日本血吸蟲果糖二磷酸醛縮酶抗體反應(yīng)模式及診斷價值的研究
發(fā)布時間：2019-04-26 21:44
【摘要】：目的觀察血吸蟲感染家兔體內(nèi)抗日本血吸蟲中國大陸株果糖二磷酸醛縮酶(Sj FBA)抗體反應(yīng)的動態(tài)變化,評價檢測抗Sj FBA抗體IgG用于血吸蟲現(xiàn)癥感染的診斷與療效考核價值。方法采用RT-PCR方法從日本血吸蟲中國大陸株cDNA中擴(kuò)增出編碼Sj FBA的開放閱讀框基因片段,然后亞克隆到表達(dá)質(zhì)粒pET28a(+)中,構(gòu)建重組表達(dá)質(zhì)粒Sj FBA-pET28a。將重組表達(dá)質(zhì)粒轉(zhuǎn)化至大腸埃希菌BL21(DE3)中,用異丙基-β-D-硫代半乳糖苷(IPTG)進(jìn)行誘導(dǎo)表達(dá),經(jīng)鎳螯合膠親和層析的方法純化重組Sj FBA蛋白。用日本血吸蟲中國大陸株尾蚴感染日本大白兔,6周后用吡喹酮與青蒿琥酯進(jìn)行治療,收集感染前和感染后2、4、6周及治療后2、4、6、8、10、12、14、16周的血樣。以重組Sj FBA為包被抗原,建立檢測抗Sj FBA抗體IgG的酶聯(lián)免疫吸附試驗方法(rSj FBA-ELISA),用于檢測家兔不同感染階段血清抗Sj FBA抗體反應(yīng)模式。分別用5、10、15和20條日本血吸蟲中國大陸株尾蚴感染小鼠,收集感染后35d的小鼠血清,采用rSj FBA-ELISA檢測抗Sj FBA抗體IgG并觀察抗Sj FBA抗體水平與感染度的關(guān)系。用rSj FBA-ELISA分別檢測血吸蟲病患者血清、健康人血清、華支睪吸蟲病和衛(wèi)氏并殖吸蟲病患者血清,觀察該方法用于血吸蟲病診斷的敏感性與特異性;使用該方法檢測治療前及治療后1年的同一血吸蟲現(xiàn)癥感染患者的配對血清,觀察抗Sj FBA抗體IgG檢測用于血吸蟲病療效考核的價值。結(jié)果血吸蟲感染家兔體內(nèi)抗Sj FBA抗體IgG水平隨感染時間的延長而逐漸增強(qiáng),感染6周達(dá)到高峰(A450值為1.364),但治療后抗Sj FBA的抗體則迅速下降,治療6-8周后抗體水平可下降至陰性閾值(A450值為0.4),而未治療組則抗體水平則始終保持在高水平,表明家兔體內(nèi)抗Sj FBA抗體反應(yīng)呈典型的短壽抗體反應(yīng)模式。不同感染度小鼠血清抗Sj FBA抗體水平隨著感染度的升高而逐漸升高,呈劑量依賴性關(guān)系。檢測抗Sj FBA抗體IgG用于血吸蟲現(xiàn)癥感染診斷的特異性為88.8%,敏感性為82.5%,陰性預(yù)測值和陽性預(yù)測值分別為78.6%和91.0%;與華支睪吸蟲病患者血清的交叉反應(yīng)率為10%,與衛(wèi)氏并殖吸蟲病患者血清的交叉反應(yīng)率為30%。治愈1年后患者血清中抗Sj FBA的抗體IgG陰轉(zhuǎn)率達(dá)100%。結(jié)論日本血吸蟲Sj FBA在宿主體內(nèi)誘導(dǎo)短壽抗體反應(yīng),應(yīng)用rSj FBA-ELISA檢測抗Sj FBA抗體IgG的敏感性和特異性均較高,具有血吸蟲病診斷與療效考核價值。
[Abstract]:Aim to observe the dynamic changes of anti-Schistosoma japonicum Chinese strain fructose diphosphate aldolase (Sj FBA) antibody reaction in rabbits infected with Schistosoma japonicum, and to evaluate the value of detecting anti-Sj FBA antibody IgG in the diagnosis and therapeutic evaluation of schistosome infection. Methods the open reading frame gene encoding Sj FBA was amplified by RT-PCR from Schistosoma japonicum Chinese strain cDNA and subcloned into the expression plasmid pET28a () to construct the recombinant expression plasmid Sj FBA-pET28a.. The recombinant expression plasmid was transformed into Escherichia coli BL21 (DE3) and induced by isopropyl-尾-D-thiogalactoside (IPTG). The recombinant Sj FBA protein was purified by Ni chelating gel affinity chromatography. Rabbits were infected with cercariae of Schistosoma japonicum in mainland China. Six weeks later, praziquantel and artesunate were treated with praziquantel and artesunate. Blood samples were collected before infection and 2,4,6,8,10,12,14,16 weeks after infection. An enzyme-linked immunosorbent assay (rSj