丁酸钠对CHO-EPO工程细胞株rhEPO表达量的影响

以稳定整合有ｐＥＤＥＰＯ的ＣＨＯＥＰＯ工程细胞株为研究对象,在无血清条件下,系统观察了０５、１０、２５和５０ｍｍｏｌ／Ｌ４个浓度的丁酸钠作用于该细胞株的情况,结果表明：丁酸钠对ＣＨＯＥＰＯ工程细胞的生长有明显的抑制作用;影响ＣＨＯＥＰＯ工程细胞ＥＰＯ表达,浓度１０ｍｍｏｌ／Ｌ可提高ＥＰＯ表达量２５倍左右,并可持续较长的一段时间;延缓ＣＨＯＥＰＯ工程细胞在无血清培养时的细胞脱落;提高ＣＨＯＥＰＯ工程细胞ＥＰＯｍＲＮＡ水平     

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。     

高产稳产聚羟基烷酸的重组大肠杆菌的构建

重组大肠杆菌Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉＨＭＳ１７４（ｐＴＺ１８ＵＰＨＢ） 含有携带聚羟基烷酸（ＰＨＡ） 合成基因（ ｐｈａＣＡＢ）＊＊ 的质粒ｐＴＺ１８ＵＰＨＢ,是很有潜力的ＰＨＡ 生产菌,但存在着质粒不稳定和不能合成３羟基丁酸（３ＨＢ） 与３羟基戊酸（３ＨＶ） 共聚物［Ｐ（３ＨＢｃｏ３ＨＶ）］ 的缺陷。将ＲＫ２ 质粒上的ｐａｒ ＤＥ 基因引入ｐＴＺ１８ＵＰＨＢ 构成质粒ｐＪＭＣ２ ,该质粒可以在宿主Ｅ．ＣｏｌｉＨＭＳ１７４ 中稳定遗传。将培养基中的磷酸盐浓度降至１８ ｍ ｍｏｌ／Ｌ,发现Ｅ．Ｃｏｌｉ ＨＭＳ１７４（ｐＪＭＣ２） 能够以丙酸为前体合成Ｐ（３ＨＢｃｏ３ＨＶ） ,其中３ＨＶ 在共聚物中的含量为５ ％ ～８ ％ 。在５Ｌ自动发酵罐中分批补料培养Ｅ．Ｃｏｌｉ ＨＭＳ１７４（ｐＪＭＣ２） ,培养基初始磷酸盐浓度为１５ ｍ ｍｏｌ／Ｌ,３０ ｈ 后每升培养液中干菌体可达４２－５ ｇ,Ｐ（３ＨＢｃｏ３ＨＶ） 占干重的７０ ％ ,其中３ＨＶ 在共聚物中的含量为４－９ ％ 。     

人铜锌超氧化物歧化酶cDNA的克隆,测序及表达

用逆转录聚合酶链反应（ＲＴＰＣＲ）,以人胎肝组织总ＲＮＡ为模板,扩增了人铜锌超氧化物歧化酶（ｈＣｕ,ＺｎＳＯＤ）的ｃＤＮＡ,并进行序列分析,将该ｈＣｕ,ＺｎＳＯＤｃＤＮＡ重组到Ｔ７启动子控制下的分泌型表达载体ｐＥＴ２２ｂ（＋）中,构建表达质粒ｐＥＴＳＯＤ,并转化大肠杆菌ＢＬ２１（ＤＥ３）。ＳＤＳＰＡＧＥ及蛋白质印迹分析表明,经１ｍｍｏｌ／Ｌ异丙基硫代βＤ半乳糖苷（ＩＰＴＧ）诱导后,可高效表达一分子量为１９ｋＤ的蛋白质,与抗人ＳＯＤ多抗有特异的免疫反应,表达量约为菌体总蛋白质的３０％,具有特异性ＳＯＤ酶活性,酶活力可达１７９７ｕ／ｍｌ培基。     

抗旱基因HDCS1的植物表达载体构建

在克隆了二棱大麦第３组ＬＥＡｃＤＮＡ,抗旱基因ＨＤＣＳ１的基因上,将其连接于ｐＢ１１２１的ＣａＭＶ３５Ｓ启动子和ＮＯＳ终止子之间,,构建了ＨＤＣＳ１的植物表达载体ｐＢＨＣ,并进行了ＰＣＲ和酶切鉴定,为进行植物抗旱基因工程研究创造了条件。     

