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Objective To observe the biological activities of human doppel (Dpl) protein transiently expressed and Dpl-like protein PrPΔ32-121 on a human neuroblastoma cell line SH-SY5Y. Methods Recombinant mammalian expression plasmids containing human PRND gene and truncated PrPΔ32-121 fragment were generated by PCR. The expression and location of Dpl and PrPΔ32-121 post-transfection were observed by IFA. The cytotoxicity was measured by MTT analysis. Cellular apoptosis was investigated by flow cytometry and Western blot. Results Both Dpl and PrPΔ32-121 protein were expressed and mainly located on the cell membrane. Remarkable cytotoxicity was detected on SH-SY5Y cells after 24 h transfection. Meanwhile, more Annexin V/PI positively-stained cells as well as lower levels of cellular pro-caspase-3 and Bel-2 were detected in the cells receiving Dpl and PrPΔ32-121 expressing plasmids. Conclusion Dpl protein transiently expressed and PrPΔ32-121 can lead to the similar neural cytotoxicity, probably triggering the cell apoptosis program.
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Objective To investigate the cell-type-specific enhancer (CTSE) in HPV16 and its variation in cervical carcinoma. Methods CTSEs were detected by polymerase chain reaction (PCR) in 58 cervical carcinoma from Shaanxi province; in addition variation of CTSEs was analyzed through single-strand conformation polymorphisms (SSCP). Results HPV16 CTSEs were detectable in 34 of 58 (57%) specimens and mutant rate was 41%(14/34) and the main mutations of chosen randomly variant CTSE (CTSEv) happened at YY1 binding sites in addition to glucocoticoid response elements (GRE). Conclusion CTSE in some specimens of Shaanxi province was obviously different from that in HPV16 wild type and variant CTSE might affect the transcriptional regulation of LCR on viral P97, which regulates over-expression of viral oncogenes in cervical carcinoma.
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Objective To investigate the relationship between erythrocyte immune function and selenium (Se)level. Methods Forty-nine Kashin-Beck patients in endemic area aged 13- 16 years were divided into two groups and were orally given either selenized yeast or sodium selenite to provide 200 μg selenium per day for 12 weeks. Erythrocyte selenium level, glutathione peroxidase activity, the rosette formation rates of red blood cells complement receptor type Ⅰ(CR1), the immune function of red blood cells, and circulating immune complexes(CIC) were determined. Results After supplementing with selenium for 12 weeks, erythrocyte selenium level, glutathione peroxidase activity, the rosette formation rates of red blood cells CR1 were significantly increased. But the difference in rosette formation rates of IC and CIC content was not significant between before and after Se supplementation. Conclusion The increase of the immune function of the erythrocyte by selenium-supplement may be one of the effective mechanisms for the prevention of Kashin-Beck disease.
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Objective To clone the fragment of hepatitis C virus(HCV) core gene and express it in E.coli.Methods The fragment of HCV core gene(approximate 366bp) was amplified by PCR and inserted into the pMD18-T vector.The cloned HCV core gene,which was confirmed by the digestion with EcoRⅠ/BamHⅠ,was subcloned into the expression vector pBV220 to construct recombinant plasmid pBV220/HCV-C.The expressed gene product was identified by SDS-PAGE and Western blotting.Results The fragment of HCV core gene was expressed successfully after temperature induction and a protein of 14 000 u was resulted.Conclusion Expression of the HCV-C gene in E.coli was achieved,which may be helpful for further studies on characterizations of HCV-C gene.
