在杂合启动子控制下乙肝病毒表面抗原SA—28融合基因在酵母中的表达？…

采用酵母杂合启动子ＰＡＤＨＺ－ＣＵＰ１或ＰＡＤＨＺ－ＧＡＰＤＨ及终止子ＴＡＤＨ１,构建了一系列酵母表达载体。在这些表达载体中插入乙肝有面抗原Ｓ－ｐｒｅＳ１融合基因ＳＡ－２８后将含乙肝病毒表面抗原的表达单元克隆至高稳定质粒ＰＨＣ１１的ＢａｍＨⅠ位点。     

聚离子亚基化合物介导的基因转移方法可用于外源基因？…

利用聚离子亚基化合物介导的基因转移方法，研究了外源β－半乳糖苷酶报告基因的瞬时表达。结果表明，聚离子亚基化合物介导的基因转移方法可用于外源基因的瞬时表达研究。而且在相同的条件下，用该方法在ＮＩＨ３Ｔ３细胞和ＰＡ３１７细胞中对报告基因转移的效率     

大肠杆菌pheA与tyrB基因的克隆与串联表达

为探讨用基因工程的手段改良苯丙氨酸的发酵菌株，采用聚合酶链反应（ＰＣＲ）的方法，从大肠杆菌总ＤＮＡ中克隆得到了编码苯丙氨酸合成途中的两个关键酶基因－即分枝酸变位酶（ＣＭ）／预苯酸脱水酶（ＰＤ）基因ｐｈｅＡ与苯丙氨酸转氨酶（ＰＡＴ）基因ｔｙｒＢ，在大肠杆菌中进行了这两个基因的单个和串联表达。ｐｈｅＡ和ｔｙｒＢ基因分别都能在λ噬菌体的ＰＲ启动子之后得到较大量的表达，在ＳＤＳ－ＰＡＧＥ上出现清晰的条带，     

杂交捕获抑制聚腺苷二磷酸核糖聚合酶基因的体外翻译

体外转录载体ｐＳＰ７２—Ｃ１２和ｐＳＰ７２—Ｃ１．４分别经ＸｂａⅠ和ＸｈｏⅠ酶切线性化后，用ＳＰ６ＲＮＡ聚合酶与Ｔ７ＲＮＡ聚合酶进行体外转录得到编码人聚腺苷二磷酸核糖聚合酶基因的ｍＲＮＡ全序列（３．７ｋｂ）及与其５’端编码区的起始密码子ＡＵＧ上游区域互补的反义ＲＮＡ片段（１．４ｋｂ）。等摩尔数的正、反义ＲＮＡ通过酚相乳浊杂交技术进行分子杂交。在网织红细胞裂解物体外翻译系统中，正义ＲＮＡ能正常地翻译出蛋白质；而反义ＲＮＡ与正义ＲＮＡ杂交可阻断翻译的进行。     

多肽在丝状噬菌体外壳蛋白上的展示

利用基因工程方法将丝状噬菌体ｆｄ基因８克隆入ｐＫＫ２３３－３质粒中,将人工合成的六个氨基酸残基的多肽基因插入到修饰后的质粒载体中,并制备了杂合噬菌体。ＳＤＳ－ＰＡＧＥ结果表明,外源多肽基因得到了正常表达,其产物展示于丝状噬菌体的外壳蛋白上。     

牛酪白基因导入大豆的研究

将带有牛酷蛋白基因ｃａｓｅｉｎＢ的植物表达载体ｐＡＳ－２,在大豆自花授粉后,用注射法导入大豆受精子房中。以质粒ｐＡＳ－２的３５Ｓ－Cａｓｅｉｎ-Ｇｕｓ扯段为模板,随机引物法标记探针,对１０３株受体植株后代的ＤＮＡ进行点杂交和Ｓｏｕｔｈｅｒｎ杂交,发现其中１株在点杂交和Ｓｏｕｔｈｅｒｎ杂交中均呈阳性。证明外源基因已整合到大豆基因组中。     

