HIV-1 integrase enzyme is a 32kDa protein encoded by HIV pol gene. It is responsible for integration of viral cDNA into host chromosomal DNA, which is indispensable for HIV replication.Since there was no functional equivalent for this enzyme in human cells, inhibition of integrase will bring little side effect to human body. Thus HIV integrase has become an attractive and rational target for therapy of AIDS after reverse transcriptase and protease.The Recent research on HIV-1integrase structure,inhibitors and new therapeutic method target at HIV integrase was reviewed.

Amino Acid Sequence ; Animals ; Calcium Channel Blockers ; pharmacology ; Calcium Channels ; drug effects ; Humans ; Molecular Sequence Data ; Structure-Activity Relationship ; omega-Conotoxins ; chemistry ; pharmacology

Animals ; Cytoplasm ; metabolism ; Endoplasmic Reticulum, Rough ; metabolism ; Golgi Apparatus ; metabolism ; Humans ; Protein Transport ; Ricin ; chemistry ; pharmacokinetics ; therapeutic use

OBJECTIVETo develop an ELISA-based method for analyzing biologic activities of HIV-1 integrase and for high throughput screening of integrase inhibitors.

METHODSAfter expression, renaturation and purification of integrase, the bioactivity of integrase and the inhibition of luffin-a were evaluated with an in vitro assay based on biotin-avidin EILSA and chemiluminescent substrates.

RESULT(1) The specific activity of the purified integrase was 54.92 units/mg of protein. (2)IC(50) (concentration causing 50% inhibition of integrase) of luffin-a was (0.63 +/- 0.026) micromol/L.

CONCLUSIONThe non-radioactive assay can be used for analysis of bioactivities and high throughput screening of inhibitors of HIV-1 integrase.

Catalysis ; drug effects ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; pharmacology ; Enzyme-Linked Immunosorbent Assay ; methods ; HIV Integrase ; chemistry ; metabolism ; Humans ; Kinetics ; Luminescent Measurements ; Ribosome Inactivating Proteins, Type 1 ; pharmacology ; Substrate Specificity

OBJECTIVETo obtain peptides binding specifically to Pisum sativum agglutinin (PSA) from a phage-displayed random peptide library.

METHODS(1) A phage-displayed random hexapeptide library was screened with PSA as target. (2) Dot blot was used to analyze the influence of the alpha-Met-D-mannoside on binding between PSA and phage-displayed peptides. (3) Three peptides (RMWSF, RYDYSY, LRLRQL) were selectively synthesized, and different concentrations were used to inhibit PSA and ConA binding to the HRP.

RESULTSThe enrichment occurred obviously after three rounds of screening. The insert sequences of amino acids, displayed on 22 phage DNAs from the third round of screening, were divided into three groups. The binding of phage-displayed peptides to PSA was specific as shown by dot blot and could be inhibited by alpha-Met-D-mannoside. LRLRQL was not dissolved in water. ARMWSF and RYDYSY inhibited binding of PSA to HRP, but failed to inhibit binding ConA to HRP.

CONCLUSIONThe binding site of peptides ARMWSF and RYDYSY is different to that of alpha-Met-D-mannoside.

Binding Sites ; Peptide Library ; Peptides ; metabolism ; Plant Lectins ; metabolism ; Protein Binding ; Recombinant Proteins ; metabolism

OBJECTIVETo extract and purify ricin from castor beans and to evaluate its anti-cancer activity.

METHODSRicin was purified from castor beans according the modified method of Nicolson and Blaustin. The lectins were extracted in 0.01 mol/L phosphate buffered saline and isolated in the 40% to 80% fraction of ammonium sulfate precipitation. The dialyzed fractionated preparation was applied with a Sepharose 4B column. The lectins were eluted with a linear lactose gradient (0.01 mol/L approximately 0.5 mol/L). Ricin was separated from the ricinus agglutinin by gel filtration on a Sephadex G-100. MTT was applied to analyze the cytotoxicity with different dosage of ricin in different cancer cell lines.

RESULTSThere was no difference between the killing effect of normal cells and that of colon cancer cells by using the high dosage of ricin (5 x 10(-8) mol/L approximately 5 x 10(-10) mol/L). However, the cytotoxicity was significant different in those cells with the low dosage of ricin (5 x 10(-11) mol/L approximately 5 x 10(-13) mol/L). Meanwhile ricin had the similar cytotoxicity to leukemia cell K562 and colon cancer cell SW480.

CONCLUSIONRicin is able to kill tumor cells selectively at low concentration, but the selectivity does not appear at high concentrations.

