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Objective:To develop a real-time PCR assays based on TaqMan chemistry for the identification of recombinant baculovirus and determination of virus physical titers in Bac-to-Bac system.Methods:The recombinant baculovirus containing human IL-18 gene was produced using Bac-to-Bac system.A 10-fold series diluted primary viral stocks were used for plaque assay and DNA extraction.Bacmid(baculovirus plasmid) was 10-fold series diluted and served as standards.Real-time PCR amplification of the IL-18 gene was performed in triplicate for each diluted recombinant virus.At the same time,plaque assays were performed using overlay agarose method.Results:The standard linear(101 to 108 copies) from quantitation was achieved with the standard curve.We also find that the "vg/ml" titer value is generally about 10 times than "pfu/ml" titer of the same recombinant virus stock.Conclusion:A TaqMan real-time PCR method is established to identify the recombinant baculovirus and determine the "vg/ml" titer of virus.The method is rapid and quantitative over a wide range of virus titers.
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AIM: To study the immune regulation of paecilomyces cicadidae total polysaccharides. METHODS: After subcutaneous injection with 200 mg/L paecilomyces cicadidae total polysaccharides in the back of the rats for 17 days, the white blood cell (WBC) counts, and the levels of lipid peroxide (LPO) and reduced glutathione (GSH) in the spleen and thymus of rats were detected. The alveolar macrophages (AM?) cultured with 10% fetal bovine serum-RPMI-1640 for 2 h in vitro , then the activity of lactate dehydrogenase (LDH) and acid phosphatase (ACP) were detected by automatic biochemistry analyzer, and the ability devouring neutral red in the AM? were examined. RESULTS: Compared with control group, the WBC, the levels of GSH in the spleen and thymus, and the activities of LDH and ACP were significantly increased and the ability of devouring neutral red was also strengthened( P
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AIM: To investigate the protective effect of crude Paecilomyces tenuipes polysaccharides(cPtPs) and Paecilomyces tenuipes polysaccharides(PtPs) in a rat model of acute hepatic necrosis induced by carbon tetrachloride(CCl4).METHODS: Wistar rats were divided into four groups(control group,CCl4 group,cPtPs+CCl4 group and PtPs +CCl4 group),the four groups were given intragastrically with normal saline,cPtPs and PtPs for 15 d,respectively.In the last two days,these groups except control group were injected peritoneally with CCl4.Serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),total bilirubin(TBIL),direct bilirubin(DBIL) and indirect bilirubin(IBIL) were detected by automatic biochemistry analyzer.Pathological changes of hepatic tissue were assessed by hematoxylin-eosine(HE) staining.The levels of superoxide dismutase(SOD) and malondialdehyde(MDA) in liver homogenate were analyzed using xanthinoxidase and thio-barbituric acid,respectively.The concentration of Ca2+ in hepatocyte mitochondria was determined by colorimetric method.The expression of ?-smooth muscle actin(?-SMA) was examined in hepatic tissue by immunohistochemical method.RESULTS: Compared with control group,serum levels of ALT,AST,TBIL,DBIL and IBIL in CCl4 group increased significantly,denaturation and necrosis implicated to the whole hepatic lobules.Compared with CCl4 group,serum levels of ALT,AST,TBIL,DBIL and IBIL in PtPs +CCl4 group decreased significantly,denaturation and necrosis located in the third region of hepatic lobules,the level of SOD increased and MDA decreased(P
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Objective To investigate the physicochemical properties,structural characters and antitumor activity of a polysaccharide PTPS-1 from Paecilomyces tenuipes.Methods PTPS-1 was obtained by DEAE-cellulose and Sephadex G-100 chromatography column.The methods of HPLC,GC,IR,and NMR were used to investigate the molecular weight,monosaccharide composition,and structural characters of PTPS-1.The CCK-8 kit was used to determine the proliferation of PTPS-1 on murine splenocyte and the supernatant of the murine macrophage with PTPS-1-stimulated on Sp2/0 cells.Results PTPS-1,whose molecular weight was 1.84?104 and purity was 98.977%,contained mannose(Man) and galactogen(Gal) as monosaccharide constitute in molar ratio of 1.3∶1.It consisted of a backbone of ?-(1→6),?-(1→2),and ?-(1→3) linkage,whose branches were made by Gal-(1→3) linkage.PTPS-1 significantly stimulated the murine splenocyte proliferation and the supernatant of macrophage inhibited the growth of Sp2/0 cells in vitro.Conclusion PTPS-1 is a polysaccharide from P.tenuipes with immunomodulatory and antitumor activity.
