含有GM-CSF或CD40L的汉滩病毒嵌合VLPs疫苗的构建及其免疫学特性研究

Hantaan viruse is the causative agent of hemorrhagic fever with renal syndrome with high mortality in China, but with no specific antiviral therapeutics available yet. The control and prevention of HTNV infection is relied on vaccination. However, the immune response induced by the currently applied vaccines is not good enough, from others and our results. Thus, there is an urgent need for developing efficient vaccine. As a new form of vaccine candidate, virus-like particles (VLPs) has been reported to be potent vaccine for a variety of viruses by eliciting potent anti-viral humoral and cellular immune responses. To develop a more effective hantaan virus vaccine, we design a chimeric HTNV VLP vaccine by incorporating GM-CSF or CD40L, which have been extensively used as effective genetic and protein adjuvants to enhance immunogenicity of antigens, into VLP as a molecular adjuvant. In this project, we modify the eukaryotic expression vector pCI-neo by replacing the promoter and insertion a stabilized integration element to improve gene expression. Then,we coexpress S、M gene of HTNV structure protein and GM-CSF or CD40L to produce HTNV chimeric VLP carrying GM-CSF or CD40L, and investigate their immunological characteristics as well as their ability of protection against hantaan virus in mouse model, laying the groundwork for the development of HTNV vaccine.

汉滩病毒（HTNV）在我国主要引起肾综合征出血热（HFRS），该病危害极为严重，目前尚缺乏特效的治疗药物，主要使用疫苗进行预防，但据文献和我们的前期研究发现，HTNV疫苗刺激机体产生免疫应答的能力较弱，因此，如何提高免疫效果是HTNV疫苗研究中亟需解决的关键问题。GM-CSF、CD40L等细胞因子作为免疫佐剂可显著提高抗原的免疫效果；近年来研究发现，病毒样颗粒（VLPs）疫苗是目前最有发展前景的候选疫苗之一，尤其是基因修饰的嵌合VLPs更具有良好的发展潜力。本项目拟在前期工作基础上，改建真核表达载体，即通过提高启动子活性、增加促进基因表达的元件，以期在真核细胞中稳定、高表达HTNV的结构蛋白，组装出HTNV VLPs，同时表达GM-CSF或CD40L，使之修饰在VLPs表面，获得HTNV嵌合VLPs ，进一步研究其免疫学特性及对感染动物的保护作用，从而为研制HTNV新型疫苗奠基础。

利用基因工程技术对真核表达载体pCI-neo进行改建，在其中引入MTX筛选标记，以利于后续筛选稳定高表达细胞株。将HTNV S和M基因分别构建入上述载体中，分别合成鼠GM-CSF-GPI和CD40L基因，并构建入上述表达M基因的载体中，与 HTNV S基因表达载体共转染CHO-K1细胞，收集细胞制备样品进行电镜观察，结果显示成功包装出病毒样颗粒；收集转染后的培养上清进行蔗糖密度梯度离心纯化、SDS-PAGE及Western-blot检测，结果显示G1、G2、NP、GM-CSF及CD40L分子量大小与预期相符，都存在于VLPs中，成功包转出了HTNV嵌合VLPs，进一步用间接免疫荧光实验证实GP与GM-CSF及CD40L共定位在细胞膜上，免疫共沉淀技术证实GP与GM-CSF及CD40L在细胞中共表达，通过以上实验证实在真核细胞中成功包装出组装正确的HTNV嵌合VLPs。