转Bt基因植物中杀虫蛋白的酶联免疫检测技术研究Ⅰ.Bt菌HD-1的杀虫晶体蛋白抗原制备

研究了培养时间对苏云金杆菌(Bacillusthuringiensis)HD-1菌株孢晶混合物的产量、杀虫晶体蛋白裂解液的种类和处理温度与时间对杀虫晶体蛋白提取率及纯度的影响。结果表明BtHD-1菌株在Bt生长繁殖平板培养基上,30℃下培养41～55h均可获得大量孢晶混合物。采用50mmol/LNa2CO3裂解液处理孢晶混合物,可获得纯度高的130kD杀虫晶体蛋白,而用40mmol/LNaOH提取的杀虫晶体蛋白中含有较多杂蛋白。所研究的处理温度和时间对杀虫晶体蛋白提取量和组成无明显影响。这些结果为杀虫晶体蛋白抗原的制备提供了依据。

Studies on the Enzyme-linked Immunosorbent Assay for Detection of Bacillus thuringiensis Insecticidal Protein Expressed in Transgenic PlantsⅠ.Preparation of Bt Strain HD-1 Insecticidal Protein (Antigen)

The effects of cultural time, lysis solution and lytic conditions on production and purity of insecticidal protein from Bacillus thuringiensis strain HD-1 were investigated to get high quality insecticidal protein as a antigen. The results are as follows: The high production of endospore and crystal protein complex can be obtained on Singer's growth and reproduction solid medium plate at 30?℃ and incubation period of 41～55?h. The best lysis solution used to treat the complex of endospore and crystal protein is 50?mmol/L Na_2CO_3. There are lots of complex protein in insecticidal protein when 40?mmol/L NaOH lytic solution is used to lytic complex of endospore and crystal protein. No significant differences in extraction yield and quality of crystal protein (MW: 130?kD) are found with different treatment of temperature and time. The result provides the scientific base for preparation of Bt HD-1 insecticidal protein as an antigen.