Optimized expression, preparation of human papillomavirus 16 L2E7 fusion protein and its inhibitory effect on tumor growth in mice

Abstract:

HPV16 L2E7 is a fusion protein used for therapeutical vaccine targeting HPV virus. To increase its expression in Escherichia coli, we optimized the codon usage of HPV16 l2e7 gene based on its codon usage bias. The optimized gene of HPV16 sl2e7 was cloned into three different vectors: pGEX-5X-1, pQE30, ET41a, and expressed in JM109, JM109 (DE3) and BL21 (DE3) lines separately. A high expression line was selected with pET41a vector in BL21 (DE3) cells. After optimization of the growth condition, including inoculation amount, IPTG concentration, induction time and temperature, the expression level of HPV16 L2E7 was increased from less than 10% to about 28% of total protein. HPV16 L2E7 protein was then purified from 15 L culture by means of SP Sepharose Fast Flow, Q Sepharose Fast Flow and Superdex 200 pg. After renaturing, HPV16 L2E7 protein with ≥95% purity was achieved, which was confirmed via SDS-PAGE gel and Western blotting. The combined use of purified HPV16 L2E7 and CpG helper has shown clear inhibition of tumor growth in mice injected with tumor cells, with six out of eight mice shown no sign of tumor. This study lays a solid foundation for a new pipeline of large-scale vaccine production.