Laboratório de Estudos Moleculares e Terapia Experimental, Departamento de Genética, Centro de Ciências Biológicas, Universidade Federal de Pernambuco, Recife, PE, Brasil.
Braz J Med Biol Res. 2011 Dec;44(12):1209-14. doi: 10.1590/s0100-879x2011007500141. Epub 2011 Oct 28.
Human papillomavirus (HPV) infection is the most common sexually transmitted disease in the world and is related to the etiology of cervical cancer. The most common high-risk HPV types are 16 and 18; however, the second most prevalent type in the Midwestern region of Brazil is HPV-33. New vaccine strategies against HPV have shown that virus-like particles (VLP) of the major capsid protein (L1) induce efficient production of antibodies, which confer protection against the same viral type. The methylotrophic yeast Pichia pastoris is an efficient and inexpensive expression system for the production of high levels of heterologous proteins stably using a wild-type gene in combination with an integrative vector. It was recently demonstrated that P. pastoris can produce the HPV-16 L1 protein by using an episomal vector associated with the optimized L1 gene. However, the use of an episomal vector is not appropriate for protein production on an industrial scale. In the present study, the vectors were integrated into the Pichia genome and the results were positive for L1 gene transcription and protein production, both intracellularly and in the extracellular environment. Despite the great potential for expression by the P. pastoris system, our results suggest a low yield of L1 recombinant protein, which, however, does not make this system unworkable. The achievement of stable clones containing the expression cassettes integrated in the genome may permit optimizations that could enable the establishment of a platform for the production of VLP-based vaccines.
人乳头瘤病毒（HPV）感染是世界上最常见的性传播疾病，与宫颈癌的病因有关。最常见的高危 HPV 类型是 16 和 18 型；然而，巴西中西部地区第二常见的类型是 HPV-33。针对 HPV 的新疫苗策略表明，主要衣壳蛋白（L1）的病毒样颗粒（VLP）诱导产生有效的抗体，从而对同一病毒类型提供保护。甲醇营养型酵母毕赤酵母是一种高效且廉价的表达系统，可使用野生型基因与整合载体稳定地生产高水平的异源蛋白。最近证明，毕赤酵母可以通过使用与优化的 L1 基因相关的附加体载体来产生 HPV-16 L1 蛋白。然而，附加体载体的使用并不适合在工业规模上进行蛋白质生产。在本研究中，载体被整合到毕赤酵母基因组中，结果显示 L1 基因转录和蛋白产生均为阳性，无论是在细胞内还是细胞外环境中。尽管毕赤酵母系统具有很大的表达潜力，但我们的结果表明 L1 重组蛋白的产量较低，但这并不会使该系统无法使用。实现包含整合到基因组中的表达盒的稳定克隆可能允许进行优化，从而能够建立基于 VLP 的疫苗生产平台。
