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David A. Bernlohr - One of the best experts on this subject based on the ideXlab platform.
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characterization of the functional interaction of adipocyte Lipid Binding Protein with hormone sensitive lipase
Journal of Biological Chemistry, 2001Co-Authors: Wenjun Shen, David A. Bernlohr, Kunju Sridhar, Yu Liang, Richard Hong, Shailja Patel, Vanita Natu, Anne Jenkins, Fredric B KraemerAbstract:Hormone-sensitive lipase (HSL) is an intracellular lipase that plays an important role in the hydrolysis of triacylglycerol in adipose tissue. HSL has been shown to interact with adipocyte Lipid-Binding Protein (ALBP), a member of the family of intracellular Lipid-Binding Proteins that bind fatty acids and other hydrophobic ligands. The current studies have addressed the functional significance of the association and mapped the site of interaction between HSL and ALBP. Incubation of homogeneous ALBP with purified, recombinant HSL in vitro resulted in a 2-fold increase in substrate hydrolysis. Moreover, the ability of oleate to inhibit HSL hydrolytic activity was attenuated by co-incubation with ALBP. Co-transfection of Chinese hamster ovary cells with HSL and ALBP resulted in greater hydrolytic activity than transfection of cells with HSL and vector alone. Deletional mutations of HSL localized the region of HSL that interacts with ALBP to amino acids 192-200, and site-directed mutagenesis of individual amino acids in this region identified His-194 and Glu-199 as critical for mediating the interaction of HSL with ALBP. Interestingly, HSL mutants H194L and E199A, each of which retained normal basal hydrolytic activity, failed to display an increase in hydrolytic activity when co-transfected with wild type ALBP. Therefore, ALBP increases the hydrolytic activity of HSL through its ability to bind and sequester fatty acids and via specific Protein-Protein interaction. Thus, HSL and ALBP constitute a functionally important lipolytic complex.
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Lipid-Binding Proteins modulate ligand-dependent trans-activation by peroxisome proliferator-activated receptors and localize to the nucleus as well as the cytoplasm.
Journal of lipid research, 2000Co-Authors: Torben Helledie, David A. Bernlohr, Marianne Antonius, Karsten Kristiansen, Ann V. Hertzel, Rikke V. Sørensen, Steen Kølvraa, Susanne MandrupAbstract:Peroxisome proliferator-activated receptors (PPARs) are activated by a variety of fatty acids, eicosanoids, and hypoLipidemic and insulin-sensitizing drugs. Many of these compounds bind avidly to members of a family of small Lipid-Binding Proteins, the fatty acid-Binding Proteins (FABPs). Fatty acids are activated to CoA esters, which bind with high affinity to the acyl-CoA-Binding Protein (ACBP). Thus, the availability of known and potential PPAR ligands may be regulated by Lipid-Binding Proteins. In this report we show by transient transfection of CV-1 cells that coexpression of ACBP and adipocyte Lipid-Binding Protein (ALBP) exerts a ligand- and PPAR subtype-specific attenuation of PPAR-mediated trans-activation, suggesting that Lipid-Binding Proteins, when expressed at high levels, may function as negative regulators of PPAR activation by certain ligands. Expression of ACBP, ALBP, and keratinocyte Lipid-Binding Protein (KLBP) is induced during adipocyte differentiation, a process during which PPARgamma plays a prominent role. We present evidence that endogenous ACBP, ALBP, and KLBP not only localize to the cytoplasm but also exhibit a prominent nuclear localization in 3T3-L1 adipocytes. In addition, forced expression of ACBP, ALBP, and KLBP in CV-1 cells resulted in a substantial accumulation of all three Proteins in the nucleus. These results suggest that Lipid-Binding Proteins, contrary to the general assumption, may exert their action in the nucleus as well as in the cytoplasm.
