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【Objective】 To construct the eukaryotic expression vector carrying the human wild-type p27 and lacking nuclear localization signal p27△NLS coding sequences， and the express them in HEK293T cells， which may contribute to investigating the different locations and roles of p27 in the cytoplasm and nucleus. 【Methods】 Total RNA was prepared from human breast cancer MCF7 cells， and cDNA was obtained by reverse transcription-polymerase chain reaction （RT-PCR）. After amplification of the p27 CDs and non-NLS fragments by PCR， full length p27WT （CDKN1B， NM_004064.5） and p27△NLS coding regions were obtained. PCR products were then subcloned into the eukaryotic expression vector pCMV-Blank. After identification with bacterial PCR， double restriction enzyme digestion and sequencing， they were defined officially as pCMV-p27WT and pCMV-p27△NLS， respectively. The recombinant plasmids were transfected into HEK293T cells by electroporation. After 48 h， the levels of p27 protein in the cytoplasm and nucleus were detected by Western blotting. 【Results】 The sequencing results showed that the sequences of p27WT and p27△NLS inserted into the plasmids were both correctly consistent with that of NM_004064.5. After transfection with pCMV-p27WT， total p27 protein expression was increased and distributed in both the cytoplasm and nucleus of HEK293T cells. After transfection with pCMV-p27△NLS， p27 protein was significantly increased and almost entirely localized in the cytoplasm of HEK293T cells. 【Conclusion】 The eukaryotic expression plasmids of human p27WT and p27△NLS coding sequences were successfully constructed and overexpressed in HEK293T cells. This research may lay a foundation for investigating the biological function of p27 in the cell cycle progression of tumor cells.
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【Objective】 To clone the full-length of human kidney and brain protein (KIBRA) coding sequence in eukaryotic expression vector and provide a model for studying the biological function of KIBRA in breast cancer cells. 【Methods】 Total RNA of human breast cancer cell line MCF7 was extracted. After reverse transcription, the full length of KIBRA (NM_001161661.2) coding region was amplified by PCR, and cloned into eukaryotic expression vector pCMV-Blank. After identification, it was defined officially as pCMV-KIBRA. Then it was transfected into MCF7 cells, and the expression of KIBRA was detected by real-time PCR and Western blotting after 48 hours. The primary, secondary and tertiary structures and post-transcriptional modification sites of KIBRA were analyzed with bioinformatics software. 【Results】 Bacterial PCR, double enzyme digestion and DNA sequencing results showed that the correct sequence of KIBRA was inserted into the vector pCMV-KIBRA. The mRNA and protein expressions of KIBRA were significantly increased in MCF7 cells transfected with pCMV-KIBRA. Bioinformatics analysis showed that KIBRA was composed of 1119 amino acids. There were 52 phosphorylation sites, 1 acetylation site and 5 ubiquitination sites, and the protein structure was mainly α-helix and random coil. 【Conclusion】 The eukaryotic expression vector of full-length of human KIBRA coding sequence was successfully constructed and overexpressed in breast cancer cell line MCF7, which can lay a foundation for studying the biological function of KIBRA in breast cancer.
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【Objective】 To construct the Rb luciferase reporter gene assay system and detect the activation ability of Rb gene for screening the targeted drugs. 【Methods】 The synthetic Rb gene sequence was annealed to form a double-stranded DNA structure and then inserted into the polyclonal site of pGL6-TA. The junction product was transformed into E.coli DH5α competent cells for expanded culture, and the constructed pGL6-Rb-Luc plasmid and pGL6-TA plasmid were transfected into HEK293 cells. The monoclonal cell line HEK293-Rb-Luc with stable expression was screened by G418, and the activation and inhibition of Rb in HEK293-Rb-Luc were tested by serum and CDK4/6 inhibitor Palbociclib. 【Results】 The sequence of Rb reaction elements in pGL6-Rb-Luc was completely correct. The recovery of serum culture significantly increased the luciferase activity in HEK293-Rb-Luc (P<0.001). Compared with 0 nmol/L, 25, 50, 75 and 100 nmol/L, CDK4/6 inhibitor Palbociclib made the inhibition rate of Rb activity rise to 6.90%, 40.23%, 50.57% and 52.07%, respectively (P<0.05). 【Conclusion】 The Rb luciferase reporter gene detection system HEK293-Rb-Luc was successfully constructed, which can effectively detect the activation level of Rb.
