Effect of mouse uroplakin II promoter on human bladder cancer cell line / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the effect of gene expression of mouse uroplakin II (UPII) promoter on human bladder cell cancer cell line.</p><p><b>METHODS</b>The mRNA expression of different cell lines was quantified by RT-PCR. Green fluorescent protein (GFP) and luciferase (Luc) were used as reporter genes. The plasmids carrying UPII or GFP were constructed and transfected into human cell lines of bladder transitional cell cancer (BIU-87), kindey cancer (GRC-1), vascular endothelium (EC), lung cancer cell line (A549) and skin fibroblast cell line (Hs27). GFP activity of cells was detected by confocual microscopy and flow cytometry (FCM). Luciferase value was measured by luminometer (microplate) and luciferase to beta-galactosidase ratios (L/G values) were used for evaluating transfection efficiency.</p><p><b>RESULTS</b>RT-PCR showed high expression level of UPII mRNA in bladder cancer cell line BIU-87, whereas low level or no expression in nonbladder cancer cell lines. The activity of GFP in bladder cancer (BIU-87) cell was higher than that in the other cell lines (5 - 10/HP versus 0 - 2/HP), with 4.34% positive cells in BIU-87 detected by FCM, but no positive cell was found in the other cell lines. L/G values indicated that the luciferase expression in human bladder cancer cells transfected with mouse UPII promoter was 1.8 - 8.2-fold as high as that in the nonbladder cell lines.</p><p><b>CONCLUSION</b>Mouse UPII promoter gene can be expressed in a tissue-specific fashion in human urinary bladder cancer. It is capable of initiating transcription of reporter genes in human bladder cancer cell line.</p>

Construction, expression and bioactivity characterization of targeting toxin DT-VEGF / 生物工程学报

Tumor rapid growth depends on neovascularization. Vascular endothelial growth factor, the main mediator during the occurrence and formation of vascularization, has specific receptors whose expression rate shows difference of orders of magnitude between tumor and the normal tissue, so it can be used to transport toxin molecules to the proliferative tumor endothelial and kill cancer cells. In our experiment, we constructed fusion protein DT-VEGF by linking diphtheria toxin's forward 389 amino acids's gene and VEGF165 via a linker. DT-VEGF is expressed in E. coli and purified. Our experiment proves in can kill vascular endothelial cells specifically, and the inhibition of neovascularization of chicken chorionic membrane is also confirmed.

Screening of new binding partners of CIKS with yeast two-hybrid system / 生物工程学报

The NF-kappaB transcription factor plays important regulatory roles in inflammation, apoptosis, immune and stress responses. IkappaB kinase (IKK) composed of two catalytic subunits and a regulator subunit, acts as a key component of NF-kappaB activation pathway through phosphorylation of IkappaB, the inhibitor of NF-kappaB. CIKS (connection to IKK and SAPK), a newly identified cellular protein, is involved in NF-kappaB and JNK activation. Although it has been known that CIKS interacts with IKK complex, and activates both IKK and SAPK when overexpressed; the underling mechanisms are poorly understood. To better understand the physiological roles of CIKS, we have screened human HeLa MATCHMAKER cDNA library for new binding partners of CIKS by using the yeast two-hybrid system with truncated CIKS (151-574) as the bait. The yeast strain AH109 was sequentially transformed with the bait plasmid and the library. The transformants were screened on SD(-Leu/-Trp/-His/-Ade/ + X-alpha-gal)selective plates. Positive clones were restreaked on SD(-Leu/-Trp / + X-alpha-gal)plates three times to allow loss of some of the AD/library plasmids while maintaining selective pressure on both the DNA-BD and AD vectors. After 3 screenings on SD(-Leu/-Trp / + X-alpha-gal), the positive clones were further verified on SD(-Leu/-Trp/-His/-Ade/ + X-alpha-gal) plates. The inserts in AD/library plasmids were amplified by PCR and PCR products were characterized by Hae III digestion to eliminate the duplicates. After screening in selective plates, the positive AD/library plasmids were rescued via transformation of E. coli HB101 and the interactions of CIKS (151-574) with positive AD/library plasmids were further assessed by yeast two-hybrid analysis. Finally, the DNA sequences of the positive AD/library plasmids were determined and BLAST analysis against the databases was performed. The BLAST results indicate that the inserts in the positive plasmids encode RIKEN cDNA 473340F03, PLAC8, CD27BP (Siva-1), CDC5L, SnRNP smB, and DVL2. CDC5L is a key component of the multi-protein complex essential for the formation of pre-mRNA splicing product and is not required for spliceosome assembly. A role for CDC5L in the cell division cycle has been precious suggested as its overexpression of this protein in mammalian cells leads to a shortening G2 phase of the cell cycle, and a negative-dominant mutant of CDC5L lacking the N-terminal activation domain delays the G2 phase cell's entry into the mitosis. It has been reported that SnRNP smB participates in pre-mRNA splicing and CD27BP (Siva-1) binds to and inhibits BCL-XL-mediated protection against UV radiation-induced apoptosis and regulates T cell homeostasis. The functions of RIKEN cDNA 473340F03, PLAC8 and DVL2 are unknown. It has been suggested that CIKS functions as a critical component for cross-talk between NF-kappaB and JNK signaling pathways. IKK subunits, which have been demonstrated to interact with CIKS, were not shown up in this experiment. We speculate that the truncated CIKS (151-574) bait may not contain the binding domain that mediates the interaction of IKK subunits with CIKS. Taken together, the above results suggest that CIKS may play a role in cell regulation through interacting with various cellular proteins. Further investigations are required to characterize these interactions.