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interaction of rat hormone sensitive lipase with adipocyte Lipid Binding Protein
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Wenjun Shen, David A. Bernlohr, Kunju Sridhar, Fredric B KraemerAbstract:Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase that functions as the rate-limiting enzyme for the mobilization of free fatty acids in adipose tissue. By using the yeast two-hybrid system to examine the potential interaction of HSL with other cellular Proteins, evidence is provided to demonstrate a direct interaction of HSL with adipocyte Lipid-Binding Protein (ALBP), a member of the family of intracellular Lipid-Binding Proteins that binds fatty acids, retinoids, and other hydrophobic ligands. The interaction was demonstrated in vitro by the Binding of ALBP to HSL translated in vitro, to HSL in extracts of HSL overexpressing Chinese hamster ovary (CHO) cells, and to HSL in extracts of rat adipose tissue. Finally, the presence of ALBP was documented in immune complexes from rat adipose tissue immunoprecipitated with anti-HSL antibodies. The HSL–ALBP interaction was mapped to an N-terminal 300-aa region of HSL that is distinct from the C-terminal catalytic domain. These results suggest that HSL-derived fatty acids are bound by ALBP to facilitate intracellular trafficking of hydrophobic Lipids.
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targeted disruption of the adipocyte Lipid Binding Protein ap2 Protein gene impairs fat cell lipolysis and increases cellular fatty acid levels
Journal of Lipid Research, 1999Co-Authors: Melanie A. Simpson, David A. BernlohrAbstract:The availability of mice containing an adipocyte Lipid-Binding Protein (ALBP/aP2) gene disruption allowed for a direct examination of the presumed role of Lipid-Binding Proteins in the mobilization and trafficking of intracellular fatty acids. Total body and epididymal fat pad weights, as well as adipose cell morphology, were unaltered in male ALBP/aP2 disrupted mice when compared to their wild-type littermates. Analysis of adipocytes isolated from wild-type and ALBP/aP2 null mice revealed that a selective 40- and 13-fold increase in the level of the keratinocyte Lipid-Binding Protein (KLBP) mRNA and Protein, respectively, accompanied the ALBP/aP2 gene disruption. Although KLBP Protein was significantly up-regulated, the total Lipid-Binding Protein level decreased 8 -fold as a consequence of the disruption. There was no appreciable difference in the rate of fatty acid influx or esterification in adipocytes of wild-type and ALBP/aP2 null animals. To the contrary, basal lipolysis decreased approximately 40% in ALBP/aP2 nulls as compared to wild-type littermates. The glycerol release from isproterenol-stimulated ALBP/aP2 null fat cells was similarly reduced by approximately 35%. Consistent with a decrease in basal efflux, the non-esterified fatty acid (NEFA) level was nearly 3-fold greater in adipocytes from ALBP/aP2 nulls as compared to wild-type animals. The significant decrease in both basal and isoproterenol-stimulated lipolysis in adipose tissue of ALBP/aP2 null mice supports the model whereby intracellular Lipid-Binding Proteins function as Lipid chaperones, facilitating the movement of fatty acids out of the fat cell.
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BIOCHEMICAL AND BIOPHYSICAL ANALYSIS OF THE INTRACELLULAR Lipid Binding ProteinS OF ADIPOCYTES
Lipid Binding Proteins within Molecular and Cellular Biochemistry, 1999Co-Authors: Melanie A. Simpson, Natalie Ribarik Coe, Vince J. Licata, David A. BernlohrAbstract:Adipocytes express two Lipid-Binding Proteins; the major one termed the adipocyte Lipid-Binding Protein or aP2 (ALBP/aP2) and a minor one referred to as the keratinocyte Lipid-Binding Protein (KLBP). In order to evaluate the potential physiological roles for these Proteins, their biochemical and biophysical properties have been analyzed and compared. ALBP/aP2 and KLBP exhibit similar Binding affinities for most long-chain fatty acids; however, ALBP/aP2 exhibits a two to three-fold increased affinity for myristic, palmitic, oleic and linoleic acids, the predominant fatty acids of adipocytes. As measured by guanidinium hydrochloride denaturation, the stability of ALBP/aP2 is nearly 3 kcal/mol greater than that of KLBP. While the pI of ALBP/ aP2 was determined to be 9.0, that of KLBP is 6.5 suggesting differing net charges at physiological pH. Analysis of surface electrostatic properties of ALBP/aP2 and KLBP revealed similar charge polarity, although differences in the detailed charge distribution exist between the Proteins. The distribution of hydrophobic patches was also different between the Proteins, ALBP/ aP2 has only scattered hydrophobic surfaces while KLBP has a large hydrophobic patch near the ligand portal into the Binding cavity. In sum, these results point out that despite the striking similarity between ALBP/aP2 and KLBP in tertiary structure, significant differences in ligand Binding and surface properties exist between the two Proteins. Hence, while it is tempting to speculate that ALBP/aP2 and KLBP are metabolically interchangeable, careful analysis suggests that the two Proteins are quite distinct and likely to play unique metabolic roles. (Mol Cell Biochem 192: 33–40, 1999)
Leonard J. Banaszak - One of the best experts on this subject based on the ideXlab platform.
