http://www.abbs.info E-mail: [email protected]

ISSN 1672-9145 Acta Biochim Biophys Sin 2005, 37(3): 153–158 CN 31-1940/Q

---

Expression of Human Papillomavirus Type 16 L1 Protein in Transgenic Tobacco Plants

Hong-Li LIU^1,2, Wen-Sheng LI¹, Ting LEI¹, Jing ZHENG¹, Zheng ZHANG³, Xiao-Fei YAN¹, Zhe-Zhi WANG³, Yi-Li WANG¹, and Lü-Sheng SI¹*

¹Institute of Cancer Research, School of Life Science & Technology, Xi’an Jiaotong University, Xi’an 710061, China;

²First Hospital of Xi’an Jiaotong University, Xi’an 710061, China;

³Shaanxi Normal University, Xi’an 710062, China

Abstract To develop a plant expression system for the production of the human papillomavirus type 16 (HPV16) vaccine, we investigated whether the HPV16 L1 protein can be expressed in tobacco plants and whether it can be used as the cheapest form of edible vaccine. The HPV16 L1 coding sequence was amplified by PCR using specific primers from the plasmid pGEM-T-HPV16 containing the template sequence, and subcloned into the intermediate vector pUCmT and binary vector pBI121 consecutively to obtain the plant expression plasmid pBI-L1. The T-DNA regions of the pBI-L1 binary vector contained the constitutive Cauliflower mosaic virus (CaMV) 35S promoter and the neomycin phosphotransferase npt II gene, which allowed the selection of transformed plants using kanamycin. The tobacco plants were transformed by co-cultivating them, using the leaf disc method, with Agrobacterium tumefaciens LBA4404, which harbored the plant expression plasmid. The regenerated transgenic tobacco plants were selected using kanamycin, and confirmed by PCR. The results of the Southern blot assay also showed that the HPV16 L1 gene was integrated stably into the genome of the transformed tobacco plants. The Western blot analysis showed that the transformed tobacco leaves could express the HPV16 L1 protein. Furthermore, it was demonstrated by ELISA assay that the expressed protein accounted for 0.034%0.076% of the total soluble leaf protein, was able to form 55 nm virus-like particles compatible with HPV virus-like particle (VLP), and induced mouse erythrocyte hemagglutination in vitro. The present results indicate that the HPV16 L1 protein can be expressed in transgenic tobacco plants and the expressed protein possesses the natural features of the HPV16 L1 protein, implying that the HPV16 L1 transgenic plants can be potentially used as an edible vaccine.

Key words human papillomavirus (HPV); virus-like particle; transgenic tobacco; plant vaccine; Agrobacterium tumefaciens; L1 major capsid protein

-----------------

Received: October 9, 2004 Accepted: January 28, 2005

This work was supported by a grant from the National Natural Science Foundation of China (No. 30070848)

*Corresponding author: Tel, 86-29-82655191; Fax, 86-29-82655499; E-mail, [email protected]