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[Preparation of antiserum to protein V of human adenovirus type 41]
Liuxin Dong1, Xiaohui Zou, Jingdong Song
1National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052. dongliuxin@163.com
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.