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     

Bcl——2基因表达对TNF及OA诱发的细胞编程死亡的不同效应

用ＴＮＦ和ＯＡ（Ｏｋａｄａｉｃａｃｉｄ）诱发人神经母细胞瘤ＳＫ细胞死亡,并证明细胞死亡为编程死亡（ＰｒｏｇｒｍｍｅｄＣｅｌｌＤｅａｔｈ,简称ＰＣＤ）。将编码Ｂｃｌ－２全长蛋白的ｃＤＮＡ植入ＰＪＸ４１ｎｅｏ载体中,使其表达由ＨＣＭＶ病毒起动子控制。形成的顺义（ｐＢｃｌ－２－Ｓ）及反义（ｐＢｃｌ－２－ＡＳ）表达质粒经转染导入ＳＫ细胞中获得稳定转染子。Ｗｅｓｔｅｒｎ印迹表明顺义转染子表达大量的２６ｋｄＢｃｌ－２蛋白,而反义转染子则不表达。增强表达的Ｂｃｌ－２蛋白能抑制由ＴＮＦ引发的ＰＣＤ,但不影响ＯＡ引发的ＰＣＤ,从而证明了Ｂｃｌ－２基因产物抗细胞死亡效应的特异性。     

国产多榔菊属两新种

国产多榔菊属两新种陈艺林（中国科学院植物研究所北京１０００９３）ＴＷＯＮＥＷＳＰＥＣＩＥＳＯＦＤＯＲＯＮＩＣＵＭＬＩＮＮ．（ＣＯＭＰＯＳＩＴＡＥ）ＦＲＯＭＣＨＩＮＡＣＨＥＮＹｉＬｉｎｇ（ＩｎｓｔｉｔｕｔｅｏｆＢｏｔａｎｙ,ｔｈｅＣｈｉｎｅｓｅＡｃ...     

猪繁殖和呼吸综合征病毒膜蛋白和核衣壳蛋白基因在大肠杆菌中的表达

以 Ｐ Ｒ Ｒ Ｓ Ｖ 弱毒株膜蛋白（ Ｍ） 和核衣壳（ Ｎ） 蛋白基因为模板,设计的一对含有 Ｅｃｏ Ｒ Ｉ 和 Ｂａｍ Ｈ Ｉ酶切位点的引物,通过 Ｒ Ｔ Ｐ Ｃ Ｒ 扩增出一约９００ ｂｐ 的 Ｍ Ｎ 基因片段,将此基因片段成功克隆于高效表达载体ｐ Ｂ Ｖ２２０ ,构建成重组质粒ｐ Ｂ Ｖ Ｍ Ｎ,导入大肠杆菌 Ｄ Ｈ５α,经温敏诱导,成功地表达了 Ｍ Ｎ 基因。表达产物经 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 电泳和 Ｗｅｓｔｅｒｎ ｂｌｏｔ 印迹分析,其分子量约３４ ｋ Ｄ,与兔抗 Ｐ Ｒ Ｒ Ｓ Ｖ 高免血清发生反应,经光密度扫描分析,表达产物量占菌体总蛋白的１２ ％ 。该研究为 Ｐ Ｒ Ｒ Ｓ 基因诊断抗原的研制奠定了基础     

大鼠酰胺化酶的信号肽引导的昆虫细胞表达外泌系统

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ＰＡＢＣｈＧＲＦ（Ｇｌｙ）、ＰＡＢＣＩＧＦＩ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、ＢａｃＰＡＩ。将重组病毒感染Ｓｆ２１细胞,ＰＡＢＣｈＧＲＦ（Ｇｌｙ）和ＰＡＢＣＩＧＦＩ均得到有效外泌表达,表达产物通过ＩｇＧＳｅｐｈａｒｏｓｅ柱可获得快速纯化。     

红细胞生成素基因在大鼠体内的表达及表达产物的生物学效应

红细胞生成素（ｅｒｙｔｈｒｏｐｏｉｅｔｉｎ,ＥＰＯ）是一种由胎儿肝脏和成人肾脏产生的多肽类生长因子,在体内的表达具有严格的组织特异性,因此,慢性肾病所引起的贫血常常难以得到有效的治疗．随着基因治疗技术的不断成熟与完善,尤其是近年一些实验室先后发现质粒...     

