土壤中苏云金杆菌HBF-1菌株毒蛋白ELISA检测方法的建立及应用
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摘要
本文以在我国分离获得的对丽金龟科幼虫高毒力的苏云金芽胞杆菌(Bacillus thuringiensis简称Bt)HBF-1为供试菌株，以其产生的杀虫晶体蛋白为检测目标，通过制备的抗Bt蛋白多克隆抗体，建立了土壤中HBF-1菌株毒蛋白含量的酶联免疫吸附分析(Enzyme-linked immunosorbent assay，ELISA)测定方法。
抗原是影响抗体特异性和效价的重要因素。为了得到高纯度的杀虫晶体蛋白，本研究采用等电点法沉淀出晶体杀虫蛋白，用聚丙烯酰胺凝胶电泳进一步纯化粗提的杀虫晶体蛋白，得到分子量约为130kDa的电泳纯杀虫晶体蛋白，作为免疫用抗原。利用该抗原分四次对新西兰白兔进行免疫，获得了高特异性的抗血清，再用辛酸—硫酸铵盐析法，获得了高免血清中的免疫球蛋白IgG，SDS-PAGE电泳结果表明，提取的IgG纯度较高，其浓度达到5.6344mg／mL。采用改良的过碘酸钠法，用辣根过氧化物酶标记IgG，制备出标记率为0.28，克分子比为0.798的酶标抗体。
通过利用直接法、间接法和双抗夹心法等ELISA方法检测土壤中Bt毒蛋白的含量，综合样品检测值的相关系数CV，EC_(50)及检测率三个方面，明确双抗体夹心ELISA法均优于其它两种方法，间接ELISA法次之，从而证明双抗夹心法是一种检测土壤中Bt毒蛋白含量较适宜的方法。明确了双抗体夹心ELISA法的最佳工作条件：抗体的包被量为7μg／mL；Bt杀虫晶体蛋白的3种提取液(Tris—硼酸、Na_2CO_3缓冲液、PBST)中，以Na_2CO_3缓冲液为最佳提取液；封闭液为0.1％明胶-PBST；酶标抗体的最适工作浓度为1：800稀释；待测样品的反应时间为37℃120min；酶标抗体的时间为37℃60min；底物显色时间为室温15min。通过对该ELISA法的特异性试验、交叉性试验和重复性试验验证，证明双抗体夹心ELISA法是一种特异、敏感、快速、操作简便的方法。
利用优化的双抗夹心ELISA系统检测花生田里Bt蛋白的消长动态，结果表明利用双抗夹心ELISA法定量测定土壤中Bt蛋白的量能够明确反映出土壤中Bt毒蛋白含量的动态变化情况。在播种期施入Bt毒蛋白，原液及2倍稀释液土深20cm的处理在3d时达到最大含量，随后为平缓下降的趋势；原液和2倍稀释液土深2cm的处理在3d时蛋白含量上升，随后下降，在21d时检测量达到最高峰，随后下降至134d检测结束，在此过程中3～14d深土层20cm的蛋白含量明显高于浅土层2cm，随后除2倍稀释液21d、31d时有所例外，其余各处理均为深土层蛋白量高于浅土层。在开花前期施入Bt毒蛋白，四处理的蛋白含量3d达到最高，随后原液和2倍稀释液土深20cm的处理急剧下降，原液和2倍稀释液土深2cm的处理下降平缓。在开花后期施入Bt毒蛋白，四处理蛋白含量3～7d达到最高，随后呈平缓下降的趋势。因此，在不同施药时期，各处理深土层20cm的Bt蛋白量高于浅土层2cm。总之，在花生开花前期和开花后期进行灌根施药，土壤中Bt蛋白表达高峰期即为铜绿丽金龟(Anomala corpulenta Motschulsky)卵高峰期和幼虫一龄期、二龄初期，能够对金龟子幼虫达到最佳防治效果。
The purpose of this research was to study on the preparation of antigen of theinsecticidal crystal protein of Bt (Bacillus thuringiensis)strain HBF-1, the polyclonalantibody and an enzyme-linked immunosorbent assay (ELISA) was developed andperformed to determine the Bt protein in soil. The specific HBF-1 strain from Bt whichshowed high toxicity to Anomala corpulenta and A. exoleta.
