Expression, purification and characterization of the Hepatitis B virus entire envelope large protein in Pichia pastoris.

The current HBsAg vaccine has performed a vital role in preventing the transmission of HBV during the past 20 years. However, a number of individuals still show no response or a low response to the vaccine. In the present study, the HBV envelope large protein gene was cloned into the eukaryotic expression vector pPIC9k and was subsequently expressed in the yeast Pichia pastoris. The HBV large protein (L protein) was produced and secreted into the medium, where some of the L protein formed particles. The soluble L protein and particles were purified by column chromatography and sucrose density gradient centrifugation. Western blot analysis demonstrated that the particle was composed of both HBV L and S protein. To compare the antigenicity of the L protein and HBsAg, rabbits were immunized with the soluble L protein and the commercially available HBV vaccine and the increasing level of antibodies was determined by ELISA. The results showed that the anti-HBsAg antibody, from rabbits injected with the L protein at a dose of 2 and 10microg, was detected on day 14, whereas rabbits vaccinated with 10 and 2microg HBsAg did not develop antibodies until day 21 and 28, respectively. The antibody level in groups inoculated with the L protein was approximately 50% higher than in the group injected with HBsAg using the same dose. Furthermore, 2microg L protein induced a significant and rapid anti-HBsAg antibody response than 10microg HBsAg. Therefore, we suggest that the L protein is an ideal candidate for a new generation HB vaccine to protect people from HBV infection.

A novel three-dimensional cadmium(II) coordination polymer incorporating 1,4-bis(1,2,4-triazol-1-ylmethyl)benzene and thiocyanate.

In the title complex, poly[cadmiumII-mu2-1,4-bis(1,2,4-triazol-1-ylmethyl)benzene-di-mu2-thiocyanato], [Cd(NCS)2(C12H12N6)]n, the CdII atom lies on an inversion centre in a distorted octahedral environment. Four N atoms from the thiocyanate and 1,4-bis(1,2,4-triazol-1-ylmethyl)benzene (bbtz) ligands occupy the equatorial positions, and two S atoms from symmetry-related thiocyanate ligands occupy the axial positions. The benzene ring of the bbtz ligand lies about an inversion centre. Single thiocyanate bridges link the CdII atoms into two-dimensional sheets containing novel 16-membered [Cd4(mu-NCS-N:S)4] rings. The bbtz ligands further link these two-dimensional sheets into an unprecedented covalent three-dimensional network for the cadmium-thiocyanate system.

A novel two-dimensional network cadmium(II) coordination polymer containing one 1,4-bis(1,2,4-triazol-1-yl)butane and double dicyanamide bridges.

In the crystal structure of the title complex, poly[mu-1,4-bis(1,2,4-triazol-1-yl)butane-di-mu-1,5-dicyanamido-cadmium(II)], [Cd(C2N3)2(C8H12N6)]n or [Cd(dca)2(btb)]n, where dca is dicyanamide and btb is 1,4-bis(1,2,4-triazol-1-yl)butane, each Cd(II) atom occupies a center of symmetry and is in a six-coordinated distorted octahedral environment. Four N atoms from four dca ligands fill the equatorial positions, and two N atoms from two btb ligands occupy the axial positions. The dca ligands adopt an end-to-end coordination mode and link the Cd(II) atoms to form a 12-membered Cd(dca)2Cd ring, and neighboring rings extend along the b axis to form a [Cd(dca)2]n chain. The btb ligands, acting as bridging bidentate ligands, link the Cd(II) atoms of adjacent one-dimensional [Cd(dca)2]n chains, forming a rhombic two-dimensional network.

[The purification of HBV full-length PreS protein in Pichia pastoris].

The Pichia pastoris strain GS115-PreS could produce a high expression level of full-length PreS protein that secreted to the supernatant after methanol induction in the fermentation. The Western blot analysis showed a single band with expected molecular mass of 48kD and that the major component of the particles was the full-length PreS protein (PreS1 + PreS2 + S) and small envelope protein (S) of 48 and 28 kD, respectively. Electron microscopy image showed PreS particles with 30 nm in diameter. The supernatants of the fermentation were desalted and concentrated. Purified PreS protein was obtained by DEAE-SFF anion exchange column chromatography and the PreS particles were obtained by ultracentrifugation and sucrose density gradient. The ELISA assay results proved that both full-length PreS protein and particles showed high immunogenicity and specificity. P/N ratio further demonstrated that the immunogenicity of the particles is higher than the full-length PreS protein.

A novel three-dimensional copper(II) coordination polymer with 1,4-bis(1,2,4-triazol-1-ylmethyl)benzene and thiocyanate.

In the title complex, poly[copper(II)-di-2-thiocyanato-2-1,4-bis(1,2,4-triazol-1-ylmethyl)benzene], [Cu(NCS)2(C12H12N6)]n, the CuII atom lies on an inversion centre in a tetragonally distorted octahedral environment. Four N atoms from thiocyanate and 1,4-bis(1,2,4-triazol-1-ylmethyl)benzene (bbtz) ligands fill the equatorial positions, and S atoms from symmetry-related thiocyanate ligands fill the axial positions. The benzene ring of the bbtz ligand lies about an inversion centre. Single thiocyanate bridges link the CuII atoms into two-dimensional sheets containing an unprecedented 16-membered [Cu4(-NCS-N:S)4] ring. The bbtz ligands further link the two-dimensional sheets into a three-dimensional network.

A novel two-dimensional cobalt(II) coordination polymer with 1,4-bis(1,2,4-triazol-1-ylmethyl)benzene.

In the crystal structure of the title complex, poly[[diazidocobalt(II)]-di-mu-1,4-bis(1,2,4-triazol-1-ylmethyl)benzene-kappa(4)N(4):N(4')], [Co(N(3))(2)(bbtz)(2)](n), where bbtz is 1,4-bis(1,2,4-triazol-1-ylmethyl)benzene (C(12)H(12)N(6)), the Co(II) atom, which lies on an inversion centre, is six-coordinated by four N atoms from four bbtz ligands and by two N atoms from two azide ligands, in a distorted octahedral coordination environment. The Co(II) atoms are bridged by four bbtz ligands to form a two-dimensional [4,4]-network.

The infinite one-dimensional chain polymer catena-poly[[bis[micro-1,1'-ethane-1,2-diylbis(1,2,4-triazole)-kappa(2)N(4):N(4')]bis[azidozinc(II)]]-di-micro-azido-kappa(4)N(1):N(1)].

In the crystal structure of the title complex, [Zn(N(3))(2)(C(6)H(8)N(6))](n) or [Zn(N(3))(2)(bte)](n), where bte is micro-1,2-bis(1,2,4-triazol-1-yl)ethane, each Zn atom is pentacoordinated in a distorted trigonal-bipyramidal coordination environment involving two N atoms from two bte ligands and three N atoms from three azide ligands. The Zn atoms are bridged by micro-1,1-azide groups and bte ligands around a centre of inversion, forming an infinite one-dimensional chain containing both four-membered Zn(micro-1,1-N(3))(2)Zn and 18-membered Zn(gauche-bte)(2)Zn rings.