甘薯储藏蛋白(sporamin)A基因在大肠杆菌中的表达研究

利用基因重组技术,将ＰＣＲ扩增获得的甘薯储藏蛋白Ａ基因的成熟肽编码区序列,插入到高效表达载体ｐＴｒｃＨｉｓＢ中,构建成重组表达质粒ｐＴＨＳＰＯ－Ａ,用限制酶切和ＰＣＲ分析均表明重组质粒与预期结果相符,用ＩＰＴＧ诱导重组质粒转化的大肠杆菌ＴＯＰ１０。菌体总蛋白经１２％ＳＤＳ－ＰＡＧＥ分析,可观察到一个大小为１０ｋＤ的蛋白质带,其分子量比预期值小。     

具全期启动子盒的杆状病毒高效表达载体的组建与应用

通过ＰＣＲ扩增，得到苜蓿丫纹夜蛾核型多角体病毒（ＡｕｔｏｇｒａｐｈａｃａｌｉｆｏｒｎｉｃａＮｕｃｌｅａｒＰｏｌｙｈｅｄｒｏｓｉｓＶｉｒｕｓ，ＡｃＮＰＶ）具早晚期启动子元件的ｐ３５基因启动子，将其插入到杆状病毒转移载体质粒ｐＳＸＩＶＶＩ＋Ｘ３多克隆位点上游，使之与ｐＳＸＩＶＶＩ＋Ｘ３质粒中的人工合成后期启动子（ＰＳｙｎ）、多角体ＸＩＶ启动子（ＰＸＩＶ）串联构成早期、晚期、极晚期能持续启动外源基因表达的转移载体质粒ｐＳＸ３５．将ｐＳＸ３５用于组建含ＨＢｓＡｇ基因并形成多角体的重组ＴｎＮＰＶ，ＨＢ－ｓＡｇ基因的表达量显著提高，表达时间亦明显提前，从而实现了外源基因在杆状病毒表达系统的全期、高效表达．ｍＲＮＡ引物延伸试验结果显示，Ｐｐ３５在重组病毒中可产生２套转录本，分别于病毒感染的早期和晚期起始ＨＢｓＡｇ基因的表达．     

沙门氏菌Ⅱ相鞭毛蛋白基因在减毒株中的表达

应用转化转导法把含有沙门氏菌Ⅱ相鞭毛蛋白基因ＦｌｊＢ＾ｅｎｘ的重组质粒ｐＨⅠ１０４导入无鞭毛的减毒株ＳＬ５９２８中获得表达;经电镜观察、玻板凝集试验及动力抑试验证实重组菌表达了相应的外源鞭毛蛋白,ＳＤＳ－ＰＡＧＥ测得表达产物分子量为５２ｋｄ：Ｗｅｓｔｅｒｎ－ｂｌｏｔ试验中表达产物可与Ｈ－２＾ｅｎｘ单因子血清发生特异性反应;体内外试验表明重组菌很稳定,口服免疫小鼠能诱导产生针对表达产物的特异性抗体。     

一种从基因数据库中识别反义snoRNA基因的新方法

报道一种从国际分子生物学数据库中识别反义ｓｎｏＲＮＡ基因的计算机新方法．介绍新方法的生物学原理及在反义ＳｎｏＲＮＡ新基因发现中的应用．     

点突变ras癌基因全cDNA真核表达重组体的构建及诱导NIH3T3细胞转化的研究

将活化癌基因Ｔ２４－ｒａｓ的全ｃＤＮＡ序列正向插入真核载体ｐＭＡＭｎｅｏ，构建成重组质粒ｐＭＡＭｎｅｏ－Ｔ２４－ｒａｓ．将该质粒转染ＮＩＨ３Ｔ３细胞，通过药物筛选，建成细胞系３Ｔ３（Ｔ２４）．Ｓｏｕｔｈｅｒｎ杂交证明外源Ｔ２４－ｒａｓ基因已整合于受体细胞染色体中．３Ｔ３（Ｔ２４）细胞表现出形态学方面的明显变化：具失去接触抑制能力，且在裸鼠体内致瘤等恶性行为．本文构建的Ｔ２４－ｒａｓ基因真核表达重组体和建立的转化细胞系可用于肿瘤的诊断、预防、治疗及抗肿瘤药物的筛选、评估等研究．     

