Control of gene expression in plant cells using a 434:VP16 chimeric protein

The herpes simplex virus type 1 VP16 polypeptide is a potent trans-activator of viral gene expression. We have tested the ability of the VP16 activation domain to activate gene expression in plant cells. A plasmid encoding a translational fusion between the full-length 434 repressor and the C-terminal 80 amino acids of VP16, was constructed. When expressed in Escherichia coli, the chimeric protein binds efficiently to 434-binding motifs (operators). For expression in plant cells, the chimeric activator gene was placed between the cauliflower mosaic virus (CaMV) 35S promoter and nos terminator sequences in a pUC-based plasmid. The 434 operators were placed upstream of a minimal CaMV 35S promoter linked to the E. coli gus reporter gene. This reporter-expression cassette was then incorporated into the same plasmid as the 434 cI/VP16 activator-expression cassette. Two control plasmids were also constructed, one encoding the 434 protein with no activator domain and the second a chimeric activator with no DNA-binding domain. The chimeric activator was tested for its ability to activate gene expression in a tobacco protoplast transient assay system. Results are presented to show that we can obtain in plant cells significant activation of gene expression that is dependent on both DNA-binding and the presence of the activator domain.     

Synthesis, cloning, and expression in Escherichia coli of a spinach acyl carrier protein-I gene

A synthetic gene of 268 bp encoding the 82 amino acid spinach acyl carrier protein (ACP)-I was constructed based on the known amino acid sequence. Two gene fragments, one encoding the amino-terminal portion and the other the carboxy-terminal portion of the protein, were assembled from synthetic oligonucleotides and inserted into the phage M13mp19. These partial gene constructions were joined and inserted into the plasmid pTZ19R. DNA sequencing confirmed the accuracy of the constructions. The synthetic gene was then subcloned into the Escherichia coli expression vector pKK233-2, under the control of the trc promoter. Western blot analysis and radioimmunoassay indicated that E. coli cells carrying this plasmid produced up to 6 mg/liter of a protein which was immunologically cross-reactive and similar in electrophoretic mobility to authentic spinach acyl carrier protein. The bacterial cells were able to attach the phosphopantetheine prosthetic group to the synthetic plant gene product allowing it to be acylated in vitro by acyl-ACP synthetase.     

人肝癌靶向性CD80基因表达重组腺病毒载体的构建及鉴定

目的:构建人肝癌靶向性CD80基因重组腺病毒载体。方法:首先,从人基因组DNA中克隆AFP增强子、启动子,利用腺病毒穿梭质粒pShuttle和pShutte-CMV,采用AFP增强子替换CMV启动子,构建含有AFP启动子和增强子的穿梭质粒,命名为pShuttle-AFP。将人CD80基因克隆到pShuttle-AFP的AFP增强子和启动子之后,构建pShuttle-AFP-CD80质粒。再将其与腺病毒骨架质粒pAdEasy-1共转化E.coliBJ5183,利用同源重组构建腺病毒质粒pAd-AFP-CD80。以获得的重组子转染AD293细胞后制备重组腺病毒,并对重组的病毒进行鉴定和病毒滴度测定。结果:测序结果与Genebank中公布的AFP启动子、核心增强子和CD80基因序列相符。双酶切结果与预期结果相符。PacI酶切结果与目的结果一致。穿梭质粒pShutte-AFP-CD80和腺病毒质粒pAd-AFP-CD80经酶切和PCR鉴定均获得期望大小的目的片段。结论:成功的构建了人肝癌靶向性pAd-AFP-CD80腺病毒载体,为CD80基因在肝癌靶向性治疗的研究奠定了基础。     

Quick selection of a chimeric T2 phage that displays active enzyme on the viral capsid

We designed a bacteriophage T2 system to display proteins fused at the N-terminus of the head protein small outer capsid (SOC) of a T2 phage. To facilitate selection of chimeric phage, a T2 phage encoding the beta-galactosidase gene (betagal) upstream of the soc gene was constructed. The phage, named T2betaGal, produces blue plaques on agar plates containing XGal. Subsequently, a plasmid encoding the target protein upstream of soc was constructed and used to transform E. coli B(E) cells. Transformed cells were infected with T2betaGal and homologous recombination between phage DNA and the plasmid resulted in a chimeric phage that produced transparent plaques due to the excision of the betagal gene. Chitosanase of Bacillus sp. strain K17 (ChoK), consisting of 453 amino acids, was used as a model target protein. Recombinant T2 phage that produced ChoK was named T2ChoK. T2ChoK was produced from T2betaGal at a recombination frequency of about 0.1%. On the other hand, the value for T2betaGal produced from wild-type T2 was 0.001 %. This new system enables us to select recombinant phage very quickly and accurately. The number of molecules of ChoK was calculated at 14.7 per single phage. Latent period and burst size were estimated for the chimeric phages.     

Expression of chemically synthesized alpha-neo-endorphin gene fused to E. coli alkaline phosphatase.

An alpha-neo-endorphin (alpha NE) gene, which we previously synthesized chemically and inserted into E. coli beta-galactosidase gene of pK013 plasmid, has been excised and fused to E. coli alkaline phosphatase (APase) gene. One of the transformants was named E15/pA alpha NE1. Under the APase gene regulation, APase-alpha NE chimeric protein was expressed at 1.3 X 10(6) molecules per cell, and accounted for about 60% of total cellular proteins. The HPLC pattern of CNBr treated E15/pA alpha NE1 was very simple reflecting the high content of the chimeric protein and low numbers of methionine residues in it. A series of genes encoding APase-alpha NE chimeric proteins in which 30 to 94 C-terminal amino acid residues were replaced by (met)-alpha NE, was cloned in E. coli. Transportation of the chimeric proteins to periplasmic space was studied. All chimeric proteins were apparently processed by signal peptidase but few, if any, was transported to the periplasmic space.