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Structural properties of the adipocyte Lipid Binding Protein.
Biochimica et Biophysica Acta, 1999Co-Authors: Amy Reese-wagoner, James R. Thompson, Leonard J. BanaszakAbstract:The adipocyte Lipid Binding Protein, ALBP (also adipocyte fatty acid Binding Protein, A-FABP, 422 Protein, aP2, and p15 Protein), is one of the most studied of the intracellular Lipid Binding Protein family. Here we sequentially compare the different sources of ALBP and describe the idea that one-third of the amino acid side chains near the N-terminal end appear to play a major role in conformational dynamics and in ligand transfer. Crystallographic data for mouse ALBP are summarized and the ligand Binding cavity analyzed in terms of the overall surface and conformational dynamics. The region of the proposed ligand portal is described. Amino acid side chains critical to cavity formation and fatty acid interactions are analyzed by comparing known crystal structures containing a series of different hydrophobic ligands. Finally, we address ALBP ligand Binding affinity and thermodynamic studies.
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Structural characterization of two synthetic catalysts based on adipocyte Lipid-Binding Protein.
Protein engineering, 1998Co-Authors: Jeramia Ory, Aram Mazhary, Hao Kuang, Ronald R. Davies, Mark D. Distefano, Leonard J. BanaszakAbstract:Adipocyte Lipid-Binding Protein (ALBP) is a small (14.5 kDa) 10-stranded beta-barrel Protein found in mammalian fat cells. The crystal structures of various holo-forms of ALBP have been solved and show the fatty acid ligand bound in a large (approximately 400 A3) cavity isolated from bulk solvent. Examination of the cavity suggests that it would be a good site for the creation of an artificial catalyst, as numerous well defined crystal structures of ALBP are available and past studies have shown the conformation to be reasonably tolerant to modification and mutagenesis. Previous work has shown ALBP to be a good Protein scaffold for exploring enantio- and stereoselective reactions; two constructs, ALBP attached to either a pyridoxamine or a phenanthroline group at C117, have been chemically characterized. Both modified Proteins have been crystallized and their structures solved and refined. The X-ray models have been used to examine the origin of the chiral selectivity seen in the products. It is apparent that these covalent adducts reduce the internal cavity volume, sterically limiting substrate interactions with the reactive groups, as well as solvent access to potential intermediates in the reaction pathway.
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INTRACELLULAR Lipid-Binding ProteinS AND THEIR GENES
Annual review of nutrition, 1997Co-Authors: David A. Bernlohr, Melanie A. Simpson, Ann V. Hertzel, Leonard J. BanaszakAbstract:Intracellular Lipid-Binding Proteins are a family of low-molecular-weight single-chain polypeptides that form 1:1 complexes with fatty acids, retinoids, or other hydrophobic ligands. These Proteins are products of a large multigene family of unlinked loci distributed throughout the genome. Each Lipid-Binding Protein exhibits a distinctive pattern of tissue distribution. Transcriptional control, regulated by a combination of peroxisome proliferator activated receptors and CCAAT/enhancer-Binding Proteins, allows for a variety of both cell and tissue-specific expression patterns. In some cells, fatty acids increase the expression of the Lipid-Binding Protein genes. Fatty acids, or their metabolites, are activators of the peroxisome proliferator-activated receptor family of transcription factors. Therefore, as the concentration of Lipid in the diet increases, the expression of Lipid-Binding Proteins coordinately increases. As revealed by X-ray crystallography, the Lipid-Binding Proteins fold into beta-barrels, forming a large internal water-filled cavity. Fatty acid ligands are bound within the cavity, occupying only about one-third of the accessible volume. The bound fatty acid is stabilized via a combination of enthalpic and entropic forces that govern ligand affinity and selectivity. Cytoplasmic Lipid-Binding Proteins are the intracellular receptors for hydrophobic ligands, delivering them to the appropriate site for use as metabolic fuels and regulatory agents.
