Zhiyong Yan

High-level expression, purification and study of bioactivity of fusion protein M-IL-2(88Arg, 125Ala) in Pichia pastoris

M-IL-2((88)Arg, (125)Ala) is a fusion protein comprising melittin genetically linked to a mutant human interleukin 2((88)Arg, (125)Ala). In this study, we constructed an expression system of M-IL-2((88)Arg, (125)Ala) in Pichia pastoris: GS115/pPICZα A/M-IL-2((88)Arg, (125)Ala), and achieved the high-level expression of the fusion protein. The maximum yield of the fusion protein M-IL-2((88)Arg, (125)Ala) reached up to 814.5mg/L, higher than the system in Escherichiacoli. The fusion protein was purified by means of ammonium sulfate fractionation, dialysis and nickel ion affinity chromatography. The molecular weight of the fusion protein is about 26kDa, conforming the theoretical value. And M-IL-2((88)Arg, (125)Ala) possesses strong antigen-specificity by Western blot detection. Bioassay results indicated that the fusion protein could directly inhibit the growth of human ovarian cancer SKOV3 cells and Hela cells in vitro. This study provides an alternative strategy for large-scale production of bioactive M-IL-2((88)Arg, (125)Ala) using P. pastoris as an expression host and paves the way to clinical practice.

Expression, Purification, and Characterization of a Functional Mutant Recombinant Human Interleukin-2

In the current study, a mutant recombinant human interleukin-2 (MhIL-2) was generated using site-directed mutagenesis. The bacteria transformed with plasmid pET15b-MhIL-2 were cultured in LB medium containing 0.6mM IPTG for 8 hours at 27°C. Approximately 90% of His-MhIL-2 was efficiently expressed in soluble form. Purification efficiency was optimized using a number of strategies, including nickel ion chelating chromatography, desalting chromatography, thrombin cleavage and Superdex 75 gel filtration chromatography. The final product had >95% purity. PBMCs, CD4+ and CD8+ T cell proliferation assays revealed that one such mutant has identical functional property to the wild-type hIL-2. In summary, we generated a mutant hIL-2 that is functionally identical to wild-type hIL-2.

Inhibition of Human Cytomegalovirus Infection by IE86-Specific Short Hairpin RNA-Mediated RNA Interference

To develop a gene therapeutic method for human cytomegalovirus (HCMV), the IE86 specific short hairpin (sh) RNA expressing vector was constructed and subsequently transfected into MRC-5 cells. After infection of these cells with HCMV AD169, expression of IE86 was reduced strikingly as compared to the control. In addition, the inhibitory effect corresponded to a decrease in viral DNA replication and the virus-induced cytopathic effect. Measurement of the virus yield demonstrated that infection of cells expressing IE86-specific shRNA resulted in suppression of the formation of infectious viral progeny. These observations indicate that IE86 can be used as an effective target against HCMV infection using RNA interference (RNAi) technology, which provides new possibilities for anti-HCMV studies.

Effect of Inducible Expressed Human Cytomegalovirus Immediate Early 86 Protein on Cell Apoptosis

Human cytomegalovirus is a common human pathogen that can cause life-threatening disease under certain conditions. During infection of host cells, the virus expresses regulatory proteins such as IE72 and IE86 that are important for viral propagation. IE86 plays a critical role in the modulation of viral replication as well as host cell cycle control and apoptosis. In this study, a Tet-On system was used to quantify the effect of IE86 on apoptosis and p53 expression. Our results indicate that IE86 inhibits tumor necrosis factor (TNF)-α induced apoptosis and that the anti-apoptotic activity of this viral protein correlates with its expression levels. In addition, IE86 did not alter the mRNA level of p53. The system developed should provide a method for functional analysis of human cytomegalovirus (HCMV) IE86 protein.

Human cytomegalovirus infection inhibits the differentiation of human hippocampus neural precursor cells into astrocytes