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AIM: To study the effects of paecilomyces cicadidae total polysaccharides on the immune function and free radical scavenging of tissues in immunosuppressed rats.METHODS: The activities of lactate dehydrogenase(LDH) and arginase(Arg) in liver,kidney,spleen,thymus of immunosuppressed rats were detected by automatic biochemistry analyzer.The content of lipid peroxide(LPO) and the reduced glutathione(GSH) level in liver,kidney,spleen,thymus of immunosuppressed rats were observed.RESULTS: The activity of the LDH,Arg and GSH level were significantly increased,and the LPO content were significantly declined in liver,kidney,spleen,thymus of immunosuppressed rats induced by cyclophosphamide.CONCLUSION: Paecilomyces cicadidae total polysaccharides promote immune function and the ability of free radical scavenging of tissues in immunosuppressed rats.
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AIM: To study the immunity modulation mechanism of achyranthes bidentata polysaccharides (ABPS). METHODS: (1) PBMC from patient with asthma or lung cancer were cultured with 800 mg/L ABPS for 18 h. IFN-? and IL-4 mRNA was detected by RT -PCR. (2) Th cells were cultured with serially diluted ABPS for 48 h and with 800 mg/L ABPS for various times. Cultured supernatant was harvested at diffevent time points and RNA was extracted, respectively. IFN-? or IL-4 transcriptional and translational levels were assay by RT -PCR and ELISA. RESULTS: (1) Positive rate of IFN-? mRNA expression in PBMC from patient with asthma or lung cancer increased from 6/25 to 14/25 (P
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AIM: To explore the immunomodulatory effect of paecilomyces cicadidae polysaccharides (PCPS).METHODS: Subcutaneous injection with 50, l00, 200 mg/kg of PCPS were given in the back of the rats everyday for l5 days. The number of white blood cells (WBC) was counted. The activities of acid phosphatase (ACP) and lactase dehydrogenase (LDH)in liver, kidney, spleen and thymus were detected by automatic biochemistry analyzer. The ability of devouring neutral red and activity of ACP, LDH, arginase in alveolar macrophages were also detected. The body weight of the rat everyday during experiment and weight of the spleen and thymus after the rats were killed were measured and wet weight index was calculated.RESULTS: The wet weight index of spleen and thymus, the activity of ACP, LDH, arginase and ability of devouring neutral red in alveolar macrophages in the test group treated with PCPS were significantly higher than those in control group. CONCLUSION: PCPS shows a significant immunomodulatory effect with the increasing counts of WBC and activation of alveolar macrophages in a dose-dependent manner.
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Objective:To express and purify a new fusion protein harboring human epidermal growth factor receptor(EGFR) binding domain and Interleukin-18(IL-18), as well as preliminary assay biological activity of recombinant proteins.Methods:Fusion protein was expressed in insect cells Spodoptera frugiperda cell (Sf9) by using Bac-to-Bac system, and an abbreviation purification procedure was used to purify fusion protein. IFN-?induction assay and EGF Receptor competitive test was used to determine fusion protein's biological activity.Results:SDS-PAGE and western blot assay showed that purified EGF-IL-18 fusion protein had high purity in 20 kD as expected and had the same antigenic specificity as human IL-18. IFN-?induction assay and EGF Receptor competitive test showed that fusion protein induced production of IFN-?in human PBMC and bound with tumor cells.Conclusion:EGF-IL-18 fusion protein has been successfully expressed and purified in insect cells and shows potential to apply in targeting therapy for tumor.