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Adipocyte Lipid-Binding Protein complexed with arachidonic acid. Titration calorimetry and X-ray crystallographic studies.
The Journal of biological chemistry, 1994Co-Authors: Judith M. Lalonde, David A. Bernlohr, M A Levenson, J J Roe, Leonard J. BanaszakAbstract:The association of the adipocyte Lipid-Binding Protein (ALBP) with arachidonic acid (all cis, 20:4 delta 5,8,11,14) and oleic acid (cis, 18:1 delta 9) has been examined by titration calorimentry. In addition, the crystal structure of ALBP with bound arachidonic acid has also been obtained. Crystallographic analysis of the arachidonic acid.ALBP complex along with the previously reported oleic acid-ALBP structure (Xu, Z., Bernlohr, D. A., and Banaszak, L. J. (1993) J. Biol. Chem. 268, 7874-7884) provides a framework for the molecular examination of Protein-Lipid association. Isothermal titration calorimetry revealed high affinity association of both unsaturated fatty acids with the Protein. The calorimetric data yielded the following thermodynamic parameters for arachidonic acid: Kd = 4.4 microM, n = 0.8, delta G = -7370 cal/mol, delta H = -6770 cal/mol, and T delta S = +600 cal/mol. For oleic acid, the thermodynamic parameters were Kd = 2.4 microM, n = 0.9, delta G = -7770 cal/mol, delta H = -6050 cal/mol, and T delta S = +1720 cal/mol. The identification of thermodynamically dominating enthalpic factors for both fatty acids are consistent with the crystallographic studies demonstrating the interaction of the fatty acid carboxylate with a combination of Arg106, Arg126, and Tyr128. The crystallographic refinement of the Protein-arachidonate complex was carried out to 1.6 A with the resultant R factor of 0.19. Within the cavity of the crystalline Binding Protein, the arachidonate was found in a hairpin conformation. The conformation of the bound ligand is consistent with acceptable torsional angles and the four cis double bonds in arachidonate. These results demonstrate that arachidonate is a ligand for ALBP. They provide thermodynamic and structural data concerning the physical basis for Protein-Lipid interaction and suggest that intracellular Lipid-Binding Proteins may mediate the biological effects of polyunsaturated fatty acids in vivo.
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X-ray crystallographic structures of adipocyte Lipid-Binding Protein complexed with palmitate and hexadecanesulfonic acid. Properties of cavity Binding sites.
Biochemistry, 1994Co-Authors: Judith M. Lalonde, David A. Bernlohr, Leonard J. BanaszakAbstract:Adipocyte Lipid-Binding Protein is a 14.6-kDa polypeptide that is responsible for the intracellular trafficking of fatty acids. Its structure previously has been solved in the apo and holo forms complexed with stearate and oleate. To examine the Binding of Lipids other than those with a carboxylate headgroup, we have determined the structure of ALBP in complex with a sulfonic acid, hexadecanesulfonic acid, and compared its structure with the natural fatty acid analog, palmitate. Crystallographic refinement led to similar models, both with R-factors of about 20% and a resolution of 1.6 A. results can be compared with earlier studies on C18 fatty acids, both saturated and unsaturated. The previously refined complexes with stearate and oleate in combination with the complexes of palmitate and hexadecanesulfonic acid demonstrate specific positions for water molecules bound in the internal cavity. Many of the water-Binding sites are present in both the apo form and the holo forms of the Protein. With ligand present, a network of 10 internalized water molecules appear to form a hydrophobic hydration region. In spite of the sp3 geometry of the sulfonic acid derivative, the headgroup occupies the same site as that of the planar carboxylate in natural fatty acids. These results demonstrate that intracellular Lipid-Binding Proteins are capable of Binding a wider variety of Lipids than previously considered and reveal the importance of interior ordered water molecules in the Binding cavity.
