论文标题:应用ELISA检测转基因棉花中Bt蛋白的研究
Development of Enzyme-linked Immunosorbent Assay (ELISA) for Determination of Transgenic Bt-cotton
论文作者
论文导师 王硕,论文学位 硕士,论文专业 微生物与生化药学
论文单位 天津科技大学,点击次数 181,论文页数 66页File Size2993K
2006-03-01论文网 http://www.lw23.com/lunwen_6668572/
Cry1Ac protein; ELISA; PCR;determination; transgenic Bt-cotton
本论文以转Bt(Bacillus Thuringiensis)抗虫基因的所表达的杀虫晶体蛋白为检测目标，建立了双抗夹心酶联免疫方法(enzyme-linked immunosorbent assay，ELISA)检测转基因棉花中表达的Bt Cry1Ac基因型蛋白。 抗原是影响抗体特异性和效价的重要因素。该实验比较两种液体培养基(1／2LB、1／2NB)，两种培养条件(28℃，150r／min和28℃，200r／min)对三种苏云金芽孢杆菌(HD-1、HD-2、HD-73)进行培养，经碱液裂解后比较等电点沉淀和高速离心方法提取Bt蛋白的产量和纯度。实验得出三种菌体在1／2NB培养基，28℃，200r／min培养三天，蛋白产量均较高，高速离心得到的蛋白收率低于等电点沉淀法，但其纯度较高，满足作为抗原的条件，因此本实验选用了HD-73菌株经高速离心方法得到的特异产Bt Cry1Ac的蛋白作为抗原。 在抗体的制备中选用将蛋白直接溶于液体缓冲液中直接进行免疫和将蛋白电泳后切下蛋白带进行免疫大白兔。结果表明，切胶免疫的大白兔其免疫反应较溶液免疫的强烈，但抗血清效价较低。实验中选择了溶液免疫得到的抗体作为第一抗体，将抗体与常用的腊根过氧化物酶(horseradish peroxidase，HRP)标记得到第二抗体即酶标抗体，从而建立了抗体-抗原-酶标抗体的双抗夹心ELISA方法，其检测限为0.005μg／mL，线性范围为0.015-0.25μg／mL。在与其它5种Bt Cry蛋白(Cry1C、Cry2A、Cry3A、Cry3Bb1、Cry9C)没有交叉反应。 在检测六种棉花样品(GK-12、GK-22、NON、抗虫棉、双价棉、石远321)时，采用了三种提取缓冲液(Tris-硼酸、Na_2CO_3缓冲液、PBST)提取样品中的Bt蛋白，得出Tris-硼酸缓冲液是较佳的Bt蛋白的样品提取缓冲液。采用聚合酶链式反应(polymerase chain reaction，PCR)方法来验证所建立的ELISA实验检测样品结果的正确性和可行性，选用了常用的花椰菜花叶病毒的35S(Cauliflower Mosaic Virus，CaMV-35S)、Cry1Ab+Cry1Ac、Cry1Ac(检测基因)和18S rRNA(内参基因)的特异性引物对六种棉花样品中的DNA进行PCR扩增，经琼脂糖凝胶电泳观察，结果表明GK-12、GK-22、抗虫棉、双价棉和石远321为转Bt Cry1Ac基因棉花，NON为非转基因棉花。
The goal of this research was to study on the preparation of antigen of Bt (Bacillus Thuringiensis) insecticidal crystal protein and a sandwich enzyme-linked immunosorbent assay (ELISA) was developed and performed to determine the Bt Cry1 Ac protein in cotton.Three species of Bt (HD-1, HD-2, HD-73) were cultured in two conditions (28 ℃, 150r/minand28 ℃, 200r/min)by two kinds of medium (1/2LB,1/2NB), the thallus were treated with lysis and then were dealed with isoelectric point deposition and high-speed centrifugation respectively. Result indicated that the yield of Bt protein was very high when three species were fermented in 1/2NB medium, 28 ℃, 200r/min for 3 days. Although the quantity of Bt protein for high-speed centrifugation was lower than that for isoelectric point deposition, it had,higher purity. Therefore, the Cry1Ac protein which produced only enriched by HD-73 was selected as antigen for developing of polyclonal antibodies.In this experiment two methods for antibody preparation were adopted, the first one used solubilization of protein sample in buffer as immunogen and the second one used emulsification of protein band obtained from polyacrylamide gels as immunogen. Results showed that the rabbits immunized by the second method showed intenser immune response in the course of immunity, however, the lower-titer antibodies was obtained compared with the first ones and antibodies from animals immunized by firsr one were chosen for ELISA and acted as the first antibody. The secondary antibody was labeled with horseradish peroxidase (HRP). The limit of detection was 0.005 ug/mL with a linear range approxiamately from 0.015-0.25 μg/ml and there was no cross-reaction with other Bt Cry proteins (Cry1C, Cry2A, Cry3A, Cry3Bb1, Cry9C).The assay was applied to determine six different cottons (GK-12, GK-22, NON, insect-resistant cotton, bivalent transgenic cotton, shiyuan 321). Three different buffers (Tris-borate buffer, sodium carbonate buffer, PBST buffer) were used for protein extraction and results showed Tris-borate buffer was the best. Meanwhile, for the validation of the ELISA test, cotton leaf samples were analyzed by polymerase chain reaction (PCR) method. The primer pairs CaMV-35S (Cauliflower Mosaic Virus, CaMV-35S), Cry1Ab + Cry1Ac, Cry1Ac as detection gene and 18S rRNA as inner gene were selected, and results by agarose gel electrophoresis showed that GK-12, GK-22, insect-resistant cotton, bivalent transgenic cotton and shiyuan 321 were positive samples and NON was negative
|