Animals ; Antineoplastic Agents, Phytogenic ; pharmacology ; Cell Line, Tumor ; Colorectal Neoplasms ; pathology ; Dose-Response Relationship, Drug ; Humans ; K562 Cells ; Male ; Mice ; Rats ; Rats, Sprague-Dawley ; Ricin ; isolation & purification ; pharmacology ; T-Lymphocytes, Cytotoxic

OBJECTIVETo clone luffin-a cDNA from the seeds of Luffa cylindrical, and to obtain bioactive recombinant luffin-a protein using the expression vector pET-44a (+) in E. coli.

METHODSThe cDNA sequence encoding luffin-a was cloned from the fresh seeds of Luffa cylindrical by RT-PCR. The target DNA fragments were sequenced after T-A cloning. The luffin-a expression plasmid was constructed by inserting the luffin-a cDNA fragment into vector pET-44a (+). Luffin-a was expressed in E. coli by addition of IPTG into final concentration 1.0 mmol/L. The recombinant luffin-a was identified by SDS-PAGE. The biological activity of luffin-a protein was evaluated by using the MTT assay in HepG2 cells following fluid-phase endocytosis.

RESULTSIn comparison with the reported luffin-a, the homology of nucleotide sequence of the cloned luffin-a gene was 99.73%, while their amino acid sequences were identical. The solubility of recombinant protein was analyzed by SDS-PAGE, and the luffin-a was mainly produced in inclusion bodies. The recombinant luffin-a, renatured by dialysis of the denatured products, showed a similar cytotoxicity to ricin A chain.

CONCLUSIONThe cDNA of luffin-a has been successfully cloned. The recombinant luffin-a protein expressed by E. coli is bioactive.

Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; Luffa ; chemistry ; Molecular Sequence Data ; Plant Proteins ; biosynthesis ; genetics ; Polymerase Chain Reaction ; Ribosome Inactivating Proteins, Type 1 ; Seeds ; chemistry

OBJECTIVETo study the expression and purification of a fusion protein of ricin A chain (RTA) and green fluorescent protein (GFP).

METHODSThe DNA sequence encoding ricin A chain was inserted into pEGFPC1 first to make the template sequence of the fusion protein. The fusion gene was amplified from the plasmid pEGFP-RTA by PCR, and directly subcloned into T vector. The fusion gene then was cloned into expression vector pET-28a(+), and the sequence was confirmed by sequencing. Expression was induced by IPTG in E. coli BL21(DE3). The fusion protein was purified by metal chelated affinity chromatography. The cytotoxicity of fusion protein was analyzed by the MTT assay in HepG2 and Hela cells.

RESULTSThe fusion protein of ricin A chain and GFP could be produced in E. coli transformed with the expression plasmid of pET-28a(+)-GFP-RTA. The molecular weight of the recombinant protein was measured by SDS-PAGE. The fusion protein showed a green fluorescence and had a similar cytotoxicity of RTA.

CONCLUSIONA recombinant fusion protein of RTA and GFP expressed in E. coli is possessed of similar biological activity of individual GFP and RTA, which could be used in study of the intracellular trafficking and translocation of RTA.

Escherichia coli ; genetics ; metabolism ; Green Fluorescent Proteins ; genetics ; HeLa Cells ; Humans ; Luminescent Proteins ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Ricin ; genetics

Animals ; Bacterial Toxins ; chemistry ; pharmacology ; therapeutic use ; Humans ; Neoplasms ; therapy ; Neurotoxins ; chemistry ; pharmacology ; therapeutic use ; Proteins ; chemistry ; pharmacology ; therapeutic use ; Toxins, Biological ; chemistry ; pharmacology ; therapeutic use

OBJECTIVETo clone and produce ribosome inactivating protein MAP30 from the seeds of Momordica charantia L(bitter melon), and to evaluate the biological activity of the recombinant protein.

METHODSThe DNA sequence encoding MAP30 was cloned from the fresh seeds of Momordica charantia by PCR, the target DNA fragments were sequenced after T-A cloning. The expression plasmid was constructed by inserting the MAP30 fragment into vector pET30a. MAP30 was expressed in E.coli by addition of IPTG into final concentration of 1.0 mmol/L. The recombinant MAP30 was identified by SDS-PAGE, and the biological activity of MAP30 protein was evaluated by using MTT assay in cancer cells and normal cells following fluid-phase endocytosis.

RESULTThe nucleotide and amino acid sequences of the cloned MAP30 were identical with those of reported MAP30. The solubility of recombinant protein was analyzed by SDS-PAGE, and the MAP30 was mainly produced in soluble form. The recombinant MAP30 showed a greater cytotoxicity to cancer cells than that to normal cells.

CONCLUSIONThe gene of MAP30 has been successfully cloned.The recombinant MAP30 protein expressed by E.coli is bioactive.

Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Momordica charantia ; chemistry ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Ribosome Inactivating Proteins, Type 2 ; biosynthesis ; genetics ; metabolism ; Seeds ; chemistry ; Transformation, Bacterial