligand 8/IL-8 release, it effectively suppressed IL-1beta-mediated expression of this neutrophil-activating CXC chemokine. Moreover, OSM synergistically blocked IL-1beta-induced CXC chemokine ligand 8 secretion in combination with the IL-6/sIL-6R complex. Thus suggesting that OSM and sIL-6R release from infiltrating neutrophils may contribute to the temporal switch between neutrophil influx and mononuclear cell recruitment seen during acute inflammation.     

Molecular and functional characterization of a soluble form of oncostatin M/interleukin-31 shared receptor

Activation of the signaling transduction pathways mediated by oncostatin M (OSM) requires the binding of the cytokine to either type I OSM receptor (leukemia inhibitory factor receptor/gp130) or to type II OSM receptor (OSMR/gp130). In the present work we have developed an enzyme-linked immunosorbent assay detecting a soluble form of OSMR (sOSMR) secreted by glioblastoma, hepatoma, and melanoma tumor cell lines. sOSMR was also present in sera of healthy individuals, with increased levels in multiple myeloma. Molecular cloning of a corresponding cDNA was carried out, and it encoded for a 70-kDa protein consisting of a half cytokine binding domain containing the canonical WSXWS motif, an immunoglobulin-like domain, and the first half of a second cytokine binding domain with cysteines in fixed positions. Analysis of the soluble receptor distribution revealed a preferential expression in lung, liver, pancreas, and placenta. sOSMR was able to bind OSM and interleukin-31 when associated to soluble gp130 or soluble interleukin-31R, respectively, and to neutralize both cytokine properties. We have also shown that OSM could positively regulate the synthesis of its own soluble receptor in tumor cells.     

腺病毒介导的人抑瘤素M基因对A375人黑色素瘤细胞的抑制作用

目的:构建抑瘤素M(OSM)重组腺病毒载体,研究其对人黑色素瘤细胞A375的抑制作用。方法:以PEGZ-OSM重组质粒为模板,通过PCR技术扩增出OSM片段,采用腺病毒载体的基因重组和体外包装技术获得表达与人OSM氨基酸序列相同的重组腺病毒子Ad-OSM,感染A375细胞,用荧光显微镜、RT-PCR、Western blot法检测OSM在A375细胞中的转录和表达;荧光显微镜观察A375细胞的形态学改变;MTT法和流式细胞术(FCM)检测Ad-OSM对A375细胞的生长抑制和细胞周期的抑制效应;半定量RT-PCR法检测OSM基因表达对A375细胞中的Bax、Bcl-2基因表达的影响。结果:基因测序和PCR分析结果显示,成功构建了Ad-OSM腺病毒表达载体;RT-PCR和Western blot法检测到OSM基因在A375细胞中的转录和表达;OSM基因的表达对A375细胞增殖有明显抑制作用,并可诱导细胞凋亡,OSM基因可通过上调细胞中Bax和下调Bcl-2基因表达诱导细胞凋亡。结论:成功构建了Ad-OSM腺病毒表达载体,感染OSM基因可明显抑制A375人黑色素瘤细胞的生长,诱导其凋亡,该现象可能是通过改变Bax、Bcl-2基因表达水平来发挥抗肿瘤作用。     

Characterization of the α-haemolysin determinant from the human enteropathogenic Escherichia coli O26 plasmid pEO5

