ABSTRACT
Objective:To construct a recombinant eukaryotic expressive vector of pEGFP-N3-M-IL-2(88 Arg,125 Ala),and to study the expression of this gene in the Glioma cell line U 87,and to detect its antitumor activities of the fusion protein M-IL-2(88 Arg,125 Ala).Methods:The target fusion gene M-IL-2 (88 Arg,125 Ala) was amplified by PCR from pPICZαA/M-IL-2 (88 Arg,125 Ala) and cloned into pEGFP-N3 vector after digestion to construct recombinant eukaryotic expressive vector pEGFP -N3-M-IL-2( 88 Arg,125 Ala).And then recombinant plasmid pEGFP-N3-M-IL-2(88Arg,125Ala) was transfected into Glioma cell U87 by LipofectamineTM2000 immediately after it was confirmed by restrictive enzyme analysis and sequencing .RT-PCR and Western blot were used to confirm expression of the fusion gene in the U87.Prohibitory effect of recombinant M-IL-2(88Arg,125Ala) protein on U87 was assessed by CCK-8 assay.Results:Restrictive analysis and sequence analysis revealed that M-IL-2(88Arg,125Ala) fusion gene was cloned into the vector pEGFP-N3 suc-cessfully,fusion gene M-IL-2(88Arg,125Ala) could express in U87 cells and could inhibit the growth of U87 cells.Conclusion:The eu-karyotic expression plasmid pEGFP-N3-M-IL-2( 88 Arg,125 Ala) was constructed and expressed in U 87 cells successfully ,the fusion protein could inhibit the growth of U87 cells.We laid a foundation for further research of gene M-IL-2(88 Arg,125 Ala).
ABSTRACT
To investigate the inhibitory effects of Ginsenoside Rb1 (GRb1) on apoptosis caused by Herpes Simplex Virus-1 (HSV-1) in Human Glioma Cells (U251),U251 cells were infected by HSV-1 at a multiplicity of infection of 5 and GRb1,GRb1+HSV-1,HSV-1 and control groups.MTT and cell apoptosis assays were used to detect the inhibitory effects of GRbl on the apoptosis of U251 cells that caused by HSV-1 infection for various concentrations of drug and virus treatments by MTT assay.We found that in the 400 μg/mL GRbl and 400 μg/mL GRbl+HSV-1 groups,MTT values were higher than control group at all times (P＜0.05).Moreover,the apoptosis rate in the 400 μg/mL GRb1+HSV-1 group was lower than the HSV-1 group (P＜0.05).These results confirmed that,at appropriate concentrations,GRb 1 could inhibit nerve cell apoptosis in HSV-1 infections.
ABSTRACT
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.
ABSTRACT
OBJECTIVE To develop a macroarray method to detect pathogens in cerebrospinal fluid.METHODS According to the bacterial 16S rRNA genes,designed 10 kinds of specific probes and a pair of universal primers that can amplify rRNA gene of all bacteria.The tailed probes were spotted onto a nylon membrane.DNA was isolated from each pathogen,and subjected to UP-PCR to amplify target fragments,which were labeled with bio-16-dUTP at the same time.All those denatured fragments were hybridized to the probes on nylon membrane and visualized by AKP labeled avidin.The sensitivity and specificity of the system were detected.A total of 32 CSF samples,which were verified the bacterial infection by the routine method,were tested by this method.RESULTS It was sensitive to 10 CFU/ml when detecting Escherichia coli.Every kind of pathogens only reacted to its corresponding probes fixed on nylon membranes,which showed high specificity.The result of identifying 32 CSF clinical specimens accorded with that of routine method.CONCLUSIONS The method can screen out common pathogens in CSF sensitively and exactly.