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<p><b>BACKGROUND</b>To study the effect of gene amplification and selection system with DHFR plus GS and DHFR or GS gene on the foreign gene expression.</p><p><b>METHODS</b>Using the N-terminal truncated hTPO(T184) gene as target gene, two plasmidsre were constructed: pDC- T184 and pGC-T184 where DHFR and GS gene were used respectively as the selective amplification marker. They were cotransfected into CHO dhfr cells to establish dual gene amplification and selection system of DHFR plus GS gen and respectively transfected to establish single gene amplification and selection system of DHFR or GS gene. Three selective methods in dual selective system to compare expression efficiency of hTPO were designed: the first method (DG) was to use drug pressure of MTX, then use MSX; the second method (GD) was reversed; the third method was simultaneously to use MTX and MSX as drug pressure.</p><p><b>RESULTS</b>DHFR+GS dual system had not only higher gene amplification efficiency but also higher level expression. There was no distinct affect in different method of drug pressure.</p><p><b>CONCLUSIONS</b>MTX plus MSX dual drug pressure in dual selection system was an efficient and simple method to increase the expression of foreign gene in mammalian cells.</p>
Subject(s)
Animals , Cricetinae , CHO Cells , Gene Amplification , Gene Expression , Glutamate-Ammonia Ligase , Genetics , Methotrexate , Pharmacology , Plasmids , Genetics , Tetrahydrofolate Dehydrogenase , GeneticsABSTRACT
<p><b>BACKGROUND</b>To explore the feasibility of cloning of the hepatocyte receptor interacting with the Pre S1 protein of HBV by two hybrid system.</p><p><b>METHODS</b>Yeast expression plasmids encoding fusion proteins of full length or portions of Pre S1 of HBV and DNA binding domain of yeast protein GAL4 were constructed and used to transform yeast reporter strain SFY526. Reporter gene product ?galactosidase activity was assayed as a measure of transcription activation in yeast. Mammalian expression plasmid encoding fusion proteins of full length Pre S1 and DNA binding domain of GAL4 was constructed and used to cotransfect hepatoma cell line Huh?7 together with CAT reporter plasmid. Cell extracts were assayed for CAT activity by thin?layer chromatography.</p><p><b>RESULTS</b>The fusion proteins of full length Pre S1 protein and GAL4 DNA binding domain present transcriptional activation function in yeast. The transcription activating sequence is localized to the 21 to 47 amino acids of Pre S1 protein Fusion proteins of full length Pre S 1 and GAL 4 DNA binding domain do not show transcriptional activation function in mammalian cells.</p><p><b>CONCLUSION</b>The transcriptional activating sequence of HBVPre S1 protein in yeast overlaps the hepatocyte receptor binding site. The transcriptional activation function of HBV Pre S1 protein in yeast may prevent researchers?from using yeast two hybrid system to clone HBV receptor interacting with Pre S1 protein. However, the Pre S1 protein does not show transcriptional activation function in mammalian cells. Mammalian two?hybrid system may be a practical method to clone the HBV hepatocyte receptor interacting with Pre S1 protein.</p>
Subject(s)
Humans , DNA-Binding Proteins , Genetics , Fungal Proteins , Genetics , Hepatitis B Surface Antigens , Genetics , Protein Precursors , Genetics , Recombinant Fusion Proteins , Genetics , Transcriptional Activation , Tumor Cells, Cultured , YeastsABSTRACT
Objective To investigate the feasibility of using synbiotics and probiotics to prevent and cure intestinal dysbacteriosis after burn. Methods Burned rats were fed with synbiotics and probiotics reagents, and the amounts of major intestinal florae in caecal contents were detected. Results The major physiological anaerobes were mostly stable, and the conditioned pathogens had no abnormal. Conclusion The micro-ecosystem regulator can quickly supplement the decreased physiological anaerobes caused by burning,and avoid the occurrence of dysbacteriosis.
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Objective To investigate the relationship between Kashin-Beck Disease(KBD) and Human Parvovirus B19(HPVB19).Methods HPVB19DNA was detected in 55 sera of KBD patients and 52 healthy in adjacent non-endemic area and 35 healthy sera in normal area using PCR and then linked the HPVB19DNA to pGEM-T vector.The nucleotide sequence was analyzed and compared with HPVB19 nucleotide sequence published by Genebank and another in Journal of virology.Results HPVB19DNA was found in 16 of 55 sera in KBD patients,and the HPVB19DNA position rate(29.09%) is significantly higher than that of the two healthy control groups(11.54%、11.42% respectively)(P＜0.05).The nucleotide sequence homologies compared with the two published nucleotide sequence were 97.75%、97%,respectively.The putative amino acid homologies compared with the tow published were 93.5%.The amino acid variation was greater than the nucleotide sequence variation because of a base insertion.Conclusion There was a close relationship between HPVB19 infection and Kashin-Beck Disease.
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The relationship between antiproliferative effect of human IFN-γ-EGF3 fusion protein and the influence of EGF receptor binding activity has been studied on A431 cell line. Antiproliferative activity of human IFN-γ-EGF3 was higher than that of its parent IFN-γ. In the 125 I-EGF receptor competition experiment, the inhibition of EGF receptor binding capacity on the target cells was observed in the treatments of human IFN-γ or IFN-γ-EGF3, but the later was more significant. Our data suggests that the antiproliferative effects by IFN-γ and its fusion protein are closely related to their EGF receptor competitions.
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Methylene blue injection has a stronger direct inactivation on VZV in vitro. When the injection was diluted from 1:16 to 1:128, which was obvious (P＜0.01 and P＜0.05). The MIC was 1:222, the IC50 was 1:135 and IC90 was 1:77. The results of microcellculture method showed when the injection was diluted from 1:16 to 1:64, it also effectively inhibited the proliferation of VZV in WISH continuous cell-lines (P＜0.01 and P＜0.05). The MIC was 1:95, the IC50 was 1:45 and the IC90 was 1:21.
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A total of twenty eight HSV isolates from the patients were typed by using HSV typespecific monoclonal antibodies (McAb). Comparison of typing results with McAb in three Sero-lminunological methods with molecular hybridization indicated 100% concordance in the results. But the typing rates were quite different among the various methods (ELISA 64%, IFA and EIA 82%, Hybridization 100%). The results demonstrate that the molecular hybrydization with HSV type-specific probes was highly sensitive and specific, and the method of EIA with HSV type-specific McAb was accurate, cheap, rapid and practical.
