宿主对巨细胞病毒免疫反应通过TAF9参与系统系红斑狼疮抗双链DNA自身抗体产生的机制研究

Human cytomegalovirus (CMV) infection is closely related to the pathogenesis of systemic lupus erythematosus (SLE), but the mechanism of CMV infection promoting the pathogenesis of SLE has not been elucidated. Anti-double-stranded DNA (dsDNA) antibody is considered to be a pathogenic autoantibody, and the reactivity of anti-CMV pp65 antibody in serum of SLE patients is related to the positive anti-dsDNA antibody, in which the B cell recognition site is the GASTSAGRKR peptide of pp65428-437. We found that TATA-box binding protein association factor 9 (TAF9) contained peptides with high homology with this epitope by blastp analysis. In this study, we aim to observe the difference of anti-CMV immunoreactive epitopes between SLE patients and healthy controls (CMV past infection), analyze whether the anti-dsDNA antibody in serum of SLE patients recognizes CMV epitope and TAF9 epitope, and confirm that CMV antigen may cross-react with TAF9. Subsequently, the TAF9 peptide was synthesized and the mice were immunized to observe whether it can stimulate the formation of anti-dsDNA and anti-TAF9 antibodies, and the cross-reactivity relationship between antibodies, and provide a basis for whether TAF9 antibody is involved in the pathogenesis of SLE. Meanwhile, anti-TAF9 antibody is detected in patients with SLE and other connective tissue diseases, and patients with SLE is followed up to determine the sensitivity and specificity of autoantibody detection in the diagnosis of SLE. We also analyze the correlation between the results of autoantibody detection and the clinical data of SLE patients to determine the relationship between autoantibody and SLE disease activity, which can provide the basis for the diagnosis and treatment monitoring of SLE.

巨细胞病毒（CMV）感染与系统性红斑狼疮（SLE）的发病密切相关，但CMV感染促使SLE发病的机制还未阐明。抗双链DNA抗体被认为是具有致病性的自身抗体，且SLE患者血清中抗CMV pp65抗体反应性与抗双链DNA抗体阳性相关。前期我们通过blastp分析发现，TATA盒结合蛋白相关因子9（TAF9）含有与该pp65抗原表位具有高度同源性的肽段。本课题分析SLE患者抗CMV免疫反应识别表位与CMV既往感染者的不同，观察SLE患者血清中抗双链DNA抗体是否识别CMV和TAF9表位，证实CMV抗原与TAF9有交叉反应。并通过合成TAF9肽段免疫小鼠后是否可以刺激抗双链DNA及抗TAF9抗体的形成，及抗体之间的交叉反应关系，为抗TAF9抗体参与SLE致病提供依据。同时病例对照及随访研究，明确抗TAF9抗体检测对SLE诊断的敏感性和特异性，及其与疾病活动度的关系，为SLE的诊断和治疗监测提供依据。

巨细胞病毒（CMV）感染与系统性红斑狼疮（SLE）的发病密切相关，但CMV感染促使SLE发病机制还未阐明。抗dsDNA抗体被认为是具有致病性的自身抗体，且SLE患者血清中抗CMV PP65抗体反应性与抗dsDNA抗体阳性相关。本课题发现SLE患者抗CMV PP65抗体免疫反应识别表位与健康对照（CMV隐性感染）识别表位不同，为CMV PP65 428-437的GASTSAGRKR肽段。通过blastp分析发现TATA盒结合蛋白相关因子9（TAF9）含有与该表位高度同源的多肽（TAF9 134-144）。SLE患者与疾病对照组、健康对照血清对CMV PP65 428-437和TAF9 134-144抗原表位的识别具有明显差异，SLE患者血清中抗CMV PP65 428-437抗体和抗TAF9 134-144抗体水平显著高于其他两组（P<0.0001），且抗TAF9 134-144抗体水平与补体3，补体4浓度呈显著负相关。SLE患者血清中抗TAF9 134-144抗体与抗CMV PP65 428-437抗体水平高度相关，r=0.7766（P<0.0001），提示CMV PP65抗原可能与TAF9有交叉反应。抗TAF9 134-144抗体水平与抗dsDNA抗体呈显著正相关，相关系数为0.2088。ROC曲线分析显示，抗TAF9 134-144抗体的曲线下面积（AUC）为0.741，最佳cutoff值为2.9706（OD值），敏感度和特异度分别为70.7%和80.3%。在评估的血清学标志物中，抗TAF9 134-144抗体的敏感度最高，具有临床应用前景。进一步用合成的CMV PP65 428-437和TAF9 134-144多肽分别免疫BALB/c小鼠，免疫后小鼠血清中可成功检测到抗CMV PP65 428-437和TAF9 134-144抗体，证明小鼠免疫成功，且免疫小鼠血清中抗dsDNA抗体、抗CMV PP65 428-437和TAF9 134-144抗体水平均明显高于对照组（P<0.0001）。免疫组小鼠尿蛋白水平明显高于对照组，提示CMV PP65 428-437和TAF9 134-144可以引起正常小鼠产生狼疮样表现。试验证实了本研究提出的假设：宿主对CMV产生的免疫反应，通过与TAF9的交叉反应参与了抗dsDNA自身抗体的产生，为阐明SLE的发病机制提供了思路。