反义PARP基因真核表达重组体的构建及其HeLa转化细胞系的建立

利用DNA体外重组技术将PARP基因cDNA部分序列反向克隆到真核表达载体pMAMneo上,构建成重组质粒pMAMneo－C1．4和pMAMneo－C0．3．将重组质粒pMAM－neo－C1．4转染HeLa细胞,经G418筛选,建成细胞系HeLa－C1．4－neo,Southern杂交结果表明,外源PARP基因cDNA部分序列已稳定整合到受体细胞基因组中,该细胞系的建立为研究聚ADP核糖基化作用在细胞内的功能打下了基础．     

降低聚腺苷二磷酸核糖聚合酶活性对外源癌基因诱导的NIH3T3转化的逆转作用研究

用ＰＡＲＰ酶抑制剂苯甲酰胺（ＢＡ）处理经ｃ－Ｈａ－Ｔ２４－ｒａｓ活化癌基因转染的ＮＩＨ３Ｔ３细胞得到转化细胞系，结果表明：经苯甲酸胺处理的细胞系，其细胞凝集、血清依赖性、形态特征等均发生了改变，呈现出正常细胞的特征．Ｓｏｕｔｈｅｒｎ杂交结果表明，已整合到细胞基因组中的外源Ｔ２４～ｒａｓ基因发生了丢失．     

Effects of CIITA antisense RNA on the expression of HLA class II molecules

To study the effect of the major histocompatibility complex class II (MHC II) transactivator (CIITA) antisense RNA on the expression of the human leukemia (HLA) class II molecules, 5′ end cDNA sequence of CIITA gene was cloned, and antisense RNA expression vector pcDNA-II was constructed. HeLa cells transfected with pcDNA-II and pcDNA3 were induced by IFN-γ for 3 d. The expression of HLA class II molecules on HeLa/pcDNA-II cells was significantly decreased, while it has no effect on the expression of HLA class I molecules. This result suggests that the CIITA antisense RNA can inhibit the expression of HLA class II molecules in HeLa cells. It also implies a promising approach to generate immune tolerance in graft transplantation.     

Raf-1 protein kinase is required for growth of induced NIH/3T3 cells

Many growth factors regulate the cytoplasmic Raf-1 protein kinase, consistent with its having a central role in transduction of growth signals. The kinase is ubiquitously expressed and can promote proliferation, presumably in a manner dependent on growth-factor receptors and membrane-associated oncogenes. We have now examined the dependence of serum- and TPA (12-O-tetradecanoylphorbol-13-acetate)-regulated NIH/3T3 cell growth on RAF-1 kinase to determine whether Raf-1 is essential for receptor signalling. We inhibited Raf-1 function by expressing c-raf-1 antisense RNA or kinase-defective c-raf-1 mutants. Antisense RNA for c-raf-1 interferes with proliferation of normal NIH/3T3 cells and reverts raf-transformed cells. In revertant cells, DNA replication induced by serum or TPA was eliminated or reduced proportionately to the reduction in Raf protein levels. Expression of a kinase-defective Raf-1 mutant (craf301) or a regulatory domain fragment (HCR) inhibited serum-induced NIH/3T3-cell proliferation and raf transformation even more efficiently. Inhibition by antisense RNA or craf301 blocked proliferation and transformation by Ki- and Ha-ras oncogenes. We conclude that raf functions as an essential signal transducer downstream of serum growth factor receptors, protein kinase C and ras.     

Ornithine decarboxylase activity is critical for cell transformation.

The enzyme ornithine decarboxylase is the key regulator of the synthesis of polyamines which are essential for cell proliferation. Expression of this enzyme is transiently increased upon stimulation by growth factors, but becomes constitutively activated during cell transformation induced by carcinogens, viruses or oncogenes. To test whether ornithine decarboxylase could be a common mediator of transformation and oncogenic itself, we transfected NIH3T3 cells with expression vectors carrying the complementary DNA encoding human ornithine decarboxylase in sense and antisense orientations. The increased expression of the enzyme (50-100-times endogenous levels) induced not only cell transformation, but also anchorage-independent growth in soft agar and increased tyrosine phosphorylation of a protein of M(r) 130K. Expression of ornithine decarboxylase antisense RNA was associated with an epithelioid morphology and reduced cell proliferation. Moreover, blocking the endogenous enzyme using specific inhibitor or synthesizing antisense RNA prevented transformation of rat fibroblasts by temperature-sensitive v-src oncogene. Our results imply that the gene encoding ornithine decarboxylase is a proto-oncogene central for regulation of cell growth and transformation.     

