Abstract: To express the group specific antigen VP6 fragment of Group A rotavirus (RV) in E.coli and therefore to provide materials for the development of the rotavirus immunoassays. Methods: By bioinformatics analysis, one conserved domain named VP61 within VP6 with strong hydrophilicity and antigenecity was picked out. The gene fragment encoding the truncated form of VP6 was amplified with PCR by using the plasmid pcDNA3.1VP6 as a template and was inserted into the GlutathioneStransferase (GST) fusion expression vector pGE5X following the confirmation of enzymatic analysis and DNA sequencing. The resultant plasmid was then introduced into E.coli for IPTG induced expression. The expression products were validated by both SDSPAGE and Western blotting. The soluble expression of the recombinant protein in E.coli was further optimized by means of different host bacteria as well as different culture conditions including IPTG concentration, temperature, medium, etc. The protein was preliminarily purified with GST affinity chromatography. Results: The fragment comprised of aa 12143 of the VP6 (nt 34429) was selected as the target peptide for prokaryotic expression after comprehensive bioinformatics analysis. The 396bp fragment encoding the peptide was amplified by PCR and the recombinant plasmid pGEX5XVP6.1 containing the VP61 gene was correctly constructed. The efficient expression of the recombinant fusion protein GST::VP61 harboring the truncated VP6 in E.coli was shown by SDSPAGE and Western blotting, which was performed with an antiRV polyclonal antibody. The 41kD recombinant fusion protein which occupied about 30% of the total cell protein was mainly expressed in an inclusion body fashion. After a series of optimization procedures, the soluble expression of the GST::VP61 was finally increased to some extent through lower culture temperature in combination with lower IPTG concentration and could be well purified by GSTSepharose 4B chromatography. Conclusions: The efficient expression and purification of the VP61 protein in E.coli makes it possible to prepare the specific monoclonal antibodies against VP6, and may lay a foundation for the development of RV specific immunoassays.

Key words: Rotavirus, Prokaryotic cells, Gene expression