家蚕微孢子虫检测及微粒子病预报研究
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摘要
微孢子虫(microspordia)是自然界普遍分布的一类细胞内专性寄生的单细胞真核生物,寄主范围从无脊椎动物到脊椎动物,包括人类,其感染经济昆虫及水产动物并能造成了不同程度的经济损失。目前已发现并报道的微孢子虫约有1400余种,分别归入160多个属~[1]。家蚕微孢子虫{Nosema bombycis)是1857年人类首次鉴定出的微孢子虫,它能够引发家蚕微粒子病,并通过胚胎垂直传播,是蚕业生产上的毁灭性病害,被列为蚕业生产国唯一的法定检疫对象。19世纪中叶,欧洲各国有多次微粒子病的流行的记录,蚕业生产损失惨重。目前,我国蚕业生产每年因微粒子病和带毒种造成的直接经济损失就高达数千万元。因此,防控微粒子病是蚕业界研究的重点和难点。目前对于微粒子病的检测主要方法有三类：显微镜检法、分子生物学检测方法和血清学检测方法。目前生产上还是使用150多年前创立的母蛾袋检法,具有直观、简单,经培训后方便掌握。不足之处是耗费大量的人力、物力,形态相近但物种不同、毒力不同的微孢子不易区分。为了解决这一生产中的问题,结合目前血清学诊断技术的新进展,我们希望研发出基于免疫胶体金层析快速检测技术的检测新方法。本研究就是以快速检测家蚕微粒子病为实验目的,制备家蚕微孢子虫的单克隆抗体,并初步探讨抗体在家蚕微孢子虫的快速检测上的可能应用。主要研究结果如下：
1.家蚕微孢子虫发芽液蛋白抗原的单克隆抗体制备及鉴定
采用差速离心和梯度密度离心纯化家蚕微孢子虫,通过碳酸钾处理,使孢子弹出极丝并释放孢原质(即发芽过程),制备发芽液蛋白抗原；用发芽液蛋白抗原以75μg／只的剂量免疫BALB／c小鼠,四次免疫后,间接ELISA法测定小鼠抗血清效价为1：6.4×10~3。取免疫小鼠的脾细胞和NS_0骨髓瘤细胞融合,融合率为97.7%,阳性率为3.2%；2次亚克隆后,得到3株稳定分泌抗体的杂交瘤细胞,分别命名为ID_6-A10,1D_6-E12和4D_2-D5。取1D_6-A1,1D_6-E12杂交瘤细胞株注入小鼠腹腔诱生腹水,获得的小鼠腹水经间接ELISA检测,抗体效价分别为1：6.4x10~5和1∶1.28×10~6,4D_2-D5,的细胞培养上清的抗体效价为1：5.12x10~2。通过Western blot对3种单抗鉴定,发现3种单抗都相同的能和发芽液蛋白中的两条蛋白有明显的杂交反应,一条约30kDa,另一条约18kDa,可能3种单克隆抗体针对同一个抗原位点。
2.针对家蚕微孢子虫孢壁蛋白SWP32的单克隆抗体的制备及鉴定
采用差速离心和梯度密度离心纯化家蚕微孢子虫,通过碳酸钾处理使孢子发芽,再通过梯度密度离心纯化孢子发芽后的空孢壳,抽提孢子的孢壁蛋白；通过SDS-PAGE电泳,挖取本实验室已经鉴定过的孢壁蛋白SWP32,再经电洗脱纯化,用纯化的SWP32以75gg／只的剂量免疫BALB/c小鼠,四次免疫后,间接ELISA法测定小鼠抗血清效价为1：5.12x 10~4。取免疫小鼠的脾细胞和NS_0骨髓瘤细胞融合,间接ELISA筛选出一株稳定分泌抗体的杂交瘤细胞株,命名为C7,注入小鼠体内诱生腹水,间接ELISA测得小鼠腹水抗体效价和细胞培养上清抗体效价分别1：1.28x10~6和1：2.56x10~3。通过Western blot对单抗C7鉴定,发现单抗C7能特异性地和SWP32蛋白结合,单抗C7的IFAT结果显示,未能观察到微孢子虫表面有荧光反应,可能是单克隆抗体C7所针对的抗原位点并未暴露在家蚕微孢子虫的表面。
3.家蚕微孢子虫单克隆抗体的初步应用
用柠檬酸三钠还原法制备胶体金,并标记家蚕微孢子虫单克隆抗体。结合免疫层析技术,用点膜的方法将待检样品点在硝酸纤维膜上,通过金标抗体在膜上流动和抗原结合,形成肉眼可见的显色反应。对处理的纯化的家蚕微孢子虫和感染的家蚕及母蛾样品进行检测。实验结果发现,用点膜的方法加样0.5μL来检测家蚕微孢子虫,纯化的家蚕微孢子虫的检出度可以达到8.0×10~6个／mL,高倍镜下约30个孢子／视野,病蚕研磨液中的检出度可以达到1.5×10~7个／mL,约60个孢子／视野。感染家蚕母蛾的研磨液,需要经过差速离心后,才能被检测出。
本研究获得了3种单克隆抗体的腹水,加上我们前期研究制备3株克隆抗体,我们从筛选出一种单抗腹水进行胶体金标记,建立的点膜法能够检出病蚕中的微孢子虫,但检出的敏感性还需提高已达到生产上的要求。
Microsporidia are a group of obligate intracellular eukaryotic parasites that are widely distributed in nature, whose host range from invertebrates to vertebrates, including human. Nowadays, about 1,400 species belonging to 160 genera of microsporidia have been reported. Nosema bombycis was firstly discovered from infected Bombyx mori in 1857. This organism can cause pebrine and vertical transmissionvia eggs and bring huge loss to silkworm feeding, so it is defined as the only disease needed for quarantine in sericulture. In Mid-19~(th)-century, pebrine outbroke in quite several European countries and resulted in heavy loss. Today, more than ten million RMB each year have been lost due to this disease in China. Thus, the identification of silkworm pebrine is very significant in sericulture. To date, the methods to diagnose the silkworm pebrine mainly contain microscopic diagnostics, molecular diagnostics and serological diagnostics, with their advantages and