本文選題：蛋白重組　切入點(diǎn)：抗中性粒細(xì)胞胞漿髓過氧化物酶抗體　出處：《安徽醫(yī)科大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：背景MPO-ANCA相關(guān)性血管炎(myeloperoxidase-specific antineutrophil cytoplasmic autoantibody associated vasculitis,MPO-AAV)在我國(guó)并不少見,主要包括顯微鏡下多血管炎(Microscopic polyangiitis,MPA)、肉芽腫性多血管炎(Granulomatosis with polyangiitis,GPA)及嗜酸性肉芽腫性多血管炎(Eosinophilic granulomatosis with polyangiitis,EGPA)。此類疾病臨床表現(xiàn)復(fù)雜多樣、進(jìn)展快,未得到及時(shí)診治的患者常因病情延誤而死亡。臨床報(bào)道、動(dòng)物實(shí)驗(yàn)以及體外實(shí)驗(yàn)證據(jù)均顯示了MPO-ANCA具有致病性,而針對(duì)其表位識(shí)別特性的研究報(bào)道顯示,MPO-ANCA不同的表位識(shí)別在AAV的不同疾病中,可能發(fā)揮不同的作用。但MPO-ANCA不同表位在MPO-AAV不同臨床損害中的作用尚未見系統(tǒng)性研究報(bào)道。目的:研究MPO-AAV患者ANCA的表位識(shí)別分布情況,探討其與臨床損害之間的相關(guān)性。方法:本研究通過構(gòu)建重組質(zhì)粒、原核表達(dá)及蛋白純化的分子生物學(xué)方法以獲得7個(gè)覆蓋全部MPO線性抗原的多肽片段,包括輕鏈(aa 165-272)、重鏈6個(gè)肽段:H4肽段(aa279-409)、H5肽段(aa341-474)、H6肽段(aa410-537)、H7(aa512-598)、H8肽段(aa538-745)及H9(aa651-745),并鑒定它們的抗原性。臨床收集初診活動(dòng)期MPO-AAV患者173例(其中,原發(fā)性MPO-AAV患者165例、PTU繼發(fā)MPO-AAV患者8例)的血清。分析MPO-AAV患者臨床損害情況。分別用所合成肽段包被ELISA反應(yīng)孔后與患者血清反應(yīng),檢測(cè)患者及健康對(duì)照血清ANCA識(shí)別MPO抗原表位肽段的特異性,分析并探討其與患者臨床多系統(tǒng)損害之間的關(guān)系。結(jié)果1.獲得人MPO蛋白及七個(gè)多肽片段的c DNA克隆,其表達(dá)的蛋白多肽具有MPO抗原性。2.正常人血清可識(shí)別H5、H6、H8、H9表位。3.8例繼發(fā)于PTU的MPO-AAV患者中,4例識(shí)別H8肽段,其中1例識(shí)別H7肽段。4.MPO-AAV患者中,有肺損較無肺損者輕鏈肽段抗體的識(shí)別陽性率、ELISA檢測(cè)OD值高,且輕鏈肽段抗體陽性患者比陰性患者的肺BVAS積分高;5.有腎損害(包括有腎功能損害、僅血尿蛋白尿)的患者和無腎損害患者H4、H5肽段抗體的陽性率差異有統(tǒng)計(jì)學(xué)意義,均為前者高于后者,6.有腎功能損害的患者H9肽段抗體陽性率以及OD值水平均高于僅尿隱血/或尿蛋白陽性患者,其余各肽段抗體陽性率比較差異均無統(tǒng)計(jì)學(xué)意義。7.有腎功能損害患者中,急性進(jìn)展性腎小球腎炎、慢性腎功能損傷患者之間比較,慢性腎功能損害患者的H4、H5、H8、H9肽段抗體OD值均高于急性腎功能損害患者。8.急性進(jìn)展性腎小球腎炎、慢性腎功能損傷患者之間比較H4、H5肽段抗體陽性率差異有顯著性,后者均較前者陽性率高。9.在急性腎功能損害患者(30例)中,輕鏈肽段抗體ELISA值和腎臟BVAS積分之間存在正相關(guān);10.有關(guān)節(jié)損傷較無關(guān)節(jié)損傷的患者H7、H8肽段抗體檢測(cè)OD值高,各肽段抗體陽性率差異無統(tǒng)計(jì)學(xué)意義。結(jié)論1.獲得MPO7個(gè)不同多肽的片段,他們保留有MPO的抗原性,可用于MPO-AAV相關(guān)的進(jìn)一步研究。2.正常人血清存在的少量ANCA識(shí)別MPO的抗原表位分散:H5、H6、H8、H9肽段(aa341-754)。3.MPO-AAV患者體內(nèi)的ANCA主要識(shí)別MPO輕鏈和重鏈兩端:輕鏈、H4、H5、H8、H9(aa165-272,aa279-474,aa538-745)的抗原表位4.相對(duì)于原發(fā)性MPO-AAV,PTU繼發(fā)性的MPO-AAV患者血清ANCA識(shí)別的MPO抗原表位比較局限,主要位Hf(aa538-598),且患者年齡相對(duì)小,病情較輕。5.MPO輕鏈檢測(cè)陽性的ANCA可能有MPO-AAV患者的肺侵害相關(guān)。6.MPO H4、H5(aa279-474)肽段檢測(cè)陽性的ANCA可能與MPO-AAV患者的腎損害相關(guān)。在腎累及的患者中,識(shí)別H9(aa538-745)的ANCA可能與腎功能損害有關(guān),在腎功能損害的MPO-AAV患者中,識(shí)別Ha、Hb、Hc(aa279-474)、H8(aa538-745)段表位的ANCA導(dǎo)致慢性腎損害可能性大,在急性進(jìn)展性腎功能損傷患者中,出現(xiàn)MPO-輕鏈(aa165-272)抗體的患者,病情更加嚴(yán)重。7.識(shí)別H7、H8肽段的ANCA抗體水平與MPO-AAV患者的關(guān)節(jié)損害有關(guān)。
[Abstract]:The background of MPO-ANCA associated vasculitis (myeloperoxidase-specific antineutrophil cytoplasmic autoantibody associated vasculitis, MPO-AAV) is not uncommon in China, mainly including the microscopic polyangiitis (Microscopic polyangiitis, MPA), granulomatous vasculitis (Granulomatosis with multiple polyangiitis, GPA) and eosinophilic granulomatous polyangiitis (Eosinophilic granulomatosis with polyangiitis, EGPA). This kind of clinical disease performance is complicated, progresses quickly, did not receive timely treatment of patients due to illness delays and death. Clinical reports, animal experiments and in vitro experiments showed evidence of MPO-ANCA pathogenicity, and the epitope recognition properties of research reports show that MPO-ANCA different epitope recognition in different diseases in AAV, may to play a different role. But different MPO-ANCA epitopes in MPO-AAV damage in different clinical The research is not yet reported. Objective: To study the MPO-AAV of patients with ANCA epitope recognition distribution, and explore the correlation between clinical damage. Methods: This study through the construction of recombinant plasmid, polypeptide fragment molecular biology method of prokaryotic expression and protein purification to obtain 7 cover all MPO linear antigens, including the light chain (AA 165-272), heavy chain 6 peptides: H4 peptide (aa279-409), H5 peptide (aa341-474), H6 peptide (aa410-537), H7 (aa512-598), H8 peptide (aa538-745) and H9 (aa651-745), and identify their antigenicity. 