應(yīng)用免疫蛋白質(zhì)組學(xué)方法鑒定弓形蟲病診斷抗原分子的研究
發(fā)布時間：2018-09-07 16:57
【摘要】：目的鑒定對弓形蟲感染具有潛在診斷價值的弓形蟲抗原分子。方法制備剛地弓形蟲RH株速殖子全蟲可溶性蛋白及弓形蟲感染小鼠腹腔蛋白,經(jīng)SDS-PAGE分離后采用半干電轉(zhuǎn)移方法將其轉(zhuǎn)移到硝酸纖維素膜上,再分別與22份弓形蟲感染者血清進(jìn)行Western blot,以22份健康人血清和其他寄生蟲病患者血清作為對照,篩選能被弓形蟲感染者血清特異性識別的蛋白組分;兩種蛋白樣本經(jīng)二維電泳分離后采用Western blot篩選能被弓形蟲感染者混合血清識別,但不能被健康人血清及其他寄生蟲病患者血清識別的特異性斑點(diǎn)。切取SDS-PAGE膠上相應(yīng)的特異性蛋白質(zhì)斑點(diǎn)進(jìn)行MALDI-TOF分析,鑒定蛋白斑點(diǎn)的基因。結(jié)果一維電泳及Western blot結(jié)果顯示,弓形蟲感染小鼠腹腔蛋白中的90、37、30、28和18ku組分分別可被部分弓形蟲感染者血清識別,其中為28ku蛋白能被22份弓形蟲感染病人血清中的15份所識別;速殖子全蟲可溶性蟲體蛋白中的90、70、67、56、48、39、37、30、28和18ku組分可被部分弓形蟲感染者血清識別,其中28ku蛋白可被22份弓形蟲感染者血清中的21份識別,22份健康人血清中有2份與28ku蛋白呈弱陽性反應(yīng),與其他寄生蟲病患者血清無交叉反應(yīng)。二維電泳W(wǎng)estern blot陽性斑點(diǎn)的飛行質(zhì)譜分析顯示,弓形蟲感染小鼠腹腔蛋白中的28ku組分為磷酸丙糖異構(gòu)酶,弓形蟲速殖子全蟲可溶性蛋白中的28ku組分為GRA2和GRA7。結(jié)論弓形蟲抗原與宿主抗體反應(yīng)具有高度異質(zhì)性,GRA2、GRA7及磷酸丙糖異構(gòu)酶等分子的弓形蟲感染者血清識別率高,此三種蛋白可作為建立具有高敏感性的弓形蟲病特異性免疫診斷方法的抗原分子。
[Abstract]:Objective to identify the antigen molecules of Toxoplasma gondii with potential diagnostic value for Toxoplasma gondii infection. Methods the soluble protein of Toxoplasma gondii RH strain and the peritoneal protein of mice infected with Toxoplasma gondii were prepared and transferred to the nitrocellulose membrane by semi-dry electric transfer after SDS-PAGE isolation. Western blot, was performed on 22 sera of Toxoplasma gondii infected with Toxoplasma gondii and 22 sera from healthy people and other parasitic patients were used as controls to screen the protein components which could be specifically recognized by the sera of Toxoplasma gondii infected persons. Two kinds of protein samples were separated by two-dimensional electrophoresis and screened by Western blot. They could be identified by mixed serum of Toxoplasma gondii but not by specific spots in healthy sera and other parasitic patients. The specific protein spots on SDS-PAGE gel were digested for MALDI-TOF analysis to identify the gene of protein spots. Results the results of one-dimensional electrophoresis and Western blot showed that the components of 90 ~ (37) ~ (30) and 18ku of Toxoplasma gondii infected mice could be recognized by some sera of Toxoplasma gondii infected by Toxoplasma gondii, of which 28ku protein could be identified by 15 out of 22 sera of patients infected with Toxoplasma gondii (Toxoplasma gondii). In soluble body protein of T. tachyzoites, the components of 90, 70, 67, 56, 48, 39, 37, 30, 28, and 18ku, can be identified in sera of some infected individuals with Toxoplasma gondii (Toxoplasma gondii, Toxoplasma gondii). Of the 22 sera of Toxoplasma gondii infected with Toxoplasma gondii, 2 of 22 sera from healthy people showed weak positive reaction with 28ku protein, but no cross reaction with other parasitic patients. FMS analysis of Western blot positive spots in Toxoplasma gondii infected mice showed that the 28ku component in the peritoneal protein of mice infected with Toxoplasma gondii was divided into propanose phosphate isomerase, and the 28ku component in the soluble protein of Toxoplasma Tachyzoites was divided into GRA2 and GRA7.. Conclusion the sera of Toxoplasma gondii infected with Toxoplasma gondii antigen and host antibody are highly heterogeneous, such as GRA2GRA7 and Propranose phosphate isomerase, and the recognition rate of Toxoplasma gondii infected with Toxoplasma is high. These three proteins can be used as antigen molecules for the establishment of highly sensitive immunodiagnostic methods for Toxoplasma gondii (Toxoplasma gondii).