disadvantages. Microscopic examination of female moths have prevalenced 100 years. Serological detection method, based on the specific and sensitive antibody-antigen reaction and the colloidal gold immunization chromatography assay, has broad prospects in the diagnostics of pebrine. In this study, we prepared monoclonal antibodies (MAbs) against N. bombycis and investigated the application of this antibody to rapid detection of silkworm pebrine. The results are as following:
1 Preparation and identification of monoclonal antibodies against germinated spores of Nosema bombycis.
The N. bombycis spore was purified by differential centrifugation and densithy gradient centrifugation. The antigen was preparated after using the potassium carbonate to trigger the germination of the spore. BALB/c mice were immunized with the dose of 75μg/mouse, and after four times'inoculation, the antibody titer in serum of mice was up to 1:6.4×103 detected by indirect ELISA.The hybridoma cell lines were prepared by fusing the NS_0 myeloma cells and the lymphocytes cells from BALB/c mice spleen using routine technology, with a cell fusion rate up to 97.7% and a positive rate reachs 3.2%. Three hybridoma cell lines to secrete monoclonal antibodies were developed by two subclones, named 1D6-A10,1D_6-E12 and 4D2-D5.The ascites antibody titer of both 1D6-A10 and1D_6-E12 and the cell-culture supernatant antibody titer of 4D_2-D5 were 1:6.4×105,1:1.28×106 and 1:5.12×102, respectively. The specificity of the McAb of 1D_6-A10, 1D6-E12 and 4D2-D5 against N. bombycis was identified by Western-blot (WB), and there were two protein bands with reaction signal, one with 31 kDa while the other with 18 kDa.2. Preparation and identification of monoclonal antibodies against Spore wall protein32(SWP32) of Nosema bombycisl.
We purified the N. bombycis spores through differential and density gradient centrifugation. The spore was promoted to germinate by potassium carbonate. Second density gradient centrifugation was used to obtain the empty spore coats. Total spore wall proteins were extracted, and SWP32 was gained as the antigen by SDS-PAGE and electroeluting. Then, BALB/c mice were immunized with dose of 75μg/mouse, and the titer reached to 1:5.12×10~4 after four times' inoculation. The spleen cells of BALB/c mice and the NSo myeloma cells were fused using PEG-1500. One hybridoma cell line, called C7, was obtained through twice subcloning. Its ascites antibody titer was 1:1.28×10~6 and cell-culture supernatant antibody titer was 1:2.56×10~3.The specificity of the McAb C7 against SWP32 protein of N. bombycis was analyzed through Western-blot (WB) and indirect fluorescent antibody test (IFAT). The WB showed only one band (SWP32) react with antibody but no signal shown in the IFAT, suggesting that the C7-target epitope may be not exhibited on the surface of spore of Nosema bombycis.
