HIV-1 integrase (IN) is a key enzyme for the viral replication. The protein-protein interaction (PPI) between HIV-1 IN and a cellular cofactor lens epithelium-derived growth factor (LEDGF/p75) is a validated target for anti-HIV drug discovery. In order to build the platform for screening inhibitor against PPI between IN and LEDGF/p75, the vector containing the LEDGF/p75 protein cDNA was constructed and expressed in Escherichia coli and the function of the LEDGF/p75 protein was assayed. The LGDGF/p75 encoding gene optimized according to the preference codon usage of E. coli, was synthesized and cloned into the expression vector pGEX-4T-1 to form a recombined plasmid, then transformed into host cell E. coli BL21 (DE3). The recombined clones were identified and confirmed by BamH I/Sal I digestion and sequencing, the successfully recombined plasmid in the host cell was induced by IPTG and the condition of the expression was optimized. The expressed protein was purified by the Ni2+ affinity chromatography column and SDS-PAGE was used to analyze the molecular weight and specificity. In addition, ELISA assay was used to analyze the function of the recombinant protein. The recombinant LGDGF/p75 was soluble, and expressed highly and stably in E. coli. The protein was proved to enhance HIV-1 IN strand transfer activity in vitro by ELISA. It will be helpful to build the platform of screening inhibitors against PPI between IN and LEDGF/p75.
Cloning, Molecular ; Escherichia coli ; metabolism ; HIV Integrase ; metabolism ; HIV-1 ; physiology ; Humans ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; Protein Binding ; Virus Replication

Objective: To reconstruct adenovirus vector of breast eukaryotic initiation factor 4E and to observe its effect on the metastasis ability of breast cancer cell line MCF-7. Methods: eIF-4E gene was constructed into adenovirus vector pAD-X by gene recombination technique, which was transformed into 293 packaged cell for high titer adenovirus. Real-time PCR was applied to detect eIF-4E gene expression. eIF-4E siRNA was applied and then transwell cabin assay was used to observe changes of invasion and motor ability of MCF-7 cells transfected with reconstruction adenovirus. Result:The finding of digestion was coincided with expected. eIF-4E gene over-expression was detected in transfected MCF-7 cells with real-time PCR. And the invasion and motor abilities of transfected MCF-7 cells were more significantly inhibited in transwell cabin assay (respectively p

Humans ; Occupational Diseases ; diagnosis

Objective To investigate change of chemokine mRNA and protein in the patients with diabetes mellitus(DM)complicated infection.Methods Fourty-eight patients with DM complicated infection were studied.Expression of mRNA and protein of monocyte chemoattractant protein-1(MCP-1)and interleukin 8 (IL-8)were measured by FQ-PCR,RT-PCR and ELISA.The relationship between hs-CRP,PMNL,IL-8 and MCP-1 were analyzed.Results Expression of mRNA and protein of MCP-1 and IL-8 were increased in the DM patients complicated infection.MCP-1 mRNA(mesured by FQ-PCR)was positively correlated with the protein(IL-8 r = 0.33;MCP-1 r = 0.88;P

Objective To determine whether left ventricular Tei Index evaluate the cardiac function and prognosis of patients with sepsis-induced cardiomyopathy (SIC).Methods A total of 86 patients with septic shock combined with SIC in the emergency department of Beijing Chaoyang Hospital affiliated to Capital Medical University from July 2014 to June 2016 were recruited and divided into non-survival group (n=35) and survival group (n=51) according to 28-day follow-up.Left ventricular Tei Index, BNP, cTNI and left ventricular ejection fraction within the first 24 h after admisson were detected and compared between the two groups.The correlations of left ventricular Tei Index to BNP, cTNI and ejection fraction were analyzed.The receiver operating characteristic curves (ROC) were constructed to analysize the value of Tei Index in evaluating the cardiac function and prognosis.Results The patientsin the non-survival group had a higher Tei Index compared with that in the survival group [(0.75±0.13) vs.(0.51±0.09), P<0.05].The Tei Index of SIC patients was significantly positively correlated with BNP and cTNI (both P<0.05), and significantly negatively correlated with ejection fraction (P<0.05).The AUC of Tei Index for predicting 28-day mortality in SIC patients was high comapred with that of BNP, cTNI and ejection fraction.Conclusion The left ventricular Tei Index has a reliable value in evaluating the cardiac function and prognosis of patients with SIC.