Screening and characterization of DNA aptamers with hTNF-alpha binding and neutralizing activity / 生物工程学报

Human tumor necrosis factor alpha (hTNF-alpha) is one of the most important inflammatory cytokines that acts as a mediator in inflammatory and immune response and plays a key role in host defense against infection. The over expression of hTNF-alpha is associated with serious consequences, such as shock, hypotension, thrombus, septicemia and even death. It has been implicated in many autoimmune and inflammatory diseases, such as rheumatoid arthritis, Crohn's disease, chronic heart failure and septic shock. Inhibiting the bio-activity of hTNF-alpha is one of the strategy for the treatment of these diseases. Compared with traditional recombinant protein drugs, small molecule drugs have many advantages, such as high affinity, low immunogenecity and low cost. Systematic evolution of ligands by exponential enrichment (SELEX) is a powerful method for the selection of oligonucleotides that bind with high affinity and specificity to target proteins. Such oligonucleotides are called aptamers, and are potential therapeutics for blocking the activity of pathologically relevant proteins. To obtain oligonucleotide aptamers specifically binding to TNF, a 40nt random DNA combinatorial library flanked by 31nt fixed sequences was chemically synthesized. The random library was amplified with PCR and subjected to selection by SELEX protocol against hTNFalpha. After incubation of the library with hTNFalpha, the mixture was blotted onto Immobilon-NC transfer membrane. The no-specific binding was washed away and the hTNFa binding aptamers were eluted and detached from the target protein. The eluted oligo nucleotides were amplified with PCR and served as the DNA library for the next round selection. After 12 rounds of such selection, the selected aptamers were cloned to pGEM-T vector. Positive clones were identified by restriction enzyme digestion and DNA sequencing. Oligo DNA were synthesized according to the sequence data and tested for their activities. Binding activity of the aptamers to hTNFalpha were detected by ELISA and dot blot with biotin-streptavidin-horseradish peroxidase system. Mouse L929 cells were used to test the anti-hTNFa activity of the DNA aptamers. The aptamers were incubated with hTNFalpha and added to the L929 cells. The results were read under microscope and with MTT staining. It was shown that these DNA aptamers bound to hTNFalpha with high affinity, and can inhibit the cytotoxicity of hTNFalpha on cell culture. The affinity of these aptamers are different and may related to their structure. These ssDNA aptamers are potential for the treatment and diagnosis of hTNFalpha related diseases.

Cloning, expression and bio-activity assay of chimeric fusion protein sTNFRII-IgG Fc / 生物工程学报

Tumor necrosis factor (TNF) is a key cytokine in immunology system and is related to many human diseases. In order to inhibit the activity of TNF, cDNA coding for soluble TNF receptor II (sTNFRII) and human IgG Fc were linked using a flexible hinge. This gene was expressed in E. coli as a chimeric protein and purified by metal chelate chromatography. The results show that the fusion protein exists in the physiological form as a dimer, has the ability to bind with TNF and inhibits the cytotoxicity of TNF on L929 cells. Contrasting to monomer sTNFRII, the chimeric protein has an improved bioactivity, and displays potential prospects for research and application.