FBA-ELISA) for detection of anti-Sj FBA antibody IgG was established by using recombinant Sj FBA as coated antigen, which was used to detect the reaction pattern of anti-Sj FBA antibody in sera of rabbits at different stages of infection. Mice were infected with 5, 10, 15 and 20 cercariae of Schistosoma japonicum (Schistosoma japonicum) Chinese mainland strain respectively. The sera of mice were collected 35 days after infection. Anti-Sj FBA antibody IgG was detected by rSj FBA-ELISA and the relationship between the level of anti-Sj FBA antibody and the degree of infection was observed. The sera of schistosomiasis patients, healthy people, clonorchiasis sinensis and paragonimiasis westerns were detected by rSj FBA-ELISA, and the sensitivity and specificity of this method for diagnosis of schistosomiasis were observed. This method was used to detect the matched sera of the same schistosomiasis infected patients before and 1 year after treatment, and to observe the value of anti-Sj FBA antibody IgG detection in evaluating the curative effect of schistosomiasis. Results the level of anti-Sj FBA antibody IgG in rabbits infected with Schistosoma japonicum increased gradually with the prolongation of infection time and reached its peak at 6 weeks after infection (A450 = 1.364). However, the level of anti-Sj FBA antibody decreased rapidly after treatment. The antibody level decreased to the negative threshold value (A450 = 0.4) after 6 weeks of treatment, while the antibody level remained high in the untreated group, indicating that the response of anti-Sj FBA antibody in rabbits showed a typical response pattern of short-lived antibody. The level of anti-Sj FBA antibody in serum of mice with different degree of infection increased gradually with the increase of infection degree, in a dose-dependent manner. The specificity, sensitivity, negative predictive value and positive predictive value of anti-Sj FBA antibody IgG for diagnosis of schistosome infection were 88.8%, 82.5%, 78.6% and 91.0%, respectively. The cross-reaction rate with clonorchiasis and paragonimiasis was 10% and 30%, respectively. One year after cure, the negative conversion rate of anti-Sj FBA antibody IgG was 100%. Conclusion Schistosoma japonicum Sj FBA can induce short-lived antibody response in the host. The sensitivity and specificity of detecting anti-Sj FBA antibody IgG by rSj FBA-ELISA are high. It is valuable to diagnose schistosomiasis and evaluate the therapeutic effect of Schistosoma japonicum.