3.Preliminary application of monoclonal antibody against Nosema bombycis.
Monoclonal antibodies were labeled with colloidal gold that was prepared by sodium citrate reduction method. According to immunization chromatography assay,we spotted the sample on the nitrocellulose membrane, and made antibody marked by colloidal gold to flow through this membrane, the labeled antibody and the antigen in sample would bind together and a red spot will form to detect N. bombycis. The results showed this method could detect the purified spores with at least 30 spores per field, the spores in the infected silkworm with more than 60 spores per field, and the spores in the silkworm moth after differential centrifugation can be detected too.
1.家蚕微孢子虫发芽液蛋白抗原的单克隆抗体制备及鉴定
采用差速离心和梯度密度离心纯化家蚕微孢子虫,通过碳酸钾处理,使孢子弹出极丝并释放孢原质(即发芽过程),制备发芽液蛋白抗原；用发芽液蛋白抗原以75μg／只的剂量免疫BALB／c小鼠,四次免疫后,间接ELISA法测定小鼠抗血清效价为1：6.4×10~3。取免疫小鼠的脾细胞和NS_0骨髓瘤细胞融合,融合率为97.7%,阳性率为3.2%；2次亚克隆后,得到3株稳定分泌抗体的杂交瘤细胞,分别命名为ID_6-A10,1D_6-E12和4D_2-D5。取1D_6-A1,1D_6-E12杂交瘤细胞株注入小鼠腹腔诱生腹水,获得的小鼠腹水经间接ELISA检测,抗体效价分别为1：6.4x10~5和1∶1.28×10~6,4D_2-D5,的细胞培养上清的抗体效价为1：5.12x10~2。通过Western blot对3种单抗鉴定,发现3种单抗都相同的能和发芽液蛋白中的两条蛋白有明显的杂交反应,一条约30kDa,另一条约18kDa,可能3种单克隆抗体针对同一个抗原位点。
2.针对家蚕微孢子虫孢壁蛋白SWP32的单克隆抗体的制备及鉴定
采用差速离心和梯度密度离心纯化家蚕微孢子虫,通过碳酸钾处理使孢子发芽,再通过梯度密度离心纯化孢子发芽后的空孢壳,抽提孢子的孢壁蛋白；通过SDS-PAGE电泳,挖取本实验室已经鉴定过的孢壁蛋白SWP32,再经电洗脱纯化,用纯化的SWP32以75gg／只的剂量免疫BALB/c小鼠,四次免疫后,间接ELISA法测定小鼠抗血清效价为1：5.12x 10~4。取免疫小鼠的脾细胞和NS_0骨髓瘤细胞融合,间接ELISA筛选出一株稳定分泌抗体的杂交瘤细胞株,命名为C7,注入小鼠体内诱生腹水,间接ELISA测得小鼠腹水抗体效价和细胞培养上清抗体效价分别1：1.28x10~6和1：2.56x10~3。通过Western blot对单抗C7鉴定,发现单抗C7能特异性地和SWP32蛋白结合,单抗C7的IFAT结果显示,未能观察到微孢子虫表面有荧光反应,可能是单克隆抗体C7所针对的抗原位点并未暴露在家蚕微孢子虫的表面。
3.家蚕微孢子虫单克隆抗体的初步应用
用柠檬酸三钠还原法制备胶体金,并标记家蚕微孢子虫单克隆抗体。结合免疫层析技术,用点膜的方法将待检样品点在硝酸纤维膜上,通过金标抗体在膜上流动和抗原结合,形成肉眼可见的显色反应。对处理的纯化的家蚕微孢子虫和感染的家蚕及母蛾样品进行检测。实验结果发现,用点膜的方法加样0.5μL来检测家蚕微孢子虫,纯化的家蚕微孢子虫的检出度可以达到8.0×10~6个／mL,高倍镜下约30个孢子／视野,病蚕研磨液中的检出度可以达到1.5×10~7个／mL,约60个孢子／视野。感染家蚕母蛾的研磨液,需要经过差速离心后,才能被检测出。
本研究获得了3种单克隆抗体的腹水,加上我们前期研究制备3株克隆抗体,我们从筛选出一种单抗腹水进行胶体金标记,建立的点膜法能够检出病蚕中的微孢子虫,但检出的敏感性还需提高已达到生产上的要求。
Microsporidia are a group of obligate intracellular eukaryotic parasites that are widely distributed in nature, whose host range from invertebrates to vertebrates, including human. Nowadays, about 1,400 species belonging to 160 genera of microsporidia have been reported. Nosema bombycis was firstly discovered from infected Bombyx mori in 1857. This organism can cause pebrine and vertical transmissionvia eggs and bring huge loss to silkworm feeding, so it is defined as the only disease needed for quarantine in sericulture. In Mid-19~(th)-century, pebrine outbroke in quite several European countries and resulted in heavy loss. Today, more than ten million RMB each year have been lost due to this disease in China. Thus, the identification of silkworm pebrine is very significant in sericulture. To date, the methods to diagnose the silkworm pebrine mainly contain microscopic diagnostics, molecular diagnostics and serological diagnostics, with their advantages and disadvantages. Microscopic examination of female moths have prevalenced 100 years. Serological detection method, based on the specific and sensitive antibody-antigen reaction and the colloidal gold immunization chromatography assay, has broad prospects in the diagnostics of pebrine. In this study, we prepared monoclonal antibodies (MAbs) against N. bombycis and investigated the application of this antibody to rapid detection of silkworm pebrine. The results are as following:
