Objective: To express and purify the TRIM5α chimaera［TRIM5α-H(R328- 332)］ protein and to explore the interaction between the TRIM5α H(R328- 332)and HIV-1gag. Methods: The plasmid pET28aTRIM5α H(R328-332) was transformed to E.coli BL21 (DE3) strain , and the expression of TRIM5α H(R328-332) protein was induced by IPTG，purified with Ni2+ chromatography.The expression and purification of TRIM5α H(R328-332) were analyzed by SDS-PAGE and Western blot,and the interaction between TRIM5α-H(R328-332) and HIV-1gag was detected by co-immunoprecipitation, His pull-down and ELISA. Results:The recombinant plasmid pET28aTRIM5α H(R328-332) was successfully expressed in E.coli. The results showed that the purified full length TRIM5α-H(R328-332) interacted with HIV-1gag protein. Conclusion: The human TRIM5α-chimaera was expressed successfully in vitro, and the study demonstrates that the human TRIM5α-chimaera interacts with HIV-1 gag in vitro.
To simplify the preparation of neuraminidase in screening influenza neuraminidase inhibitors, the neuraminidase gene of H5N1 influenza A virus was optimized for high expression in mammalian cells and cloned into pcDNA4/TO vector. The recombinant plasmids were transfected into T-rex 293 cells to establish stable cell lines, in which the expression of neuraminidase was induced by tetracycline. Unlike from virions, the preparation of neuraminidase became conveniently and safely from these stable cell lines, which would facilitate developing high throughput assay to screen neuraminidase inhibitors. More than 3000 natural extracts and herbal components were screened in this study. Baicalin and baicalein were found to inhibit oseltamivir-sensitive and oseltamivir-resistant neuraminidase at similar level, furthermore, their anti-influenza activity was confirmed by plaque assay and virus inhibition assay.
Eukaryotic expression plasmid pCI-neo-mdr1 which contains human multidrug resistance gene 1(mdr1), was constructed and transferred into human hepatocarcinoma HepG2 cells by use of liposome. G418 was used to screen the cells successfully with mdr1 and the selected cells was named HepG2/mdr1 Morphological and biological properties of HepG2/mdr1 cells were observed. The results show that the constructed HepG2/mdr1 cell line was high efficient and stationary in the expression of mdr1. The work was valuable and desirable for the establishment of multidrug resistant cell models, and for the study of MDR in human hepatoma. Furthermore, the work also provided a perfect model for the research of relationship between insulin resistance and MDR in hepatocarcinoma cells.To construct the eukaryotic expression plasmid pCI-neo-mdr1 containing multidrug resistance gene 1(mdr1), and transfer into human hepatocarcinoma cell line HepG2 cell by use the liposome, screening human hepatoma multidrug resistance (MDR) cell line HepG2/mdr1 with G418， and the biological property of HepG2/mdr1 cells was observed. The results showed that the HepG2/mdr1 cell line that we constructed is highly efficient and stationary in performance and the goal gene expression. This work was valuable and desirable for the study of MDR phenomenon and reversing measure of MDR, and exploring establish of MDR hepatocarcinoma cell line. Furthermore, The work also provided a perfect model cell for the research of relationship between insulin resistance and MDR in hepatocarcinoma cells.
In order to further study mouse embryonic stem cells(ES cells), lentiviral vector PLL-IRES-Nanog-Neo was constructed and mouse ES cells overexpressed nanog gene were cultured. At first, mouse nanog primers with Nhe I and Xho I restricted enzyme were designed according to nanog mRNA sequence. Nanog gene fragment was amplified by RT-PCR with mRNA as templates from ES cells treated with Trizol reagent. pcDNA3.1-Nanog vector were construted after nanog DNA fragments were digested with Nhe I and Xho I. Nanog fragment was confirm to original sequence by DNA sequence and then lentiviral vector PLL-IRES-Nanog-Neo was constructed. Mouse ES cells overexpressed nanog by mediation of lentiviral were cultured on mouse fetal fibroblast feeders after 2 weeks under G418 media and examined according to gowth characteristics. Results were showed that 918 bp nanog fragments were expressed in mouse ES cells mediated by lentiviral vector PLL-IRES-Nanog-Neo, mouse nanog- ES cells were taken on mass-like image and positve with alkaline phosphatase staining and Oct4 and SSEA1 immunocytochemistry under no LIF condition in the media. It is concluded that mouse ES cells Elevated nanog gene expression by mediation of lentiviral were constucted and cultured.
DHCR24 can protect cells from apoptosis induced by oxidative stress, but the detail mechenism is unknow. Our previous data obtained from in vivo experiments shown that DHCR24 might scavenge intracellular H2O2. In this study, we examined whether DHCR24 could directly decompose H2O2. We constructed the 3 plasmids including pIVEX2.4a－DHCR24（Wild Type）, pIVEX2.4a-ORD(the C-terminal deletion mutant, 1-395, including entire putative oxidareductive domain), and pIVEX2.4a-CTR（232-516, lacking the ORD, but it contains the entire C-terminal region,which has no similarity to known protein domains..We syhthesized all the His-tagged DHCR24 and its mutant in E.coli by the RTS 500 systerm, and purified them by Ni column. When applied to the H2O2-scavenging assay, WT (1-516) exhibited significant scavenging activity, ORD (1-395) preserved the activity, whereas CTR (232-516) lost it, indicating the important role of ORD in H2O2 decomposition. To explore the physiological role of antioxidatic function of DHCR24, we investigated the cellular location of DHCR24 utilizing the double immunocytochemical staining. The results demonstrated the DHCR24 is localized to endoplasmic reticulum ( ER ), indicating that the location on the ER of DHCR24 might be involved in its antioxidatic activity.
To decrease the oxygen content in the cell is a key method to improve hydrogen production in Chlamydomonas reinhardtii. To achieve this goal, we developed a new approach by transforming the leghemoglobin gene lba, which has high affinity to oxygen, into the chloroplast of C. reinhardtii to get a low dissolved oxygen in the cell and result into improvement of H2ase activity and H2 yield. The results showed that lba was successfully transformed into the chloroplast of C. reinahrdtii strain 849 and did not affect its growth significantly. This work paved the road for further regulation of lba expression in the chloroplast to improve of hydrogen production of C. reinahrdtii.
A chitosanase-producing bacterium S-1 was isolated from Lianyungang seaside. It was identified as Bacillus sp. based on its morphological, the physiological and biochemical properties and 16S rDNA sequence analysis. According to the information about chitosanases sequence deposited in NCBI, a pair of degenerated primers was designed and a partial sequence of chitosanase gene was obtained by polymerase chain reaction (PCR) using Bacillus sp. S-1 genome DNA as the template. A genome walking library was constructed followed as the protocol provided by CLONTECH Company. The flanking sequences of the 5' and 3' terminal was obtained by genome walking method and two-step PCR technique. After overlapped and confirmed, the full-length sequence of chitosanase from Bacillus sp. S-1 was achieved (Accession number: EU924147) and it contained 1362 bp coding 453 amino acids. The characterization of the deduced amino acid was analysis using the bioinformatics method.