人乳头瘤病毒16型-L1真核表达质粒的构建和表达以及实验动物对裸DNA的免疫反应

Recombinant plasmid pcDNA-L1 was constructed by inserting HPV16-L1 gene fragment into eukaryotic expression plasmid pcDNA3.1. pcDNA-L1 was transfected into mammalian cells Cos-7 and the expression of HPV16-L1 protein was testified by immunohistochemistry(IHC). Then the recombinant plasmid was directly injected into the quadriceps muscle of BALB/c mice. Antibodies against HPV16-L1 in the immunized mice were positively detected by immunodot and IHC at 28 and 41 days after the last immunization.     

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。     

Features of transfection and expression of cDNA for the human dystrophin gene, delivered into muscles of mdx mice using MF-2 synthetic microspheres]

The number of dystrophin-positive myofibers (DPM), that appeared in different skeletal muscles of mdx mice after a single injection of synthetic microspheres containing the full-length human dystrophin cDNA within the pHSADy expressing vector into femoral quadriceps muscle, was examined on cryostat sections. Injection of 25 micrograms cDNA resulted in the occurrence of 1, 2.4, 5.8 and 4.8% of DPM in the treated muscle in 1, 7, 21, and 60 days after the injection respectively. 7, 21, and 60 days after the treatment, these values comprised 2.1, 4.3 and 1% in the same muscle of the contralateral leg, and 5.5, 8.4, and 1% in the gluteal muscle. Expression of the full-length human dystrophin (427 kDa) in the muscle of the transfected mdx mice was observed. The presence of the transfected construction in skeletal muscles, heart, brain, lungs, and fetuses was demonstrated PCR. Utilization of the FISH technique with biotinilated pHSADy construct as a DNA probe showed that 7 days after the injection, the MF-2 microspheres were present in 70% of myoblast nuclei, in 64% of nuclei of gluteal muscles, and in 62% of the contralateral quadriceps nuclei. 21 days after the treatment, these values were 41, 29, and 45%, respectively. The MF-2 microsphere were detected in the nuclei of the blood, brain, heart, and lung cells, as well as in the placenta and tissues of 18-day-old fetuses. Our results demonstrated the high efficiency of microsphere-mediated transfer of gene constructs into cell nuclei, their long-term intranuclear persistence, and the ability to direct expression for at least 2 months after injection. The MF-2 microspheres attract special interest in respect to the targeted delivery of gene constructs into the nuclei.     

Gene suture--a novel method for intramuscular gene transfer and its application in hypertension therapy

In this report, reporter gene beta-galactosidase (LacZ) was chosen to compare two different intramuscular gene transfer methods, direct injection and gene suture. Evidence showed that gene suture can produce a higher foreign gene express efficiency in skeletal muscle compared with the direct injection method. The highly efficient eukaryotic expressing vectors of human atrial natriuretic factor (ANF) were constructed (pcD2/pAdVAntage/hANF and pcDNA3/hANF), and in vivo ANF gene delivery was performed by intramuscular gene suture. The effects of ANF gene transfer on blood pressure and renal sodium and water excretion were studied in three models of hypertensive animals. Results showed that a marked decrease of mean arterial pressure (MAP) and a significant increase of urine volume and urinary sodium excretion was produced in rats receiving the hANF construct due to the local expression of ANF and its secretion into plasma. Taken together, these results indicate that gene suture may represent a novel gene delivery modality in gene therapy.     

Gene therapy for rat renal anemia with implantation of erythropoietin-transgenic myoblasts

To investigate whether an erythropoietin (EPO) gene-based therapy could serve as an alternative to the repeated injection of rhEPO in treatment to renal anemia, the genetically modified myoblasts of rats, named Myo/ EPO, were implanted through intramuscular injection to model rats with renal anemia. The hemoglobin (Hb) and hematocrit (HCT) of the rats increased from (92. 5±3.0) g/L and 0.29 ±0.04 to the peak values of (103.8 ±5.0) g/L and 0. 32 ±0. 04 respectively 14 d after implantation, and sustained the pre-implantation level for 90 d. Otherwise, the control rats implanted with Myo/X, which carried the parent retroviral vector, gradually became severe in anemia. The PCR detection for hEPO cDNA in the rat muscle adjacent to injection sites indicated that the Myo/EPO cells survived for a long period in the muscle of rats. The results primarily demonstrate that myoblast gene transfer of EPO is effective for the treatment of rat renal anemia.     

