利用諾羅病毒拆開之P蛋白質自我組裝形成P顆粒呈現抗原

Abstract

諾羅病毒外鞘P蛋白質具有三個表面環狀結構適合作為抗原嵌入位。嵌有外來抗原的P蛋白質組裝成P粒子後，可大幅提高外來抗原的免疫原性，是一個近年來被廣泛研究，極具潛力的疫苗平台。更重要的是P蛋白質可以利用大腸桿菌或嗜甲醇酵母菌Pichia pastoris進行大量生產奠定了產業化的基礎。然而，現今以兩端固定在P蛋白質上的方式呈現抗原，卻可能因外來抗原的結構特殊，或體積龐大而破壞P蛋白質本身的結構，進而阻礙P粒子的正常組裝，這可能就是現在以諾羅病毒做為多抗原呈現平台時，多以呈現小片段胜肽為主的原因。因此，本研究嘗試將P蛋白質由第2個環狀結構的胺基酸位置拆成兩段（分別為PN及PC），分別利用兩個甲醇誘導的啟動子各自表現後，利用SDS-PAGE及西方墨點法進行胞內或胞外蛋白質的生產確認，再搭配蛋白質原態電泳、Ni-NTA pull down及Ni-NTA純化及膠體過濾法證實拆開的P蛋白質（PN及PC）可以重新組裝成完整的P蛋白質，更可以進一步組裝成P粒子。將來，利用拆開的P蛋白質呈現外來抗原時，可藉由一端游離的形式攜帶抗原，降低抗原大小及種類對P蛋白質結構造成的壓力，使諾羅病毒P粒子多抗原呈現平台更具彈性及應用性。

As a vaccine platform, norovirus P particle not only can present antigens in a highly repetitive manner to enhance the immunogenicity of the foreign antigen, but also have three surface loops for antigens insertion in a P protein. Moreover, P protein can be produced in many host systems including E.coli and Pichia pastoris. However, the most common way to present whole protein antigen with two terminus linked to P protein might disrupt the structure of P protein, and consequently lead to the difficulty of P particle assembly. This might be the cause of difficulties of presenting large antigen or antigen with spatially distant N and C termini. In order to overcome this obstacle, P protein was split into two parts from surface loop 2, so that antigen was presented with only one terminus fused, to decrease the conformational stress on P protein. Here we showed that the split P proteins (PN and PC) were successfully produced by P. pastoris and can be scaled-up by a 5-L fermenter. Ni-NTA pull-down assay, native PAGE and gel filtration data together showed that PN and PC interact with each other and could form multimers. The establishment of this split P protein vaccine platform might improve the flexibility of presenting different antigens.