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AIM: To investigate whether and how N, N-dimethylsphingosine (DMS) plays a role in modulating the adhesion of monocytes to vascular endothelial cells, and identify whether human umbilical vein endothelial cell line EA.hy926 take place of the vascular endothelial cells.METHODS: Adhesion ratio was measured by flow cytometry, and immunohistochemistry was used to detect the expression of ICAM-1 and P-selectin in HUVEC: EA.hy926 cells after the effect of DMS. RESULTS: DMS inhibited the adhesion of monocytes to HUVEC: EA.hy926 cells in a time-dependent and concentration-dependent manner by reducing the expression of ICAM-1 and P-selectin. CONCLUSIONS: DMS reduced adhesion molecule expression in vascular endothelial cells. DMS may be an important contributor to reduce adhesion ratio, suggesting that DMS plays a negative role in proinflammatory and immune functions of the modified vascular endothelial cells during atherosclerosis and restenosis.
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AIM: To identify the down-regulated genes in adult rat cardiac fibroblasts (CF) stimulated with angiotensin Ⅱ (AngⅡ). METHODS: Suppression subtractive hybridization (SSH) was performed between the CF stimulated by AngⅡ (driver) and unstimulated CF (tester) to generate subtractive cDNA library. The library was screened with dot blots hybridization to further verify the differentially expressed cDNA clones. Partial positive clones were sequenced and BLAST analyzed. RESULTS: Seventeen down-regulated genes related to intracellular signal transduction, transcriptional repression, deposition of fibrous matrix and cellular cytoskeletal rearrangement, and 4 new expression sequence tags (EST) were acquired. CONCLUSION: SSH is a powerful technique with high sensitivity for the detection and clone of down-regulated genes expressed in CF induced by AngⅡ, which is helpful to clarify the mechanism of cardiac remodeling.
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Objective To observe the biological activities of human doppel(Dpl) protein transiently expressed and Dpl-like protein PrP?32-121 on a human neuroblastoma cell line SH-SY5Y.Methods Recombinant mammalian expression plasmids containing human PRND gene and truncated PrP?32-121 fragment were generated by PCR.The expression and location of Dpl and PrP?32-121 post-transfection were observed by IFA.The cytotoxicity was measured by MTT analysis.Cellular apoptosis was investigated by flow cytometry and Western blot.Results Both Dpl and PrP?32-121 protein were expressed and mainly located on the cell membrane.Remarkable cytotoxicity was detected on SH-SY5Y cells after 24 h transfection.Meanwhile,more Annexin V/PI positively-stained cells as well as lower levels of cellular pro-caspase-3 and Bel-2 were detected in the cells receiving Dpl and PrP?32-121 expressing plasmids.Conclusion Dpl protein transiently expressed and PrP?32-121 can lead to the similar neural cytotoxicity,probably triggering the cell apoptosis program.
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Objective To clone and express the ns5a gene of hepatitis C virus(HCV) 1b strain DY.Methods By using the prokaryotic cell gene engineering,HCV ns5a gene was amplified with nested PCR from the plasmid HCV17 of HCV 1b strain DY full-length gene and inserted into the cloning pMD18-T vector.The cloned HCV ns5a gene was separated and subcloned into expression vector pET-28a and induced by IPTG in E.coli.BL21.The expressed product was identified by SDS-PAGE and Western-blot methods.Results Recombinant expression plasmid pet-28a-ns5a was constructed and expressed successfully.Conclusion HCV ns5a gene was cloned and expressed.This might be helpful for further studies on the nature and biological properties of the ns5a gene.
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It is critical to construct safe,efficient and controllable vectors in gene therapy of tumor.The vectors available include viral and non-viral vectors.However,there are numerous problems to be solved.The current progresses focus on studies of safety,high efficiency and combination of multifunctional gene.
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In order to mutate the BamHI C fragment of Herpes Simplex Virus 1 (HSV-1), the thymidin kinase gent-mini Mu phage DNA (TK-mM) was randomly inserted into the BamHI C fragment in the plasmid pRB 132 with DNA recombination technique. The six recombinant plasmids carrying the BamHI C fragment of HSV-1 and inserted TK-mM were isolated with DNA hybridization. The results of restriction endonucleasc analysis showed that the BamHI C fragment has been mutated by insertion of TK-mM system, and the insertional sites of TK-mM located in the six recombinant plasmids were different.
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BamHI C fragment of Herpes Simples Virus type 1 (HSV-1) DNA has been subcloned by recombinant DNA technique and the cloning system of 8 small fragments of the BamHI C fragment was established. At the same time. the restriction enzyme mapping of the BamHI C fragment has also been achieved.