转Bt基因植物中杀虫蛋白的酶联免疫检测技术研究Ⅱ.Bt杀虫晶体蛋白抗体的制备

用碳酸钠裂解液(50mmol/LNa2CO3,50mmol/LEDTA,pH10)从BacillusthuringiensisHD-1的孢晶混合物中分离提取杀虫蛋白的基础上,进一步用聚丙烯酰胺凝胶制备电泳技术纯化杀虫蛋白,获得了高纯度的杀虫蛋白作为抗原,用于免疫家兔制备抗体。比较了不同抗原注射剂量的免疫效应和免疫过程中(不同注射时间)抗体生成量的变化。结果表明,供试的二种抗原注射剂量对抗体的效价无明显影响,随着免疫次数的增加抗体效价不断上升,最终获得了用琼脂双扩散测定效价为1/120的抗血清。Bt杀虫蛋白的免疫原性低。抗血清中的抗体免疫球蛋白(Ig),首先经硫酸铵分级沉淀粗提,再用DEAE-52纤维素柱层析提取纯化,获得了高纯度的IgG,为抗体的酶标记和ELISA检测杀虫蛋白等研究提供了高特异性的Bt抗体。研究的结果为Bt菌HD-1杀虫蛋白抗体的制备提供了依据。

Studies on the Enzyme-linked Immunosorbent Assay for Detection of Bacillus thuringiensis Insecticidal Protein Expressed in Transgenic Plants Ⅱ.Preparation of Polyclonal Antibody to Bt Insecticidal Protein

The Bacillus thuringiensis HD-1 mixture of endospore and crystal protein were treated with Na_2CO_3 solution (50?mmol/L Na_2CO_3, 50?mmol/L EDTA, pH 10) to get the insecticidal protein. The extracted protein was purified by SDS-PAGE preparation electrophoresis. The highly purified protein with molecular weight of 130?kD was used as an antigen. The antigen was emulsified with Freud's complete adjuvant (first injection) or Freud's incomplete adjuvant (second to fourth injection) and injected into New Zealand white rabbits. The first injection was subcutaneous injection on the back of rabbit. The second to fourth booster injections were intramuscular injection on legs. After 10 days of each injection. The rabbits were bled and check the titre using agar gel double diffusion. Two immunizing dosage of antigen (750?μg and 375?μg, each rabbit and time) were tested to assay the effect of antigen dosage on antibody production. After 10 days of the fourth injection, the immunized rabbits were bled thorough heart. The antiserum from blood of immunized rabbit was collected and stored at 4?℃ or -20?℃. The results show no effect of the tested immunizing dosage on the quality and titre of antibody. The titre of antibody increases with the time of booster injection and reaches 1/120 by using agar gel double diffusing test. Purification of antibodies is conducted by ammonium sulfate precipitation technique and DEAE cellulose chromatographic fractionation. High quality IgG is obtained and used to be labeled by enzyme. The research results provide scientific base.