In this study, the crystal protein was obtained by isoelectric point deposition, and theextracted protein was purified by SDS-PAGE. The highly purified protein with molecularweight of 130 kDa was used as antigen to immune New Zealand Rabbit for four times, andthen specific antibody was acquired. Mainly immunoglobuling (IgG) of rabbit anti-Bt waspurified by caprylic acid-ammonium sulphate precipitation. The result of SDS-PAGEsuggested that it's one simple, efficient, rapid and low cost method for the purification ofIgG from antiserum, and the concentration of the purified IgG was 5.6344mg/mL. With thesodium periodate method antibody was labeled by HRP to make enzyme-labelled antibody.After tested, In the combination of enzyme-labelled antibody, HRP/Ab was 0.798, thecombination rate of enzyme was 0.28.
In this research, Direct—ELISA, ID—ELISA, DAS—ELISA were experimented toassay the content of Bt protein in soil, Through comparison of CV, EC_(50) and the rate ofdetection, DAS—ELISA is the best method, and the DAS—ELISA for detection of the Btprotein was established. Through the specificity test blocking test, cross test, andduplication test, we can see clearly that the method of double sandwich ELISA is veryspecific, sensitive and stable. It offers that the assay has a promising application future.
The method of DAS—ELISA was applied to detect Bt protein in the peanut field. Theresult showed that the content of the Bt protein which was detected by this method canincarnate the dynamics of the Bt protoxin in soil. when manuring the fermentation liquid ofHBF-1 in sowing time, concerned the sample of original liquid and double diluted liquid in the depth of 20cm, the detection of Bt protein reached the peak on the 3rd day, and then gotinto slowly degradation stage. Concerned the sample of 2cm, the dynamics of Bt protoxinis not stable before the 21st day, and reached the peak on the 21st day, and then slowlydegraded till 134th day, this experimentation was over. The result showed that in the periodof 3rd～14th day, the detection of Bt protoxin in the depth of 20cm is significantly higherthan that in the depth of 2cm, and then except the sample of double diluted in the period of21st～31st, the Bt protoxin in the depth of 20cm is more than 2cm. When manuring thefermentation liquid of HBF-1in flowering early time, the detection of protoxin reached thepeak on the 3rd day, and then the content ofprotoxin in the depth of 20cm degraded rapidly,though in the depth of 2cm the protoxin degraded slowly. When manuring the fermentationliquid of HBF-1 in flowering later time, the detection of Bt protoxin reached the peak onthe 3rd～7th, and then degraded all along with lower speed.
Finally, it will be a best control to the larvae of scarbaeoid beetles when manuring thefermentation liquid of HBF-1 in flowering early time or later time, because in this periodthe expressing levels of Bt protoxin reached the peak and in time at peak egg stage, theduration of the 1st instar and 2nd instar of Anomala corpulenta Motschulsky.
抗原是影响抗体特异性和效价的重要因素。为了得到高纯度的杀虫晶体蛋白，本研究采用等电点法沉淀出晶体杀虫蛋白，用聚丙烯酰胺凝胶电泳进一步纯化粗提的杀虫晶体蛋白，得到分子量约为130kDa的电泳纯杀虫晶体蛋白，作为免疫用抗原。利用该抗原分四次对新西兰白兔进行免疫，获得了高特异性的抗血清，再用辛酸—硫酸铵盐析法，获得了高免血清中的免疫球蛋白IgG，SDS-PAGE电泳结果表明，提取的IgG纯度较高，其浓度达到5.6344mg／mL。采用改良的过碘酸钠法，用辣根过氧化物酶标记IgG，制备出标记率为0.28，克分子比为0.798的酶标抗体。
通过利用直接法、间接法和双抗夹心法等ELISA方法检测土壤中Bt毒蛋白的含量，综合样品检测值的相关系数CV，EC_(50)及检测率三个方面，明确双抗体夹心ELISA法均优于其它两种方法，间接ELISA法次之，从而证明双抗夹心法是一种检测土壤中Bt毒蛋白含量较适宜的方法。明确了双抗体夹心ELISA法的最佳工作条件：抗体的包被量为7μg／mL；Bt杀虫晶体蛋白的3种提取液(Tris—硼酸、Na_2CO_3缓冲液、PBST)中，以Na_2CO_3缓冲液为最佳提取液；封闭液为0.1％明胶-PBST；酶标抗体的最适工作浓度为1：800稀释；待测样品的反应时间为37℃120min；酶标抗体的时间为37℃60min；底物显色时间为室温15min。通过对该ELISA法的特异性试验、交叉性试验和重复性试验验证，证明双抗体夹心ELISA法是一种特异、敏感、快速、操作简便的方法。
