基于cDNA表达库梯级组分免疫高通量筛选抗内脏利什曼病疫苗候选分子

Visceral leishmaniasis (VL), caused by infection with Leishmania donovani or L. infantum, represents a serious clinical and public health problem in endemic regions and is rapidly emerging as an opportunistic infection in HIV patients. In most of endemic areas there is no effective strategies or measures for control of the disease. 80 to 90% of infected individuals do not develop clinical symptoms and past infection (including clinical recovery from visceral leishmaniasis) leads to robust immunity against reinfection. The fact provides a biological rationale for the development of VL vaccines, suggesting that in principle vaccination is feasible. Despite the obvious need and considerable effort, however, there is no effective and safe vaccine approved for human use against VL. Similarly, no effective vaccine has been developed for dogs, which serve as a major reservoir for VL. A number of candidate vaccine molecules against experimental murine leishmaniasis have been identified. However, these molecules demonstrated only partial protection. Therefore, screening new candidate vaccine molecules become sticking point for developing vaccine against VL. Host resistance to Leishmania infection is associated with the preferential induction of a specific Th1 cell response, requiring effective activation of macrophages, dendritic cells (DCs), and antigen-specific CD4+ and CD8+ T cells. It is likely that effective vaccination against a complex parasitic infection such as VL would require a multivalent vaccine containing a number of candidate molecules. .Expression library immunization (ELI) as a high-throughput technology is a novel protocol for the systemic screening of any given genome to identify potential vaccine candidates at will, by using the immune system. ELI is a potent technology to discover new vaccines and also generate genomic vaccines with amplified, multivalent immunostimulatory capacities. The main advantage of ELI is twofold; it can serves as platform for screening candidate vaccine molecules providing a rapid screening protocol for an entire genome and the readout of screening is often protection, an end goal for vaccine development..In this study we will construct two Leishmania infantum cDNA libraries using promastigotes and amastigotes, respectively. By sequential in vivo DNA immunization with fractions from the cDNA expression library and parasite challenge followed by further fractionation of the plasmid pools into smaller groups, we will identified a small of candidate vaccine molecules with protection and analyze the production of cytokines elicited by these candidate vaccine molecules. The identified molecules will become target molecules for developing vaccine for protection of human and dogs. The results of the study will contribute to understanding mechanism of immune protection against VL and strategy for development of vaccine against VL.

内脏利什曼病（又称黑热病）是一种人畜共患病，在国内外导致了严重的临床和公共卫生问题，在大部分流行区目前还没有有效的防制策略和措施，应用疫苗防制不仅是最理想的防制策略，而且研制有效的疫苗具有理论基础。而目前没有疫苗可用，原因是目前的疫苗靶分子只有部分保护作用，需要筛选更有效靶分子。观察表明只有Th1型细胞免疫反应能赋予寄主对利什曼原虫感染的抗性，而利什曼原虫是较复杂的生物体，因此，需要筛选多个能协同作用赋予寄主保护力的疫苗靶分子。表达库免疫技术是一种高通量快速筛选任何基因组以鉴定疫苗靶分子的技术，且能观察多个分子协同诱导免疫保护作用。本研究拟建立利什曼原虫cDNA表达库，通过亚库及其梯级组分免疫BALB/c小鼠，并进行攻击感染，观察分析其诱导的免疫保护作用和细胞因子生产，最终筛选出疫苗候选分子，为研制有效的犬用和人用疫苗提供分子基础，也有助于理解内脏利什曼病免疫保护机制，为疫苗研究提供对策。

按计划完成研究工作。应用婴儿利什曼原虫（L.infantum）毒力株MCAN/CN/08/GS（分离于甘肃人-犬共患型黑热病流行区）无鞭毛体mRNA构建了婴儿利什曼原虫无鞭毛体cDNA表达库，文库效率为2.1×106个/ml；插入片段长度范围在0.5kb~4.0kb间，cDNA文库的平均插入片段长度为1.18kb，插入效率为87.5%。将构建的文库涂布LB板进行培养（每块板视为一亚库），收集各亚库所有菌落进行培养，抽提DNA,接种BALB/c小鼠，对接种鼠应用婴儿利什曼原虫无鞭毛体进行攻击感染，4周后将鼠处死，确定虫荷。统计分析表明接种鼠和对照鼠虫荷无显著差异。本研究没有筛选到具有保护作用的亚库，可能的原因是由于初筛是应用一个亚库中全部重组克隆的DNA去免疫小鼠，由于克隆数较多致目的基因被稀释，从而致有免疫保护作用的分子表达量过少而不能诱导有效的免疫反应。

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