1 Preparation and identification of monoclonal antibodies against germinated spores of Nosema bombycis.
The N. bombycis spore was purified by differential centrifugation and densithy gradient centrifugation. The antigen was preparated after using the potassium carbonate to trigger the germination of the spore. BALB/c mice were immunized with the dose of 75μg/mouse, and after four times'inoculation, the antibody titer in serum of mice was up to 1:6.4×103 detected by indirect ELISA.The hybridoma cell lines were prepared by fusing the NS_0 myeloma cells and the lymphocytes cells from BALB/c mice spleen using routine technology, with a cell fusion rate up to 97.7% and a positive rate reachs 3.2%. Three hybridoma cell lines to secrete monoclonal antibodies were developed by two subclones, named 1D6-A10,1D_6-E12 and 4D2-D5.The ascites antibody titer of both 1D6-A10 and1D_6-E12 and the cell-culture supernatant antibody titer of 4D_2-D5 were 1:6.4×105,1:1.28×106 and 1:5.12×102, respectively. The specificity of the McAb of 1D_6-A10, 1D6-E12 and 4D2-D5 against N. bombycis was identified by Western-blot (WB), and there were two protein bands with reaction signal, one with 31 kDa while the other with 18 kDa.2. Preparation and identification of monoclonal antibodies against Spore wall protein32(SWP32) of Nosema bombycisl.
We purified the N. bombycis spores through differential and density gradient centrifugation. The spore was promoted to germinate by potassium carbonate. Second density gradient centrifugation was used to obtain the empty spore coats. Total spore wall proteins were extracted, and SWP32 was gained as the antigen by SDS-PAGE and electroeluting. Then, BALB/c mice were immunized with dose of 75μg/mouse, and the titer reached to 1:5.12×10~4 after four times' inoculation. The spleen cells of BALB/c mice and the NSo myeloma cells were fused using PEG-1500. One hybridoma cell line, called C7, was obtained through twice subcloning. Its ascites antibody titer was 1:1.28×10~6 and cell-culture supernatant antibody titer was 1:2.56×10~3.The specificity of the McAb C7 against SWP32 protein of N. bombycis was analyzed through Western-blot (WB) and indirect fluorescent antibody test (IFAT). The WB showed only one band (SWP32) react with antibody but no signal shown in the IFAT, suggesting that the C7-target epitope may be not exhibited on the surface of spore of Nosema bombycis.
3.Preliminary application of monoclonal antibody against Nosema bombycis.
Monoclonal antibodies were labeled with colloidal gold that was prepared by sodium citrate reduction method. According to immunization chromatography assay,we spotted the sample on the nitrocellulose membrane, and made antibody marked by colloidal gold to flow through this membrane, the labeled antibody and the antigen in sample would bind together and a red spot will form to detect N. bombycis. The results showed this method could detect the purified spores with at least 30 spores per field, the spores in the infected silkworm with more than 60 spores per field, and the spores in the silkworm moth after differential centrifugation can be detected too.
引文
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