NSP4, as the diarrhea-related protein of rotavirus, is becoming an attractive candidate for vaccine development. To compare the immunogenicity of NSP4 from different genetic groups, we constructed eukaryotic expression plasmids comprising the NSP4 genes from four different genetic types using the pCI vector. The recombinant vectors were designated as pCI-97B6, pCI-97S36, pCI-97S34 and pCI-97SZ8, respectively. Following the conformation of the transient expression of the constructs in 293 cells, the plasmids were respectively subjected to the 5 round i. m. inoculation of BALB/c mice. The specific antibodies against NSP4 as well as the IgG1/IgG2a subclasses of immunoglobulin in mice sera were examined with indirect ELJSA after each immunization. The results showed that the immunization of plasmids expression NSP4s could elicit not only humoral but also cellular immunity, but the humoral immune response is dominant. There is a difference of immunogenecity among the NSP4 of different genetic type. Further studies were needed to focus on the relationship between the immunogenicity and protection effect.

Sixteen compounds were isolated from the aerial parts of Euphorbia sikkimensis by means of various chromatographic techniques such as silica gel, Sephades LH-20 and RP-18, and their structures were elucidated as naringenin (1), kaempferol (2), quercetin (3), kaempferol-3-O-alpha-L-arabinopyranoside (4), quercetin-3-O-alpha-L-arabinopyranoside (5), quercetin-3-O-(2"-galloyl)-alpha-L-arabinopyranoside (6), 5alpha, 8alpha-epidioxy-(22E, 24R)-ergosta-6,22-dien-3beta-ol (7), stigmast-5-ene-7-one-3beta-ol (8), 3beta-hydroxy4a, 14alpha-dimethyl-5alpha-ergosta-8, 24(28)-dien-7-one(9), beta-sitosterol (10) , 10-cucurbitadienol( 1) , scopoletin(12) , ethyl gallate(13), p-hydroxybenzaldehyde (14), 3 betahydroxybenzeneethanol( 15) ,and 2,4-dihydroxy-6-methoxy-acetophenone (16) on the basis of spectroscopic data analysis. All the compounds are isolated from this plant for the first time, and compounds 1, 4-8, 15 are obtained from Euphorbia species for the first time.
Chromatography ; Euphorbia ; chemistry ; Organic Chemicals ; analysis ; isolation & purification

Mitogen-activated protein kinases (MAPKs) are important signaling transduction components well conserved in eukaryotes and play essential roles in various physiological, developmental and hormonal responses in plant. In the present study, a MAPK gene, designated as DoMPK4 (GenBank accession No. JX297597), is identified from a rare endangered medicinal orchid species D. officinale using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The full length cDNA of DoMPK4 is 1 518 bp in length and encoded a 369 aa protein with a molecular weight of 42.42 kD and an isoelectric point of 5.55. DoMPK4 protein contained a serine/threonine protein kinase active site (158-170), a MAP kinase site (71-174), and eight conserved motifs. DoMPK4 had a transmembrane (214-232) but no signal peptide. Multiple sequence alignment showed that DoMPK4 shared high identities (74.9%-80.6%) with MAPK proteins from various plants. Phylogenetic analysis demonstrated that DoMPK4 belonged to group A of the MAPK evolutionary tree, and is closely related to monocots. Real time quantitative PCR (qPCR) analysis revealed that DoMPK4 is differentially expressed among the five organs including leaf, stem, root, seed, and protocorm-like body (PLB). The transcription level of DoMPK4 is the highest in the PLBs with 17.65 fold, followed by seeds, roots, and stems with 5.84, 2.28, and 1.64 fold, respectively. The progressive enhancement of DoMPK4 transcripts in the developing PLBs compared to that in the germinating seeds, suggests a role of DoMPK4 during the development of embryogenic PLBs formation in D. officinale.
Amino Acid Sequence ; DNA, Complementary ; genetics ; DNA, Plant ; genetics ; Dendrobium ; enzymology ; genetics ; Gene Expression Regulation, Plant ; Mitogen-Activated Protein Kinases ; genetics ; metabolism ; Phylogeny ; Plant Leaves ; metabolism ; Plant Proteins ; genetics ; metabolism ; Plant Roots ; metabolism ; Plant Stems ; metabolism ; Seeds ; metabolism ; Sequence Alignment