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Objective@#To investigate the clinical and epidemiological characteristics of Coronavirus HKU1 (Human CoV-HKU1) and NL63 (Human CoV-NL63) in children with acute respiratory tract infection in Nanjing.@*Methods@#From August 2009 to July 2011, 1 286 respiratory samples were collected from the outpatient and hospitalized children in the Children′s Hospital of Nanjing Medical University. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect HCoV-HKU1 and NL63 genes, besides, positive samples were used for common respiratory virus screening. The positive amplification products were cloned, sequenced, homologous and phylogenetic analysis was conducted by molecular biological method .@*Results@#The detection rate of HCoV-HKU1 was 1.1% (14/1 286), the positive sequences shared a 98.2%-100% nucleotide identity with the HCoV-HKU1 strains and mixed infection rate was 92.9%. The main clinical diagnoses were bronchitis, bronchopneumonia and bronchiolitis. The clinical manifestations were cough, fever, wheezing. The detection rate of HCoV-NL63 was 1.5% (19/1 286), the positive sequences shared a 95.6%-100% nucleotide identity with the HCoV-NL63 strains and mixed infection rate were 63.2%. The main clinical diagnosis were acute upper respiratory tract infection, bronchitis, bronchopneumonia. The clinical manifestations were fever, cough, expectoration. No deaths were found in both HCoV-HKU1 and NL63 infections.@*Conclusions@#From August 2009 to July 2011, HCoV-HKU1 and NL63 were detected in children with respiratory tract infection in Nanjing area. HCoV-HKU1 infected cases were lower respiratory tract infection, epidemic in winter and spring, infected cases were mainly under 1 years of age, HCoV-NL63 infected cases including upper respiratory and lower respiratory tract infection, epidemic in the season of summer and autumn. The infected cases were mainly at the age rank from 1 year to 3 years. The clinical manifestations of children infected with coronavirus HKU1 and NL63 are not specificity.
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Objective To compare the clinical effect and safety evaluation of three different dose regimens for treating children with viral encephalitis.Methods Totally 126 cases treated in Xi'an Central Hospital from January 2010 to December 2015 were randomly divided into observation group 1 (ganciclovir combined with gangliosides,42 cases),observation group 2 (ganciclovir combined with gamma globulin,43 cases),and control group (39 cases).The clinical effect and levels of NSE,inflammatory cytokine were compared in the three groups.Results The total effective rate in observation group 1 was 95.24％ and that of observation group 2 was 93.02％,which were significantly higher than that of control group (79.48％).The disappearance time of headache,fever,convulsions,clouding of consciousness,meningeal irritation sign,cerebrospinal fluid abnormalities,and length of stay in observation groups (both 1 and 2)were significantly shorter than those in control group (P ＜ 0.05);After therapy,the levels of NSE in three groups were obviously decreased compared with those before therapy (P ＜ 0.05),and those in observation group were significantly lower than the control group (P ＜ 0.05);the levels of inflammatory cytokine in all three groups were obviously decreased compared with those before therapy (P ＜ 0.05),and that of observation group 1 had no statistical difference with the normal group,whereas that in control group was significantly higher than the normal group (P ＜ 0.05).Conclusion Ganciclovir combined with gangliosides as well as ganciclovir combined with gamma globulin were both effective methods in treating children with viral encephalitis and could decrease levels of inflammatory cytokine.Ganciclovir combined with gangliosides could effectively repair nerve damage,which deserves clinical expansion.
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Objective:To compare the clinical effect and safety of three different regimens treating children with severe pneumonia.Methods:120 cases treated in our hospital from January,2012 to January,2016 were randomly divided into the observation group 1 (dopamine combined with dobutamine,42 cases),observation group 2 (dopamine combined with phentolamine,40 cases),control group (38 cases).The clinical effect and levels of inflammatory cytokine were compared between the three groups.Results:The total effective rate in the observation group 1 was 90.48％ and that of observation group 2 was 87.5％,which were significantly higher than that of the control group (63.16％).The disappearance time of pulmonary rales,cough,dyspnea,pyretolysis and length of stay in the observation group (both 1,2) were significantly shorter than those of the control group (p ＜0.05).After therapy,the level of serum IL-6,IL-8,CRP and TNF-α in all the three groups were obviously decreased compared with those of before therapy (p＜0.05),and those of the observation group were significantly lower than the control group (p ＜ 0.05).Conclusion:Dopamine combined with dobutamine as well as dopamine combined with phentolamine were both effective methods in treating children with severe pneumonia,which were significantly better than conventional therapy.