AbstratHCMV is a major cause of congenital brain disease in humans, and its neuropathogenesis is not yet fully understood. The objective of the present study is to investigate the effect of human cytomegalovirus (HCMV) infection on human hippocampus neural precursor cell (NPCs) differentiation in vitro. Fetal hippocampus tissue was dissociated mechanically and then cultured in proliferation medium with EGF and bFGF. The identification and purity of the NPCs were confirmed by using immunofluorescence to detect the expression of the NPCs marker-Nestin. To drive NPCs differentiation, bFGF and EGF were withdrawn from the medium and replaced with FBS (10%). HCMV AD169 (MOI=5) was added into the differentiation medium at the onset of the differentiation. After 7 days of differentiation, in order to confirm whether NPCs are permissive for HCMV infection, immunofluorescence was used to stain for the presence of immediate early (IE) and late (pp65) HCMV proteins in the infected cells. The effects of HCMV infection on NPCs’ differentiation was observed by detecting the ratio of nestin and GFAP positive cells with confocal microscopy and immunofluorescence. The data showed that 95%±8% of the cells (passage 4–8) cultured were Nestin positive which suggested that majority of the cells were NPCs. On day 7 postinfection, most of the infected cells were IE and PP65 positive. The percentage of Nestin-positive cells were 93%±10% and 50%±19% (t=6.03, p<0.01) and those of GFAP-positive cells were 55±17% and 81%±11% (t=3.77, p<0.01) in HCMV treated and control groups respectively. These findings indicate that NPCs are HCMV permissive cells and HCMV (AD 169) infection suppresses the differentiation of Hippocampus-genetic human NPCs into astrocytes. These effects may provide part of the explanation for the abnormalities in brain development associated with congenital HCMV infection.

Effect of Human Cytomegalovirus Infection on Nerve Growth Factor Expression in Human Glioma U251 Cells

OBJECTIVE To explore the change of endogenic nerve growth factor (NGF) expression in human glioma cells infected with human cytomegalovirus (HCMV). METHODS U251 cells were cultured in RPMI 1640 culture medium and infected with HCMV AD169 strain in vitro to establish a cell model of viral infection. Morphologic changes of U251 cells were observed under inverted microscope before and after infection with HCMV. Expression of NGF gene and protein of cells was detected by RT-PCR and Western blotting before and after infection with HCMV. RESULTS The cytopathic effects of HCMV-infected cells appeared on day 5 after infection. However, differential NGF expression was evident on day 7. NGF expression was decreased significantly in U251 cells on day 7 after infection in comparison with control group (P < 0.05). CONCLUSION HCMV can down-regulate endogenous NGF levels in human glioma cell line U251.

Using the SELDI ProteinChip system to detect changes in protein expression in Vero cells after infection

Human herpes simplex virus 1 (HSV-1) causes facial, ocular, and encephalitic disease and is associated with latent infection and cancer. Here, we developed a means of studying the pathogenesis of HSV-1 infection at the protein level by using the SELDI Protein Chip to detect changes of protein expression in Vero cells cultured in vitro. After infection with HSV-1 and culture for 12, 24 or 48 h, cells were harvested and lysed. IMAC3 arrays were applied to SELDI-TOF-MS to detect proteomic differences before and after infection. The chip detected a series of differentially expressed protein peaks. Inte-restingly, both peaks at 16 912 Da and 17 581 Da corresponded precisely with the molecular mass of ISG15, which may participate in antiviral activity during the process of infection. Thus, the results we obtained can serve as a basis to study the pathogenesis of HSV-1 and the interaction between the virus and its host. In addition, they can help in the discovery of new therapeutic targets for treatment of HSV-1 infection.

Apatinib suppresses cell growth and metastasis and promotes antitumor activity of temozolomide in glioma

Apatinib, a small-molecule multi-targeted tyrosine kinase inhibitor, is widely used to treat various types of solid tumors. In the present study, it was investigated whether apatinib has therapeutic potential for glioma. Cell Counting Kit-8 and colony formation assays were utilized to determine the cell viability of p53- and EGFR-mutated U251MG glioma cells, and wild-type U-87MG ATCC glioma cells. Furthermore, apoptosis, and the invasion and migration abilities of glioma cells were investigated by flow cytometry, invasion assays and wound-healing assays. The potential of the combination of apatinib with temozolomide (TMZ) for glioma therapy was also investigated. The results demonstrate that apatinib significantly inhibited cell proliferation and colony formation through promoting cell apoptosis in p53- and EGFR-mutated and wild-type glioma cells. Cell invasion and migration abilities were notably decreased following treatment with apatinib. Overall, the present study indicates a synergistic antitumor effect of apatinib and TMZ in glioma, and presents a basis for the use of apatinib in glioma treatment.

HCMV Infection Depress NGF Expression in Human Glioma Cells

Human cytomegalovirus (HCMV) is the most common cause of congenital infection, resulting in birth defects such as microcephaly. In this study, RT-PCR and Western Blotting were performed to quantify the regulation of endogenic nerve growth factor expression in neuroglia cells by HCMV infection. The results showed that basal, endogenous NGF expression in U251 was unchanged during early HCMV infection. NGF expression is strongly down-regulated during the latent phase of infection. These results suggest that HCMV can depress the NGF expression in U251 cells.