[Prokaryotic Expression and Immunogenicity Analysis of a Fusion Protein Containing Cell Epitopes of Mycobacterium tuberculosis Rv2660c, Rv2460c, Rv3875 and Rv3804c Genes].

OBJECTIVE: To analyse the immunogenicity of a fusion protein containing cell epitopes of Mycobacterium tuberculosis genes Rv2660c, Rv2460c, Rv3875 and Rv3804, and to evaluate the feasibility of using it as a novel target antigen for developing multi-stage TB vaccines. METHODS: Cell epitopes of Rv2660c, Rv2460c, Rv3875 and Rv3804c were fused in series to form a new antigen gene (named msv). Then msv was cloned into the prokaryotic expression vector pEASY-Blunt E1. The fusion protein msv was expressed by pEASY-Blunt E1 under the induction of isopropyl-ß-d-thiogalactoside (IPTG). Purified the protein by affinity chromatography and identified the protein by SDS-PAGE and Western blot. To evaluate the immunogenicity of the protein, the mice were immunized with the purified fusion protein, and the titer of the antibody in mice serum was evaluated by ELISA. Besides, splenocytes of immunized mice were separated and splenocytes proliferation was determined under the stimulation of the protein. RESULTS: The prokaryotic expression plasmid carrying msv gene was constructed successfully and msv protein could be expressed by the plasmid under the induction of IPTG. SDS-PAGE and Western blot results confirmed that a purified protein (relative molecular mass was 41.3×103) was obtained. ELISA result indicated that the titer of the antibody in msv immunized mice serum was about 1:81 920.The spleen lymphocyte proliferation assay showed that after immunization with msv protein, significant proliferation of antigen-sensitized lymphocytes was observed. CONCLUSIONS: The fusion protein msv was successfully expressed and purified, which can induce humoral and cellular immunity in mice. It may be used as an antigen component for the development of TB vaccine in the future.

Printed circuit board ion trap mass analyzer: its structure and performance.

An ion trap (IT) mass analyzer can be simply built with low cost material-the printed circuit board (PCB). A printed circuit board ion trap (PCBIT) can perform ion trapping, mass analysis, and tandem mass spectrometry as a conventional ion trap mass analyzer. In a PCBIT, each PCB electrode was fabricated to specially designed patterns with several separate electric strips. The strips' electrodes were insulated from each other and applied with different voltages during the experiment. Therefore, the electric field distribution inside the ion trap region may be adjusted and optimized by simply adjusting the voltage on each strip. The performance of the PCBIT can also be optimized since the property of an ion trap is strongly dependent on the field distribution. The fabrication, operation, and performance of the PCBIT are described and characterized in this paper. A prototype PCBIT was built with two pairs of 64 mm × 12 mm PCB rectangular plates and one pair of 10 mm × 10 mm stainless steel square plates. A mass analysis with a resolving power of over 1500 and a mass range of around 3000 Th was observed. The mass-selected isolation and collision-induced dissociation (CID) of ions were also tested using the homemade PCBIT system. The adjustable electric field distribution, simple structure, and low cost of PCBIT make it certainly suitable for the further miniaturization of the portable mass spectrometer.

Studies on the non-covalent interactions between cyclodextrins and aryl alkanol piperazine derivatives by mass spectrometry and fluorescence spectroscopy.

The non-covalent complexes of alpha- and beta-cyclodextrins (alpha-, beta-CDs) with two aryl alkanol piperazine derivatives (Pipe I and Pipe II) have been studied by electrospray ionization mass spectrometry (ESI-MS) and fluorescence spectroscopy. The ESI-MS experimental results demonstrated that Pipe I can conjugate to beta-CD and form 1:1 or 1:2 stoichiometric non-covalent complexes, and Pipe II can only form 1:1 complexes with alpha- or beta-CD. Fluorescence spectra indicated that the fluorescence intensities of Pipe I and Pipe II can be enhanced by increasing the content of beta-CD. The mass spectrometric titration experiments showed that the dissociation constants K(d1) were 5.77 and 9.52 x 10(-4) mol L(-1) for the complexes of alpha-CD with Pipe I and Pipe II, respectively, revealing that the binding of alpha-CD-Pipe I was stronger than alpha-CD-Pipe II. The K(d1) and K(d2) values were 9.81 x 10(-4) mol L(-1) and 1.11 x 10(-7) (mol L(-1))(2) for 1:1 and 1:2 complexes of Pipe I with beta-CD, respectively. The K(d) values obtained from fluorescence spectroscopy were in agreement with those from ESI-MS titration.