猪树突状细胞Clec9A受体鉴定及其介导抗原靶向递呈研究

Clec9A, also designated as DNGR-1, is a C-type lectin receptor expressed in mouse CD8+ Dendritic cell(DC).This subset of DC has been shown to cross-present antigen to CD4+ and CD8+ T lymphocytes, and then facilitate both the humoral and cellular immune responses.Clec9A has been found in DC of human, cow and other animals, but still unknown in pig. In this study, we attempt to clone and express Clec9A gene of pig DC, and then develop both monoclonal antibody(McAb) and camel sigle heavy chain antibody(ScAb) against Clec9A.The DC phenotype of pig that expressing Clec9A will be explored using the FITC labeled McAb and ScAb,and some other commercial antibodies of DC receptors. In order to construct the anti-Clec9A genetically engineered antibodies,the gene sequences coding for H and L chains of McAb, and ScAb will be cloned and sequenced. The gene sequence of foreign epitopes will be linked with CH1 domain of heavy chain of McAb and inserted into baculovirus expression vector together with coding sequence of L chain of McAb located under different protomer.Also, the gene sequence of foreign epitopes will be linked with C-terminal of ScAb coding sequence and inserted into baculovirus expression vector. The epitopes targeting anti-Clec9A McAb and ScAb will be expressed by infecting insect cells with two recombinant baculoviruses.The binding property of these two epitope targeting antibodies to Clec9A receptor will be detected in vitro by flow cytometry and confocal laser scanning microscope. And then, these two epitope-tagged antibodies will be used to inoculate pigs to evaluate the efficiency of antigen targeting to DC cell. The humoral and cellular immune responses of inoculated pigs will be detected and compared with DC non-targeting epitope vaccine. This study will provide very valuable information on antigen-targeting molecular vaccine research in future.

Clec9A，又叫DNGR-1，是新发现的树突状细胞（DC）C型凝集素类受体，主要表达于类似小鼠CD8+ DC表面，已证明该类DC可以通过MHC-I与II类分子交互递呈抗原于CD4+ 与CD8+ T细胞,从而促进体液免疫与细胞免疫的协同应答。在人与牛等多种动物均发现了同类受体，但在猪体还未见报道。本研究通过克隆表达猪Clec9A受体基因，研制其单克隆抗体（McAb）与骆驼重链抗体（ScAb），鉴定表达Clec9A受体的猪DC类型。然后，将病毒抗原表位嵌合于McAb重链CH1区之后，与轻链基因共同插入双表达杆状病毒载体；同样将抗原表位融合于ScAb抗体C端，于昆虫细胞表达抗原靶向性的McAb与ScAb，通过体外结合Clec9A受体试验，以及用携带抗原表位的靶向性抗体免疫猪，测定产生的特异性体液与细胞免疫应答水平，评价Clec9A受体介导抗原靶向递呈效果，为抗原靶向性分子疫苗研究奠定基础。

树突状细胞（DCs）是专职的抗原递呈细胞，在启动与控制获得性免疫应答方面发挥着关键性的作用。在人与小鼠体内的研究表明，表达Clec9A（又称为DNGR-1）C型凝集素类受体和趋化因子受体XCR1的DC细胞具有抗原交互递呈功能，可以通过MHC-I与II类分子交互递呈抗原于CD4+ 与CD8+ T细胞，从而促进体液免疫与细胞免疫的协同应答。在人、小鼠与牛等多种动物均发现了表达Clec9A的DC，但在猪体还未见报道，因此本研究的目的是确定猪与牛体内表达该受体的DC表型，研究受体介导的抗原靶向免疫效果。研究初期，通过基因组、mRNA以及蛋白层面综合研究发现猪Clec9A是一个假基因，因而，将研究重点调整为鉴定牛外周血表达Clec9A和XCR1的DCs表型，并研究受体介导的抗原靶向免疫效果。研究表明，牛Clec9A与XCR1共表达于同一类DC细胞，其表型为CD26+CADM1+CD205+MHCⅡ+CD11c+CD11b-CD4-CD8-CD163-lin- DC细胞，代表cDCs的一类，而非浆性DCs（pDCs）。牛外周血中cDCs可进一步划分为CD26+CD172a-CD11c+MHCⅡ+lin- DC和CD26-CD172a+CD11c+MHCⅡ+lin- DC，分别代表cDC1和cDC2。小鼠源XCL1对牛XCR1+DC细胞具有趋化作用。牛XCL1的C-C结是发挥趋化作用的关键功能域。用牛XCR1的配体XCL1融合O型FMDV多表位重组蛋白OB7免疫牛，评价了XCL1介导抗原靶向免疫效果。结果表明，XCL-OB7可显著增强O型FMDV特异性中和抗体应答水平，具有增强体液免疫的效果。利用合成肽制备了牛XCR1与Clec9A胞外区的单克隆抗体，筛选到了三株Clec9A的单抗，体外与体内试验表明可特异性结合Clec9A受体。选择其中G7-E2株单抗杂交瘤，测定了其抗体基因序列，表达了完整小鼠源工程抗体和与牛IgG恒定区融合的嵌合抗体，体外验证均可特异性结合Clec9A受体，为抗体介导的抗原靶向递呈研究奠定了基础。