Non-viral gene carrier mediated short hairpin RNA interference for inhibition of tumor cells growth

A tumor-targeting gene vector G250mAb-PEI-PEG has been prepared by modification of polyethylenimine （PEI） with polyethyleneglycol （PEG） and G250, a monoclonal antibody against the G250 antigen on tumor cell surface. The transfection efficiency was as high as 70% in G250 positive HeLa cells, whereas the transfection efficiency was relatively low （30%） in normal NIH3T3 cells. A plasmid encoding the short hairpin RNA （shRNA） specific for nucleostemin gene （NS） was efficiently transfected into the HeLa cells with this nonviral gene vector. RNA interference down-regulated the expression of NS gene in HeLa cells, inhibited cells proliferation and induced apoptosis. However, the growth and activity of the NIH3T3 cells were not affected under the same treatment. These results indicate that the reported nonviral gene vector, G250mAb-PEI-PEG, can target and efficiently deliver genes into HeLa cells, and has the potential for the cervical cancer treatment.     

An elaborate pathway required for Ras-mediated epigenetic silencing

The conversion of a normal cell to a cancer cell occurs in several steps and typically involves the activation of oncogenes and the inactivation of tumour suppressor and pro-apoptotic genes. In many instances, inactivation of genes critical for cancer development occurs by epigenetic silencing, often involving hypermethylation of CpG-rich promoter regions. It remains to be determined whether silencing occurs by random acquisition of epigenetic marks that confer a selective growth advantage or through a specific pathway initiated by an oncogene. Here we perform a genome-wide RNA interference (RNAi) screen in K-ras-transformed NIH 3T3 cells and identify 28 genes required for Ras-mediated epigenetic silencing of the pro-apoptotic Fas gene. At least nine of these RESEs (Ras epigenetic silencing effectors), including the DNA methyltransferase DNMT1, are directly associated with specific regions of the Fas promoter in K-ras-transformed NIH 3T3 cells but not in untransformed NIH 3T3 cells. RNAi-mediated knockdown of any of the 28 RESEs results in failure to recruit DNMT1 to the Fas promoter, loss of Fas promoter hypermethylation, and derepression of Fas expression. Analysis of five other epigenetically repressed genes indicates that Ras directs the silencing of multiple unrelated genes through a largely common pathway. Last, we show that nine RESEs are required for anchorage-independent growth and tumorigenicity of K-ras-transformed NIH 3T3 cells; these nine genes have not previously been implicated in transformation by Ras. Our results show that Ras-mediated epigenetic silencing occurs through a specific, complex, pathway involving components that are required for maintenance of a fully transformed phenotype.     

Prevention of chemical carcinogenesis using glutathine S-transferase-pi (GST-pi)

In order to explore whether the protective function of GST-pi can prevent transformation in vitro, NIH3T3 cells and carcinogen glycidyl methacrylate (GMA) have been used in cell transformation study. NIH3T3 cells have been transfected with GST-pi cDNA inserted retrovirus vector, pXT1, and then G418 resistant clones have been analyzed by Southern and Northern analyses. NIH3T3/pXGST clones that stably express GST-pi and control cells, untransfected NIH3T3 and NIH3T3/pXT1, have been treated three times discontinuously with GMA. 1.287% of untransfected NIH3T3 and 1.197% of NIH3T3/pXT, cells obtained a transformation pheno-type, forming type Ⅲ transformed clones, which could grow in soft agar and form fibrosarcoma in nude mice. In comparison, the transformation rate is only 0.007% in NIH3T3/pXGST cells, which could not grow in soft agar and formed no tumor in vivo. The results showed that expression of exogenous GST-pi in NIH3T3 do protect NIH3T3 cells from GMA induced transformation in vitro, which provides an evidence that GST-pi may play a role in preventing chemical car-cinogenesis.