Construction and characterization of calreticulin-HBsAg fusion gene recombinant adenovirus expression vector

AIM: To generate recombinant adenoviral vector con-taining calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine.METHODS: CRT and HBsAg gene were fused using polymerase chain reaction (PCR), endonuclease diges-tion and ligation methods. The fusion gene was cloned into pENTR/D-TOPO transfer vector after the base pairs of DNA (CACC) sequence was added to the 5′ end. Adenoviral expression vector containing CRT-HBsAg fusion gene was constructed by homologous recombinan-tion. The human embryo kidney (HEK) 293A cells were transfected with linearized DNA plasmid of the recombi-nant adenoviral vector to package and amplify recombi-nant adenovirus. The recombinant adenovirus titer was characterized using the end-dilution assay. The expres-sion of the CRT/HBsAg fusion protein in Ad-CRT/HBsAg infected 293A cells was detected by Western blotting.RESULTS: The CRT-HBsAg fusion gene was char-acterized by PCR and sequencing and its length and sequence were confirmed to be accurate. The CRT-HB-sAg fusion gene recombinant pENTR/D-TOPO transfer vector was constructed. The recombinant adenoviral vector, Ad-CRT/HBsAg, was generated successfully. The titer of Ad-CRT/HBsAg was characterized as 3.9 × 1011 pfu/mL. The CRT-HBsAg fusion protein was ex-pressed by HEK 293A cells correctly. CONCLUSION: CRT/HBsAg fusion gene recombinant replication-defective adenovirus expression vector is constructed successfully and this study has provided an experimental basis for further studies of Hepatitis B vi-rus gene therapy.

EFFECT OF THE PHASE STRUCTURE EVOLUTION ON THE PROPERTIES OF FILMS FORMED FROM PBA/P(St-co-MMA) COMPOSITE LATEX

A group of heterogeneous latexes poly(butyl acrylate)/poly(styrene-co-methyl methacrylate)(PBA/P(St-co-MMA)) were prepared by a semi-continuous seeded emulsion polymerization process under monomer starved conditions.The glass transition temperature(T_g)and the mechanical properties of the film formed from the composite latex changed with the evolution of the particle morphology.A photon transmission method was used to monitor the phase structure evolution of films which were prepared from core-shell PBA/P(S...

Did Civilian Granaries Affect Social Stability？ Empirical Evidence from the Qing Dynasty （1817-1856）

Based on historical panel data of social instability, civilian granary and climate shocks in the 17 Provinces of the Qing from 1817 to 1856, this paper examines the correlation between social instability and civilian granary holdings. It shows that the civilian granary system reduced social instability in the Qing Dynasty, while climate shocks raised social instability. Moreover, this paper shows that civilian granaries mitigated the effects of climate shocks on social instability.

DFT Studies on Detonation Properties, Pyrolysis Mechanism, and Stability of the Nitro and Hydroxyl Derivatives of Benzene

Ninety-one nitro and hydroxyl derivatives of benzene were studied at the B3LYP/6-31G＊ level of density functional theory. Detonation properties were calculated using the Kamlet-Jacobs equation. Three candidates （pentanitrophenol, pentanitrobenzene, and hexanitrobenzene） were recommended as potential high energy density compounds for their perfect detonation performances and reasonable stability. The pyrolysis mechanism was studied by analyzing the bond dissociation energy （BDE） and the activation energy （Ea） of hydrogen transfer （H--T） reaction for those with adjacent nitro and hydroxyl groups. The results show that Ea is much lower than BDEs of all bonds, so when there are adjacent nitro and hydroxyl groups in a molecule, the stability of the compound will decrease and the pyrolysis will be initiated by the H--T process. Otherwise, the pyrolysis will start from the breaking of the weakest C--NO2 bond, and only under such condition, the Mulliken population or BDE of the C--NO2 bond can be used to assess the relative stability of the compound.