美国利用转基因蛋白质抑制流感病毒传播

流感病毒经过遗传漂变会周期性地变得更具毒性，严重威胁全球公共健康。通常，抵抗新流感菌株的疫苗需要数月的时间完成开发、测试和生产等环节，同时它们对已患流感者毫无作用。因此，当致命性流感突然出现和快速传播时，疫苗的研制和发送所造成的长时间延误让人们束手无策，而人们缺乏免疫能力又加速了流感病毒在公众间的传播。

Increasing Accumulation Level of Foreign Protein in Transgenic Plants Through Protein Targeting

Targeting of the synthesized polypeptide in the cells is an important research field in modern cell biology. Cowpea trypsin inhibitor (cpti) gene has been modified and a fusion protein gene (sck) was produced by fusing a signal peptide sequence at cpti 5' end and an endoplasm reticulum (ER) retention signal peptide at cpti3' end respectively. The signal peptide can direct the newly synthesized polypeptide into ER, while ER retention signal can make the protein retained in the ER and its derivative protein body. ELISA test indicated that the accumulation level of foreign CpTI protein in sck transgenic tobacco (Nicotiana tabacum L.) was two times higher than cpti transgenic tobaccos and some individuals were four times higher. At the same time, sck transgenic tobacco has a high resistance to Lepidoptera pest due to the increased accumulation level of foreign CpTI protein. The strategy of foreign protein targeting can be used to increase the accumulation level of foreign protein in transgenic plants and can be widely applied to other related research field in plant genetic engineering.

Characterization of Function of Three Domains in Dishevelled-1: DEP Domain is Responsible for Membrane Translocation of Dishevelled-1

Wnt signaling plays an important role in embryogenesis and tumorgenesis. Although the mechanism about how Wnts transduce their signaling from receptor frizzled (Fz) to cytosol has not been understood, dishevelled (Dvl) protein was considered as the intersection of Wnt signal traffic. In this study, we characterized the function of three domains (DIX,PDZ and DEP) of Dvl-1 in canonical Wnt signal transduction and Dvl-1 membrane translocation. It was found both DIX and DEP domain were sufficient to block Wnt-3a-induced LEF-1 transcriptional activity and free cytosol β-catenin accumulation; whereas PDZ domain and a functional mutant form of DEP domain (DEP-KM) had no effect on canonical Wnt signaling. In addition, when cotransfected with Fz-7, DEP domain, but not DIX, PDZ or DEP-KM, translocated and co-localized with Fz-7 to the plasma membrane, which was similar to Dvl-1. Furthermore, it was DEP domain that could block Fz-7-induced membrane translocation of Dvl- 1 via a possible competitive mechanism. These results strongly suggest that DEP domain is responsible for the membrane translocation of Dvl-1 protein upon Wnt signal stimulation.

A novel protein-DNA interaction involved with the CpG dinucleotide at -30 upstream is linked to the DNA methylation mediated transcription silencing of the MAGE-A1 gene

To understand the DNA-methylation mediated gene silencing mechanisms, we analyzed in cell culture of the promoter function of the MAGE-A1 gene, which is frequently demethylated and over-expressed in human hepatocellular carcinoma. We have established the correlation of the DNA methylation of the promoter CpG island with expression status of this gene in a panel of the established liver cancer cell lines. The crucial CpG dinucleotide(s) within the minimal promoter subjected to the control mediated by DNA methylation with profound biological functions was also delineated.Furthermore, a novel sequence-specific DNA-protein interaction at the -30 CpG dinucleotide upstream of the gene was found having a vital part to play in the DNA methylation mediated transcription silencing of the MAGE-A1 gene. Our results would not only provide new insights into the DNA methylation mediated mechanisms over transcription of the MAGE-A1 gene, but also pave the way for further defining the cross-talk among DNA methylation, histone modification and chromatin remodeling in detail.

Techniques for Detecting Functional Protein Expression in Transgenic Plants

With the development of plant genetic engineering techniques, numerous genetically modified plants have been generated. At the same time, the technologies for detecting transgenic organisms get improved constantly, which also promotes the scientific identification, evaluation and commercial cultivation of transgenic plants. In this review, we evaluate various detection methods for transgenic plants at the level of protein expression.