The zinc-regulated transporters (ZRT), iron-regulated transporter (IRT)-like protein (ZIP) plays an important role in the growth and development of plant. In this study, a full length cDNA of ZIP encoding gene, designed as DoZIP1 (GenBank accession KJ946203), was identified from Dendrobium officinale using RT-PCR and RACE. Bioinformatics analysis showed that DoZIP1 consisted of a 1,056 bp open reading frame (ORF) encoded a 351-aa protein with a molecular weight of 37.57 kDa and an isoelectric point (pI) of 6.09. The deduced DoZIP1 protein contained the conserved ZIP domain, and its secondary structure was composed of 50.71% alpha helix, 11.11% extended strand, 36.18% random coil, and beta turn 1.99%. DoZIP1 protein exhibited a signal peptide and eight transmembrane domains, presumably locating in cell membrane. The amino acid sequence had high homology with ZIP proteins from Arabidopsis, alfalfa and rice. A phylogenetic tree analysis demonstrated that DoZIP1 was closely related to AtZIP10 and OsZIP3, and they were clustered into one clade. Real time quantitative PCR analysis demonstrated that the transcription level of DoZIP1 in D. officinale roots was the highest (4.19 fold higher than that of stems), followed by that of leaves (1.12 fold). Molecular characters of DoZIP1 will be useful for further functional determination of the gene involving in the growth and development of D. officinale.
Amino Acid Sequence ; Cloning, Molecular ; Dendrobium ; chemistry ; classification ; genetics ; metabolism ; Gene Expression Regulation, Plant ; Iron ; metabolism ; Membrane Transport Proteins ; chemistry ; genetics ; metabolism ; Molecular Sequence Data ; Phylogeny ; Plant Proteins ; chemistry ; genetics ; metabolism ; Plants ; chemistry ; classification ; genetics ; Sequence Alignment ; Zinc ; metabolism

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate in mavalonic acid pathway, which is the first committed step for isoprenoid biosynthesis in plants. However, it still remains unclear whether HGMR gene plays a role in the isoprenoid biosynthesis in Dendrobium officinale, an endangered epiphytic orchid species. In the present study, a HMGR encoding gene, designed as DoHMGR1 (GenBank accession JX272632), was identified from D. officinale using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods, for the first time. The full length cDNA of DoHMGR1 was 2 071 bp in length and encoded a 562-aa protein with a molecular weight of 59.73 kD and an isoelectric point (pI) of 6.18. The deduced DoHMGR1 protein, like other HMGR proteins, constituted four conserved domains (63-561, 147-551, 268-383 and 124-541) and two transmembrane motifs (42-64 and 85-107). Multiple sequence alignment and phylogenetic analyses demonstrated that DoHMGR1 had high identity (67%-89%) to a number of HMGR genes from various plants and was closely related to Vanda hybrid cultivar, rice and maize monocots. Real time quantitative PCR (qPCR) analysis revealed that DoHMGR1 was expressed in the three included organs. The transcripts were the most abundant in the roots with 2.13 fold over that in the leaves, followed by that in the stems with 1.98 fold. Molecular characterization of DoHMGR1 will be useful for further functional elucidation of the gene involving in isoprenoid biosynthesis pathway in D. officinale.
Base Sequence ; Cloning, Molecular ; DNA, Complementary ; genetics ; Dendrobium ; enzymology ; genetics ; Gene Expression Regulation, Plant ; Hydroxymethylglutaryl CoA Reductases ; genetics ; metabolism ; Molecular Weight ; Phylogeny ; Plant Leaves ; enzymology ; genetics ; Plant Roots ; enzymology ; genetics ; Plant Stems ; enzymology ; genetics ; Plants, Medicinal ; enzymology ; genetics ; Sequence Alignment ; Sequence Homology, Amino Acid