利用优化的双抗夹心ELISA系统检测花生田里Bt蛋白的消长动态，结果表明利用双抗夹心ELISA法定量测定土壤中Bt蛋白的量能够明确反映出土壤中Bt毒蛋白含量的动态变化情况。在播种期施入Bt毒蛋白，原液及2倍稀释液土深20cm的处理在3d时达到最大含量，随后为平缓下降的趋势；原液和2倍稀释液土深2cm的处理在3d时蛋白含量上升，随后下降，在21d时检测量达到最高峰，随后下降至134d检测结束，在此过程中3～14d深土层20cm的蛋白含量明显高于浅土层2cm，随后除2倍稀释液21d、31d时有所例外，其余各处理均为深土层蛋白量高于浅土层。在开花前期施入Bt毒蛋白，四处理的蛋白含量3d达到最高，随后原液和2倍稀释液土深20cm的处理急剧下降，原液和2倍稀释液土深2cm的处理下降平缓。在开花后期施入Bt毒蛋白，四处理蛋白含量3～7d达到最高，随后呈平缓下降的趋势。因此，在不同施药时期，各处理深土层20cm的Bt蛋白量高于浅土层2cm。总之，在花生开花前期和开花后期进行灌根施药，土壤中Bt蛋白表达高峰期即为铜绿丽金龟(Anomala corpulenta Motschulsky)卵高峰期和幼虫一龄期、二龄初期，能够对金龟子幼虫达到最佳防治效果。
The purpose of this research was to study on the preparation of antigen of theinsecticidal crystal protein of Bt (Bacillus thuringiensis)strain HBF-1, the polyclonalantibody and an enzyme-linked immunosorbent assay (ELISA) was developed andperformed to determine the Bt protein in soil. The specific HBF-1 strain from Bt whichshowed high toxicity to Anomala corpulenta and A. exoleta.
In this study, the crystal protein was obtained by isoelectric point deposition, and theextracted protein was purified by SDS-PAGE. The highly purified protein with molecularweight of 130 kDa was used as antigen to immune New Zealand Rabbit for four times, andthen specific antibody was acquired. Mainly immunoglobuling (IgG) of rabbit anti-Bt waspurified by caprylic acid-ammonium sulphate precipitation. The result of SDS-PAGEsuggested that it's one simple, efficient, rapid and low cost method for the purification ofIgG from antiserum, and the concentration of the purified IgG was 5.6344mg/mL. With thesodium periodate method antibody was labeled by HRP to make enzyme-labelled antibody.After tested, In the combination of enzyme-labelled antibody, HRP/Ab was 0.798, thecombination rate of enzyme was 0.28.
In this research, Direct—ELISA, ID—ELISA, DAS—ELISA were experimented toassay the content of Bt protein in soil, Through comparison of CV, EC_(50) and the rate ofdetection, DAS—ELISA is the best method, and the DAS—ELISA for detection of the Btprotein was established. Through the specificity test blocking test, cross test, andduplication test, we can see clearly that the method of double sandwich ELISA is veryspecific, sensitive and stable. It offers that the assay has a promising application future.
The method of DAS—ELISA was applied to detect Bt protein in the peanut field. Theresult showed that the content of the Bt protein which was detected by this method canincarnate the dynamics of the Bt protoxin in soil. when manuring the fermentation liquid ofHBF-1 in sowing time, concerned the sample of original liquid and double diluted liquid in the depth of 20cm, the detection of Bt protein reached the peak on the 3rd day, and then gotinto slowly degradation stage. Concerned the sample of 2cm, the dynamics of Bt protoxinis not stable before the 21st day, and reached the peak on the 21st day, and then slowlydegraded till 134th day, this experimentation was over. The result showed that in the periodof 3rd～14th day, the detection of Bt protoxin in the depth of 20cm is significantly higherthan that in the depth of 2cm, and then except the sample of double diluted in the period of21st～31st, the Bt protoxin in the depth of 20cm is more than 2cm. When manuring thefermentation liquid of HBF-1in flowering early time, the detection of protoxin reached thepeak on the 3rd day, and then the content ofprotoxin in the depth of 20cm degraded rapidly,though in the depth of 2cm the protoxin degraded slowly. When manuring the fermentationliquid of HBF-1 in flowering later time, the detection of Bt protoxin reached the peak onthe 3rd～7th, and then degraded all along with lower speed.
Finally, it will be a best control to the larvae of scarbaeoid beetles when manuring thefermentation liquid of HBF-1 in flowering early time or later time, because in this periodthe expressing levels of Bt protoxin reached the peak and in time at peak egg stage, theduration of the 1st instar and 2nd instar of Anomala corpulenta Motschulsky.
引文
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