173 cases of newly diagnosed activities MPO-AAV patients (including 165 patients with primary MPO-AAV were PTU, MPO-AAV patients 8 cases). The serum analysis of clinical impairment of patients with MPO-AAV. Respectively with the synthetic peptide coated by ELISA reaction hole after reaction with serum, detection of patients and healthy controls serum ANCA identification of MPO antigen. A specific peptide analysis, and to explore their relationships with patients with multisystem damage. Results in the 1. C DNA clone of MPO protein and seven peptide fragments, the expression of the protein polypeptide has the antigenicity of MPO.2. in normal human serum can be identified by H5, H6, H8, H9 epitope.3.8 cases after in PTU MPO-AAV patients, 4 cases of identification of H8 peptide, H7 peptide recognition including 1 cases of.4.MPO-AAV patients, the positive rate of loss is no recognition of pulmonary lung injury were light chain peptide antibody, ELISA staining, and the light chain peptide antibody positive patients than in patients with negative pulmonary BVAS integral 5.; kidney damage (including renal damage, only hematuria and proteinuria) in patients with and without renal damage in patients with H4, there were statistically significant differences in the positive rate of H5 peptide antibody, were higher, the positive rate of patients with H9 peptide antibody of 6. had renal damage and the OD value was higher than only urine occult blood or / Urine protein positive patients, the positive rate of the peptide antibody showed no significant difference.7. with renal damage in patients with acute progressive glomerulonephritis, comparison between patients with chronic renal injury, H4, patients with chronic renal damage in H5, H8, H9 peptide antibody OD value was higher than that of acute renal injury patients.8. in acute glomerulonephritis, comparison between H4 patients with chronic renal injury, there was significant difference in the positive rate of H5 peptide antibody, which were higher than the positive rate of patients with high.9. in acute kidney injury (30 cases), there is a positive correlation between BVAS and renal integral light chain peptide antibody of ELISA; 10. there is no joint injury of joint damage in patients with H7, H8 peptide antibody detection was high, there was no significant difference in the positive rate of peptide antibody. Conclusion MPO7 1. different peptide fragments, they retain antigenicity of MPO, can be used for A small amount of ANCA identification of MPO further study of MPO-AAV related.2. of normal human serum are epitopes: H5, H6, H8 dispersion, H9 peptide (aa341-754) in patients with.3.MPO-AAV ANCA recognition MPO light chain and heavy chain ends: light chain, H4, H5, H8, H9 (aa165-272, aa279-474, aa538-745) the antigen epitope of 4. relative to the primary MPO-AAV and secondary PTU MPO-AAV in serum of patients with ANCA to identify the epitopes of MPO is limited, mainly Hf (aa538-598), and the age of patients is relatively small, mild light chain.5.MPO ANCA may have MPO-AAV positive patients with lung damage related.6.MPO H4, H5 (aa279-474) peptide detection of renal damage in ANCA positive patients may be associated with MPO-AAV. The renal involvement in patients with H9 (aa538-745) on identification of ANCA may be associated with impairment of renal function in renal damage in patients with MPO-AAV, Hb, Hc recognition Ha (aa279-474), H8 (aa538-745) segment table a ANCA caused the possibility of chronic renal damage. In patients with acute progressive renal impairment, patients with MPO- light chain (aa165-272) antibodies were more severe..7. recognized H7 and H8 peptide ANCA antibody level was associated with joint damage in MPO-AAV patients.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R593.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 李光富,張兆松,王新軍,李春玲,王勇,季e，

本文編號(hào)：1594392