[Preparation of antiserum to protein V of human adenovirus type 41].

OBJECTIVE

The fastidious property of human adenovirus type 41 (Ad41) may be resulted from inadequate expression of protein V (pV), the minor core protein of adenovirus, in packaging cells. In this report, we prepared antiserum to pV of Ad41 and for study the mechanism of Ad41 fastidiousness.

METHODS

Coding sequence of pV was amplified by PCR with the genome DNA of wild Ad41 (NIVD103) as template, and cloned into pET30a(+) vector to generate a recombinant plasmid called pET-pV. His-tag-fused pV was expressed in pET-pV-transformed E. Coli strain BL21(DE3) by adding the inducer of Isopropy beta-D-1-Thiogalactopyranoside (IPTG) and purified with the method of immobilized metal ion affinity chromatography (IMAC). Antiserums to pV were collected from pV inclusion bodies-immunized mice and evaluated by Western blot.

RESULTS

The sequencing assay showed that the cloned pV gene was highly homologous with that of Ad41 Tak strain, and there were only three residues changed in the corresponding amino-acid sequence. pV was expressed as inclusion bodies or in soluble form in BL21(DE3) cells under inducing condition of 1 mmol/L IPTG, 37 degrees C, 4 h or 0.5 mmol/L IPTG, 25 degrees C, 8 h, respectively. Antiserums to pV from most immunized mice were highly effective for Western blot assay. After infected with equivalent Ad41, 293E12, an Ad41 E1B55K-transfected 293 cell line, expressed more pV than 293 cells.

CONCLUSION

We successfully prepared antiserums to Ad41 pV and it could be used in Western blot assay to study the fastidious property of Ad41.