Expression and Characterization of Hepatitis B Surface Antigen in Transgenic Potato Plants

N. Ya. Shulga¹, E. B. Rukavtsova², M. A. Krymsky³, V. N. Borisova³, V. A. Melnikov⁴, V. A. Bykov¹, and Ya. I. Buryanov^2*

¹Sechenov Moscow Medical Academy, ul. Bol'shaya Pirogovskaya 2/6, Moscow 119881, Russia; fax: (7-095) 248-0181

²Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, pr. Nauki 6, Pushchino 142290, Moscow Region, Russia; fax: (8-27) 33-0527; E-mail: buryanov@fibkh.serpukhov.su

³Combiotech AOZT, ul. Miklukho-Maklaya 16/10, Moscow 117997, Russia; fax: (7-095) 330-7429; E-mail: sales@combiotech.com

⁴Academy of Medical and Technical Sciences, ul. Kasatkina 3, Moscow 129301, Russia

^* To whom correspondence should be addressed.

Received April 26, 2004; Revision received June 3, 2004

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Transgenic potato plants expressing the gene of hepatitis B surface antigen (HBsAg) under the control of the double promoter of 35S RNA of cauliflower mosaic virus (CaMV 35SS) and the promoter of patatin gene of potato tubers have been obtained. Biochemical analysis of the plants was performed. The amount of HBsAg in leaves, microtubers, and tubers of transgenic potatoes growing in vitro and in vivo was 0.005-0.035% of the total soluble protein. HBsAg content reached 1 µg/g in potato tubers and was maximal in plants expressing the HBsAg gene under the control of CaMV 35SS promoter. In transgenic plants expressing HBsAg gene under the control of tuber-specific patatin promoter, HBsAg was found only in microtubers and tubers and was absent in leaves. Western blot analysis of HBsAg eluted from immunoaffinity protein A-Sepharose matrix has been performed. The molecular weight of HBsAg peptide was approximately 24 kD, which is in agreement with the size of the major protein of the envelope of hepatitis B virus. Using gel filtration, it was determined that the product of HBsAg gene expression in potato plants is converted into high-molecular-weight multimeric particles. Therefore, as well as in recombinant HBsAg-yeast cells, assembling of HBsAg monomers into immunogenic aggregates takes place in HBsAg-transgenic potato, which can be used as a source of recombinant vaccine against hepatitis B virus.

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KEY WORDS: transgenic potato, hepatitis B surface antigen, gene expression, synthesis, immunoassay, electrophoresis, gel filtration

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