Gene therapy for rat renal anemia with implantation of erythropoietin-transgenic myoblasts

To investigate whether an erythropoietin (EPO) gene-based therapy could serve as an alternative to the repeated injection of rhEPO in treatment to renal anemia, the genetically modified myoblasts of rats, named Myo/ EPO, were implanted through intramuscular injection to model rats with renal anemia. The hemoglobin (Hb) and hematocrit (HCT) of the rats increased from (92.5 ±3.0) g/L and 0.29±0.04 to the peak values of (103.8 ±5.0) g/L and 0. 32 ±0. 04 respectively 14 d after implantation, and sustained the pre-implantation level for 90 d. Otherwise, the control rats implanted with Myo/X, which carried the parent retroviral vector, gradually became severe in anemia. The PCR detection for hEPO cDNA in the rat muscle adjacent to injection sites indicated that the Myo/EPO cells survived for a long period in the muscle of rats. The results primarily demonstrate that myoblast gene transfer of EPO is effective for the treatment of rat renal anemia.     

中国人红细胞生成素cDNA克隆与高效表达

以中国人正常胎肝为原料提取总RNA,逆转录合成cDNA与pCR3载体相连接,构建成pE／C表达质粒。序列鉴定结果表明,cDNA序列与国内外已报导的相比,除第2个密码子的第3个碱基系引物设计所致差异外,其余部分完全相同。利用脂质转染法将质粒转入中国白鼠卵巢细胞,获得能高效稳定表达重组人红细胞生成素的工程细胞。     

Optimizing hyaluronidase dose and plasmid DNA delivery greatly improves gene electrotransfer efficiency in rat skeletal muscle

Transfection of rat skeletal muscle in vivo is a widely used research model. However, gene electrotransfer protocols have been developed for mice and yield variable results in rats. We investigated whether changes in hyaluronidase pre-treatment and plasmid DNA delivery can improve transfection efficiency in rat skeletal muscle. We found that pre-treating the muscle with a hyaluronidase dose suitable for rats (0.56 U/g b.w.) prior to plasmid DNA injection increased transfection efficiency by >200% whereas timing of the pre-treatment did not affect efficiency. Uniformly distributing plasmid DNA delivery across the muscle by increasing the number of plasmid DNA injections further enhanced transfection efficiency whereas increasing plasmid dose from 0.2 to 1.6 µg/g b.w. or vehicle volume had no effect. The optimized protocol resulted in ~80% (CI95%: 79–84%) transfected muscle fibers with a homogenous distribution. We also show that transfection was stable over five weeks of regular exercise or inactivity. Our findings show that species-specific plasmid DNA delivery and hyaluronidase pre-treatment greatly improves transfection efficiency in rat skeletal muscle.     

小鼠EVL 基因的克隆和真核表达载体的构建

目的:构建小鼠EVL(Ena/VASP like)基因的真核表达载体,为深入研究EVL的功能奠定基础.方法:采用PCR方法,从小鼠cDNA文库中,扩增出1245bp的EVL编码区片段,经电泳、胶回收后连接入pMD- 18T载体中,测序鉴定正确.用BamHI和HincⅡ双酶切,定向克隆EVL编码区片段到真核表达载体pcDNA3.1中,用限制性内切酶酶切鉴定重组质粒正确后.将重组质粒转染入HELA细胞中,以RT-PCR检测EVL的mRNA的表达,以Western Blot检测EVL蛋白的表达.结果:酶切鉴定结果显示小鼠EVL编码区基因被成功克隆入真核表达载体pcDNA3.1中;RT-PCR和Western Blot结果以及免疫荧光染色显示Hela细胞中有EVL的mRNA和蛋白的表达.结论:成功获得pcDNA3.1 -EVL的真核表达载体,为进一步深入研究EVL蛋白的功能奠定了基础.