J. Barrett - One of the best experts on this subject based on the ideXlab platform.
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characterisation and properties of an intracellular Lipid Binding Protein from the tapeworm moniezia expansa
FEBS Journal, 1997Co-Authors: J. Barrett, Nahid Saghir, Katie Clarke, Anna Timanova, Peter M. BrophyAbstract:The tapeworm Moniezia expansa contains an extremely abundant cytoplasmic Lipid-Binding Protein (LBP). It is a small Protein consisting of 66 amino acids with a molecular mass of 7943 ± 1.5Da. The amino acid sequence has been established by Edman degradation and confirmed by PCR analysis. The Moniezia LBP shows no sequence similarity with any previously described Binding Protein, but does show similarity with antigen B from Echinococcus glanulosus and Echinococcus multilocularis and with Taenia crassiceps antigen. The predicted structure for Moniezia LBP shows four helices and a putative tyrosine kinase site on the loop between helix 1 and 2. Each of the four helices has a well defined hydrophobic face. Studies with fluorescent probes suggest a single hydrophobic Binding site. Results indicate that the single tryptophan residue in the molecule (Trp41) is involved in ligand Binding, and calculation of the Stern-Volmer quenching constant shows that Trp41 is in a relatively hydrophobic environment.
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Characterisation and Properties of an Intracellular Lipid‐Binding Protein from the Tapeworm Moniezia expansa
FEBS Journal, 1997Co-Authors: J. Barrett, Nahid Saghir, Katie Clarke, Anna Timanova, Peter M. BrophyAbstract:The tapeworm Moniezia expansa contains an extremely abundant cytoplasmic Lipid-Binding Protein (LBP). It is a small Protein consisting of 66 amino acids with a molecular mass of 7943 ± 1.5Da. The amino acid sequence has been established by Edman degradation and confirmed by PCR analysis. The Moniezia LBP shows no sequence similarity with any previously described Binding Protein, but does show similarity with antigen B from Echinococcus glanulosus and Echinococcus multilocularis and with Taenia crassiceps antigen. The predicted structure for Moniezia LBP shows four helices and a putative tyrosine kinase site on the loop between helix 1 and 2. Each of the four helices has a well defined hydrophobic face. Studies with fluorescent probes suggest a single hydrophobic Binding site. Results indicate that the single tryptophan residue in the molecule (Trp41) is involved in ligand Binding, and calculation of the Stern-Volmer quenching constant shows that Trp41 is in a relatively hydrophobic environment.
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a novel Lipid Binding Protein from the cestode moniezia expansa
Biochemical Journal, 1995Co-Authors: D Janssen, J. BarrettAbstract:A Lipid-Binding Protein (LBP) has been purified from the cytosol of the cestode Moniezia expansa. The native LBP was found to be an oligomer of approx. 250 kDa, consisting of 11 kDa monomers. The LBP bound saturated and unsaturated fatty acids, but not their CoA derivatives, with KD values in the range 0.68-7.8 microM. Cholesterol, dihydroergosterol, bilirubin and retinoids were also bound, but alpha-tocopherol, bile acids, alk-2-enals and alka-2,4-dienals were not. Evidence suggests that there are two Binding sites per subunit, each with different specificities. The fluorescent fatty acid 11-[(5-dimethylaminonaphthalene-1-sulphonyl)amino]undecanoic acid (DAUDA) and retinol both showed an additional high-affinity Binding site with a density of approximately 0.1 per subunit, suggesting specific Binding to the oligomer. The amino acid composition of Moniezia LBP was distinct from that of previously characterized fatty acid-Binding Proteins (FABPs). The Protein was not N-terminally blocked and yielded a unique amino acid sequence, unrelated to that of any known FABP; there was also evidence of microheterogeneity. Polyclonal antibodies raised to the Moniezia Protein did not cross-react with mammalian, nematode or digenean FABP. The Gibbs free energy for Protein folding (13.02 kJ/mol; 3.1 kcal/mol), determined by urea denaturation, was identical for both the native and ligand-bound Moniezia LBP. CD spectra showed that the Moniezia Protein contained 36% alpha-helix and that the secondary structure underwent only minor changes on ligand Binding. Moniezia LBP binds a range of anthelmintics, with KD values again in the range 0.66-7.3 microM. It is possible that, in helminths, Binding Proteins may play a role in determining the specificity and site of action of anthelmintics.
