基于恶性疟原虫染色体精准转录组的裂殖子相关基因功能研究

Malaria accounts for an enormous burden of disease globally, with Plasmodium falciparum accounting for the majority of malaria. Artemisinin resistance and lack of effective vaccines threaten the control of malaria. The whole genome sequences, transcriptomics and proteomics have provided critical information on the expression profiles of malarial proteins during the parasite’s life cycle..However, the current transcriptomic data of P. falciparum is derived from a previous DNA microarray, which based on an incomplete P. falciparum genome sequences and RNA extracted from imprecisively synchronized parasites. Compared to DNA/cDNA microarray and RNA-seq, realtime-PCR is more accurate and used widely for verification and replenishment of the P. falciparum transcriptomic data..During infection with Plasmodium spp., the merozoite form of the parasites invades red blood cells and replicates inside them. It is during the blood-stage of infection that malaria disease occurs. Therefore, understanding merozoite invasion, and targeting merozoite proteins by novel vaccines and therapeutics have been important areas of research. Several merozoite surface proteins show strong potential as malaria vaccines. However, of the ~100 vaccine candidates currently under investigation, more than 60% are based on only four parasite antigens. New antigen candidates are urgently needed. .The discovery of plasmodium antigens tended to depend on infected serum from human or animals in the previous studies. Most antigens recognized by this approach were immunodominant or possessed immunodominant regions made of repeats. Although immunodominant antigens induce very strong antibody responses, recent studies have shown that they did not offer adequate protection when tested as subunit vaccines in clinical trials, suggesting that these antigens might be used by the parasites for immune evasion, such as CSP and AMA1..In our previous study, we screened the transcriptome of P. falciparum 3D7 parasites from different stages of its life cycle. Analysis of chromosome 14 transcriptome showed a batch of 87 genes that had stage-specific high-level expression in the merozoite stage, compared to the other stages, including ring-stage, trophozoites and immature schizonts. In the 87 genes, 64 genes are unkown. These genes maight play important roles in the invasion progress of merozoites into human erythrocytes. In the present project, we plan to identify P. falciparum antigens by functional analysis, instead of antibody response using immune serum. The 64 unknown genes of merozoite will be cloned using gateway technology, and related recombinant proteins and polyclonal antibodies will be prepared to study their roles in parasite replication and related mechanism. Gene knockout will also be carried out using CRISPR/Cas9 technology to find out the critical genes in the invasion processes. Furthermore, the antibody response of all these antigens in serum from P. falciparum infected human beings will be examined to analyze the relationship between immunogenicity and related protection of P. falciparum antigens. Then, chimeras of P. beighei ANKA with homologous genes replaced by indicated P. falciparum genes will be constructed. Mice vaccinated with recombinant P. falciparum proteins will be challenged with the corresponding chimera parasites to evaluate the value of the proteins as potential vaccine antigens. These studies will discover potential antigen candidates of malaria vaccines and provide evidence for the value of immunodominant and non-immunonominant antigens in malaria vaccine design.

恶性疟原虫是致病力最强和危害最为严重的寄生虫。近年来，随着青蒿素抗药虫体的出现及疟疾疫苗的缺乏，对疟原虫防治的研究越发紧迫。病原完整的基因组序列和高通量组学研究技术，为深入研究疟原虫的生物学机理奠定了基础。本项目在开展恶性疟原虫染色体精准转录组分析过程中，发现恶性疟原虫第14号染色体上的很多基因（约11%）在裂殖子期呈现特异性高表达。这些基因多为未知功能基因，推测可能直接或间接参与恶性疟原虫裂殖子入侵红细胞的过程。本项目拟利用分子生物学结合免疫学的方法，研究这些基因编码蛋白的功能，揭示其与裂殖子发育繁殖和细胞入侵的相关性。研究结果对揭示疟原虫病原生物学机理和研制新型抗疟药物及疫苗具有重要意义。

恶性疟原虫裂殖子入侵红细胞是红内期虫体引起人体致病乃至致命的首要环节，因此裂殖子入侵红细胞相关分子的研究至关重要，且裂殖子蛋白是疟疾红内期疫苗的首选抗原靶标。现有疟原虫转录组数据库数据不完整，且其数据不准确无法直接采纳。为了发掘新型裂殖子入侵关键蛋白，本项目首先采用实时定量PCR的方法对对恶性疟原虫实验室标准株PF3D7第14号染色体全部基因进行了精准转录检测，进而深入挖掘了恶性疟原虫各个生长发育时期基因的表达规律及表达特征。恶性疟原虫第14号染色体精准转录组的获得，为相关科研人员进行疟原虫研究提供了准确完整的研究数据。然后，项目以这些转录组数据为基础，深入挖掘入侵关键时期—裂殖子期的特异性高表达基因，针对这些基因编码蛋白的功能进行研究。免疫印迹和免疫荧光检测结果显示大部分新型裂殖子蛋白均在裂殖子期和晚期裂殖体期高表达，且主要表达在裂殖子表面。这些蛋白质不仅能够结合人或小鼠的红细胞，还具有肝素结合功能。其中5个蛋白的多抗加入恶性疟原虫培养体系后，能够显著抑制疟原虫的入侵和增殖。这些裂殖子新型蛋白可能在恶性疟原虫入侵红细胞过程中发挥关键作用，是有价值的疟疾疫苗候选抗原。继而，项目进一步设计制备了恶性疟裂殖子抗原多肽微阵列芯片，对37个裂殖子期关键蛋白质的两千多个多肽进行了微阵列检测，完成了恶性疟原虫红细胞入侵相关蛋白质的B细胞表位分析，发现了免疫优势抗原的特征性信息。进一步的体外保护性实验显示免疫优势抗原可能是疟原虫吸引宿主体液免疫的武器，保护疟原虫蛋白质功能性位点不被宿主免疫系统攻击，从而帮助疟原虫实现免疫逃逸。综上所述，本项目完成了恶性疟原虫第14号染色体基因精准转录组检测分析，据此深入挖掘裂殖子新型蛋白质，并对新型裂殖子蛋白的功能和关键裂殖子抗原的抗原性进行了深入研究，相关研究结果对新型疟疾疫苗设计制备提供了理论依据和有价值的候选抗原。