【作者單位】： 江蘇省寄生蟲病分子生物學(xué)重點實驗室衛(wèi)生部重要寄生蟲病預(yù)防與控制技術(shù)重點實驗室江蘇省血吸蟲病防治研究所;中國疾病控制中心傳染病預(yù)防控制所傳染病診斷室;
【基金】：國家傳染病重大專項(No.2012ZX1004-220) 國家自然科學(xué)基金項目(No.81201316,30972581) 江蘇省自然科學(xué)基金項目(No.BK2012544) 江蘇省衛(wèi)生廳血地寄科技項目(No.X201429,X201402)
【分類號】：R383.24
[Abstract]:Aim to observe the dynamic changes of anti-Schistosoma japonicum Chinese strain fructose diphosphate aldolase (Sj FBA) antibody reaction in rabbits infected with Schistosoma japonicum, and to evaluate the value of detecting anti-Sj FBA antibody IgG in the diagnosis and therapeutic evaluation of schistosome infection. Methods the open reading frame gene encoding Sj FBA was amplified by RT-PCR from Schistosoma japonicum Chinese strain cDNA and subcloned into the expression plasmid pET28a () to construct the recombinant expression plasmid Sj FBA-pET28a.. The recombinant expression plasmid was transformed into Escherichia coli BL21 (DE3) and induced by isopropyl-尾-D-thiogalactoside (IPTG). The recombinant Sj FBA protein was purified by Ni chelating gel affinity chromatography. Rabbits were infected with cercariae of Schistosoma japonicum in mainland China. Six weeks later, praziquantel and artesunate were treated with praziquantel and artesunate. Blood samples were collected before infection and 2,4,6,8,10,12,14,16 weeks after infection. An enzyme-linked immunosorbent assay (rSj FBA-ELISA) for detection of anti-Sj FBA antibody IgG was established by using recombinant Sj FBA as coated antigen, which was used to detect the reaction pattern of anti-Sj FBA antibody in sera of rabbits at different stages of infection. Mice were infected with 5, 10, 15 and 20 cercariae of Schistosoma japonicum (Schistosoma japonicum) Chinese mainland strain respectively. The sera of mice were collected 35 days after infection. Anti-Sj FBA antibody IgG was detected by rSj FBA-ELISA and the relationship between the level of anti-Sj FBA antibody and the degree of infection was observed. The sera of schistosomiasis patients, healthy people, clonorchiasis sinensis and paragonimiasis westerns were detected by rSj FBA-ELISA, and the sensitivity and specificity of this method for diagnosis of schistosomiasis were observed. This method was used to detect the matched sera of the same schistosomiasis infected patients before and 1 year after treatment, and to observe the value of anti-Sj FBA antibody IgG detection in evaluating the curative effect of schistosomiasis. Results the level of anti-Sj FBA antibody IgG in rabbits infected with Schistosoma japonicum increased gradually with the prolongation of infection time and reached its peak at 6 weeks after infection (A450 = 1.364). However, the level of anti-Sj FBA antibody decreased rapidly after treatment. The antibody level decreased to the negative threshold value (A450 = 0.4) after 6 weeks of treatment, while the antibody level remained high in the untreated group, indicating that the response of anti-Sj FBA antibody in rabbits showed a typical response pattern of short-lived antibody. The level of anti-Sj FBA antibody in serum of mice with different degree of infection increased gradually with the increase of infection degree, in a dose-dependent manner. The specificity, sensitivity, negative predictive value and positive predictive value of anti-Sj FBA antibody IgG for diagnosis of schistosome infection were 88.8%, 82.5%, 78.6% and 91.0%, respectively. The cross-reaction rate with clonorchiasis and paragonimiasis was 10% and 30%, respectively. One year after cure, the negative conversion rate of anti-Sj FBA antibody IgG was 100%. Conclusion Schistosoma japonicum Sj FBA can induce short-lived antibody response in the host. The sensitivity and specificity of detecting anti-Sj FBA antibody IgG by rSj FBA-ELISA are high. It is valuable to diagnose schistosomiasis and evaluate the therapeutic effect of Schistosoma japonicum.
【作者單位】： 江蘇省寄生蟲病分子生物學(xué)重點實驗室衛(wèi)生部重要寄生蟲病預(yù)防與控制技術(shù)重點實驗室江蘇省血吸蟲病防治研究所;中國疾病控制中心傳染病預(yù)防控制所傳染病診斷室;
【基金】：國家傳染病重大專項(No.2012ZX1004-220) 國家自然科學(xué)基金項目(No.81201316,30972581) 江蘇省自然科學(xué)基金項目(No.BK2012544) 江蘇省衛(wèi)生廳血地寄科技項目(No.X201429,X201402)
【分類號】：R383.24
【參考文獻(xiàn)】
相關(guān)期刊論文 前4條
1 雷正龍;鄭浩;張利娟;朱蓉;郭家鋼;李石柱;王立英;陳朝;周曉農(nóng);;2010年全國血吸蟲病疫情通報[J];中國血吸蟲病防治雜志;2011年06期
2 王s，
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