Peter M. Brophy - One of the best experts on this subject based on the ideXlab platform.
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characterisation and properties of an intracellular Lipid Binding Protein from the tapeworm moniezia expansa
FEBS Journal, 1997Co-Authors: J. Barrett, Nahid Saghir, Katie Clarke, Anna Timanova, Peter M. BrophyAbstract:The tapeworm Moniezia expansa contains an extremely abundant cytoplasmic Lipid-Binding Protein (LBP). It is a small Protein consisting of 66 amino acids with a molecular mass of 7943 ± 1.5Da. The amino acid sequence has been established by Edman degradation and confirmed by PCR analysis. The Moniezia LBP shows no sequence similarity with any previously described Binding Protein, but does show similarity with antigen B from Echinococcus glanulosus and Echinococcus multilocularis and with Taenia crassiceps antigen. The predicted structure for Moniezia LBP shows four helices and a putative tyrosine kinase site on the loop between helix 1 and 2. Each of the four helices has a well defined hydrophobic face. Studies with fluorescent probes suggest a single hydrophobic Binding site. Results indicate that the single tryptophan residue in the molecule (Trp41) is involved in ligand Binding, and calculation of the Stern-Volmer quenching constant shows that Trp41 is in a relatively hydrophobic environment.
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Characterisation and Properties of an Intracellular Lipid‐Binding Protein from the Tapeworm Moniezia expansa
FEBS Journal, 1997Co-Authors: J. Barrett, Nahid Saghir, Katie Clarke, Anna Timanova, Peter M. BrophyAbstract:The tapeworm Moniezia expansa contains an extremely abundant cytoplasmic Lipid-Binding Protein (LBP). It is a small Protein consisting of 66 amino acids with a molecular mass of 7943 ± 1.5Da. The amino acid sequence has been established by Edman degradation and confirmed by PCR analysis. The Moniezia LBP shows no sequence similarity with any previously described Binding Protein, but does show similarity with antigen B from Echinococcus glanulosus and Echinococcus multilocularis and with Taenia crassiceps antigen. The predicted structure for Moniezia LBP shows four helices and a putative tyrosine kinase site on the loop between helix 1 and 2. Each of the four helices has a well defined hydrophobic face. Studies with fluorescent probes suggest a single hydrophobic Binding site. Results indicate that the single tryptophan residue in the molecule (Trp41) is involved in ligand Binding, and calculation of the Stern-Volmer quenching constant shows that Trp41 is in a relatively hydrophobic environment.
Li Mu-lan - One of the best experts on this subject based on the ideXlab platform.
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CONSTRUCTION OF DNA VACCINE OF ATP SYNTHASE Lipid-Binding Protein-LIKE Protein GENE OF SCHISTOSOMA JAPONICUM AND ITS EXPRESSION IN EUKARYOTIC CELLS
Modern Preventive Medicine, 2008Co-Authors: Li Mu-lanAbstract:Objective To clone and construct the recombinant plasmid containing ATP synthase Lipid-Binding Protein-like Protein gene of schistosoma japonicum(SjAslp)and transfer it into mammalian cells to express ATP synthase Lipid-Binding Protein-like Protein.Methods By polymerase chain reaction(PCR)technique,SjAslp was amplified from the constructed recombinant plasmid pBC SK+/SjAslp,and inserted into cloning vector pUCm-T.Then,SjAslp was subcloned into eukaryotic expression vector pcDNA3.1(+).After identifying it by PCR,restrictive enzymes digestion and DNA sequencing,the recombinant plamid was transfected into HeLa cells using electroporation,and analyzed the expression of the recombinant Protein by immunocytochemistry assay.Results The specific gene fragment 558bp was successfully amplified.The DNA vaccine of SjAslp was successfully constructed.Immunocytochemistry assay showed that SjAslp was expressed in HeLa cells and located in cytoplasm.Conclusion SjAslp gene can express in eukaryotic system,which lay the foundation for development of the SjAslp DNA vaccine against schistosoma.