Cloning and GST－fused expression in E. coli of mouse β－1,4－galactosyltransferase

b-1,4-galactosyltransferase (b4Gal-T) (EC 2.4.1.38) plays a multifunctional role in many aspects of normal cell physiology. By now, several dozens of b4Gal-T genes have been cloned, separated from mouse, chick, bovine, human, etc. This paper presents the cloning and GST-fused expression of mouse b4Gal-T gene in Escherichia coli (E. coli). The target gene was cloned by PCR, followed by identification by DNA sequencing and expression in E.coli with isopro-pyl-b-D-thiogalactoside (IPTG) gradient concentrations, products of which were separated on SDS-PAGE showing that the target protein had the same molecular weight as that of mouse b4Gal-T. The transcriptional product of b4Gal-T gene was proved by Western hybridization analysis to be due to GST-fusion.

Molecular Modification of a HSV-1 Protein and Its Associated Gene Transcriptional Regulation

The molecular modifications of Herpes Simplex Virus Type I （HSV-1） proteins represented by acetylation and phosphorylation are essential to its biological functions. The cellular chromatin-remodeling/ assembly is involved in HSV-1 associated gene transcriptional regulation in human cells harboring HSV-1 lytic or latent infections. Further investigation on these biological events would provide a better understanding of the mechanisms of HSV-1 viral gene transcriptional regulation

RAT GDNF GENE TRANSFECTION AND EXPRESSION OF ITS mRNA AND PROTEIN IN SCHWANN CELLS

Objective To investigate the possibility of the transfection of glial-cell line derived neurotro-phic factor (GDNF) gene into Schwann cells (SCs). Methods SCs cultures from sciatic nerves of neonatal rats were established. A recombinant retrovirus vector containing GDNF gene was constructed and transferred into SCs. Expression levels of GDNF mRNA and protein were respectively identified with reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry. Determination of GDNF synthesis rates from Retro. pLNCX2-GDNF-transduced SCs (GDNF-SCs) in vitro by enzyme-linked immunoassay sensitive assay (ELISA). Biololgical activity of conditioned medium from GENF-SCs was analysed by co-culture with rat motoneurons. Results Transfection of GDNF gene into SCs lead to significantly enhanced expression of GDNF mRNA and protein. The rate of GDNF secreted by GDNF-SCs was also enhanced(5. 1-fold) ,and more motoneurons survived co-cultured with conditioned medium of GNDF-SCs than with that of normal SCs. Conclusion GNDF gene transfection may be a better way to graft SCs promoting regeneration and repairing demyelination in PNS and CNS.

Characterization of 5'-proximal sequence of mouse GABAtransporter gene(GAT-1)

The cDNA molecule encoding the mouse GABA transporter gene (GAT-1) was used as probe for selecting GAT-1 gene from mouse genomic library. A positive clone, harboring the whole open reading frame of the GAT-1 protein and designated as MGABAT-G, was fished out from the library, the 5’ proximal region and nitron 1 were sequenced and analysed, and low homology was found in the above region between GAT-1 genes from mouse and human except some short conserved sequences. The DNA-protein interactions between DNA fragments containing the conserved sequences in the 5’ proximal region and nuclear proteins from different tissues of mouse were studied by means of gel-shift assay, and Southern-Western blot. The results indicate a possible positive-negative regulation mode controlling the expression of the mouse GAT-1 gene.

Ectopic bone formation of human bone morphogenetic protein-2 gene transfected goat bone marrow-derived mesenchymal stem cells in nude mice

Objective: To evaluate the osteogenic potential of bone morphogenetic protein (BMP)-2 gene transfected goat bone marrow-derived mesenchymal stem cells (MSCs). Methods: Goat bone marrow- derived MSCs were transfected by Adv-human bone morphogenetic protein (hBMP)-2 gene(Group 1), Adv-beta gal transfected MSCs (Group 2)and uninfected MSCs(Group 3). Western blot analysis, alkaline phosphatase staining, Von Kossa staining and transmission electron microscopy were adopted to determine the phenotype of MSCs. Then the cells were injected into thigh muscles of the nude mice. Radiographical and histological evaluations were performed at different intervals. Results: Only Adv-hBMP-2 transfected MSCs produced hBMP-2. These cells were positive for alkaline phosphatase staining at the 12th day and were positive for Von Kossa staining at the 16th day after gene transfer. Electron microscopic observation showed that there were more rough endoplasmic reticulum, mitochondria and lysosomes in Adv-hBMP-2 transfected MSCs compared to MSCs of other two groups. At the 3rd and 6th weeks after cell injection, ectopic bones were observed in muscles of nude mice of Group 1. Only fibrous tissue or a little bone was found in other two groups. Conclusions: BMP-2 gene transfected MSCs can differentiate into osteoblasts in vitro and induce bone formation in vivo.