【作者單位】： 衛(wèi)生部寄生蟲病預(yù)防與控制重點(diǎn)實(shí)驗(yàn)室 江蘇省寄生蟲病分子生物學(xué)重點(diǎn)實(shí)驗(yàn)室 江蘇省血吸蟲病防治研究所;中國疾病預(yù)防控制中心 傳染病預(yù)防控制所診斷室;
【基金】：國家重大科技專項(xiàng)(No.2012ZX10004220) 國家自然科學(xué)基金項(xiàng)目(No.81201316,30972581,30471515) 江蘇省自然科學(xué)基金項(xiàng)目(No.BK2012544,BK2008110)
【分類號】：R531.8
[Abstract]:Objective to identify the antigen molecules of Toxoplasma gondii with potential diagnostic value for Toxoplasma gondii infection. Methods the soluble protein of Toxoplasma gondii RH strain and the peritoneal protein of mice infected with Toxoplasma gondii were prepared and transferred to the nitrocellulose membrane by semi-dry electric transfer after SDS-PAGE isolation. Western blot, was performed on 22 sera of Toxoplasma gondii infected with Toxoplasma gondii and 22 sera from healthy people and other parasitic patients were used as controls to screen the protein components which could be specifically recognized by the sera of Toxoplasma gondii infected persons. Two kinds of protein samples were separated by two-dimensional electrophoresis and screened by Western blot. They could be identified by mixed serum of Toxoplasma gondii but not by specific spots in healthy sera and other parasitic patients. The specific protein spots on SDS-PAGE gel were digested for MALDI-TOF analysis to identify the gene of protein spots. Results the results of one-dimensional electrophoresis and Western blot showed that the components of 90 ~ (37) ~ (30) and 18ku of Toxoplasma gondii infected mice could be recognized by some sera of Toxoplasma gondii infected by Toxoplasma gondii, of which 28ku protein could be identified by 15 out of 22 sera of patients infected with Toxoplasma gondii (Toxoplasma gondii). In soluble body protein of T. tachyzoites, the components of 90, 70, 67, 56, 48, 39, 37, 30, 28, and 18ku, can be identified in sera of some infected individuals with Toxoplasma gondii (Toxoplasma gondii, Toxoplasma gondii). Of the 22 sera of Toxoplasma gondii infected with Toxoplasma gondii, 2 of 22 sera from healthy people showed weak positive reaction with 28ku protein, but no cross reaction with other parasitic patients. FMS analysis of Western blot positive spots in Toxoplasma gondii infected mice showed that the 28ku component in the peritoneal protein of mice infected with Toxoplasma gondii was divided into propanose phosphate isomerase, and the 28ku component in the soluble protein of Toxoplasma Tachyzoites was divided into GRA2 and GRA7.. Conclusion the sera of Toxoplasma gondii infected with Toxoplasma gondii antigen and host antibody are highly heterogeneous, such as GRA2GRA7 and Propranose phosphate isomerase, and the recognition rate of Toxoplasma gondii infected with Toxoplasma is high. These three proteins can be used as antigen molecules for the establishment of highly sensitive immunodiagnostic methods for Toxoplasma gondii (Toxoplasma gondii).
【作者單位】： 衛(wèi)生部寄生蟲病預(yù)防與控制重點(diǎn)實(shí)驗(yàn)室 江蘇省寄生蟲病分子生物學(xué)重點(diǎn)實(shí)驗(yàn)室 江蘇省血吸蟲病防治研究所;中國疾病預(yù)防控制中心 傳染病預(yù)防控制所診斷室;
【基金】：國家重大科技專項(xiàng)(No.2012ZX10004220) 國家自然科學(xué)基金項(xiàng)目(No.81201316,30972581,30471515) 江蘇省自然科學(xué)基金項(xiàng)目(No.BK2012544,BK2008110)
【分類號】：R531.8
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