The 157-kb conjugative plasmid pEO5 encoding α-haemolysin in strains of human enteropathogenic Escherichia coli (EPEC) O26 was investigated for its relationship with EHEC-haemolysin-encoding plasmids of enterohaemorrhagic E. coli (EHEC) O26 and O157 strains. Plasmid pEO5 was found to be compatible with EHEC-virulence plasmids and did not hybridize in Southern blots with plasmid pO157 from the EHEC O157:H7 strain EDL933, indicating that both plasmids were unrelated. A 9227-bp stretch of pEO5 DNA encompassing the entire α- hly CABD operon was sequenced and compared for similarity to plasmid and chromosomally inherited α- hly determinants. The α- hly determinant of pEO5 (7252 bp) and its upstream region was most similar to corresponding sequences of the murine E. coli α-hly plasmid pHly152, in particular, the structural α- hly CABD genes (99.2% identity) and the regulatory hly R regions (98.8% identity). pEO5 and α-hly plasmids of EPEC O26 strains from humans and cattle were very similar for the regions encompassing the structural α- hly CABD genes. The major difference found between the hly regions of pHly152 and pEO5 is caused by the insertion of an IS 2 element upstream of the hly C gene in pHly152. The presence of transposon-like structures at both ends of the α- hly sequence indicates that this pEO5 virulence factor was probably acquired by horizontal gene transfer.     

Oncostatin M is a differentiation factor for myeloid leukemia cells.

Oncostatin M (OSM) is a 28-kDa glycoprotein produced by stimulated macrophages and T lymphocytes that inhibits the proliferation of a number of different cell lines derived from solid tumors. Analysis of both amino acid sequence and gene structure has demonstrated that OSM is a member of a cytokine family that includes leukemia inhibitory factor (LIF), IL-6, and granulocyte colony-stimulating factor (G-CSF). We demonstrate that, like LIF, IL-6 and G-CSF, OSM can induce the differentiation of the myeloblastic M1 murine leukemia cells into macrophage-like cells. The morphologic and functional changes induced by OSM are more similar to those observed with LIF and IL-6 than those induced with G-CSF. OSM can also induce the differentiation of the histiocytic U937 human leukemia cells in the presence of granulocyte-macrophage CSF, a property shared with LIF and IL-6. In murine M1 cells, binding of labeled OSM is completely inhibited by excess LIF or OSM, reflecting the binding of OSM to the high affinity form of the murine LIF receptor. In contrast, the binding of labeled OSM to human U937 leukemia cells is inhibited by OSM, but the inhibition by LIF is significantly less. These results suggest that, in human leukemia cells, OSM may act through the LIF receptor and an OSM-specific receptor. The existence of an OSM-specific receptor was confirmed by both growth inhibition and competition binding assays on A375 human melanoma cells. The growth of human A375 cells was inhibited by OSM and IL-6 but not LIF or G-CSF. Neither LIF, G-CSF, nor IL-6 could compete with the binding of labeled OSM to A375 cells.     

Oncostatin M induces tissue-type plasminogen activator and plasminogen activator inhibitor-1 in Calu-1 lung carcinoma cells

Oncostatin M (OSM) is a glycoprotein cytokine that is produced by activated T-lymphocytes, monocytes, and macrophages. In a DNA synthesis assay, OSM reduced tritiated thymidine incorporation by 53% in Calu-1 lung carcinoma cells. Radiolabeled cDNAs from untreated Calu-1 cells and 30-h OSM-treated cells were used to probe duplicate nylon membrane cDNA expression arrays. This study revealed OSM-mediated expression of mRNAs encoding tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1). Northern blot analysis showed that the steady-state level of tPA mRNA is nearly undetectable in Calu-1 cells. Exposure of these cells to OSM for 30 h increased tPA mRNA expression by 20-fold and PAI-1 mRNA expression by 5-fold. Exposure of these cells to other gp130 receptor family cytokines, including leukemia inhibitory factor (LIF), interleukin-6 (IL-6), and IL-11, do not significantly affect DNA synthesis or induction of tPA/PAI-1. Western blot studies demonstrated that OSM mediates a marked increase in secretion of the tPA protein. Secreted tPA was present in the conditioned medium almost exclusively as tPA/PAI-1 complexes. Inhibitor studies demonstrated that OSM-mediated induction of tPA and PAI-1 mRNAs is largely dependent upon activation of the MEK1/2 pathway. The JAK3/STAT3 pathway potentially serves a secondary role in these regulatory events.