抗牛胰蛋白酶(TRY)抗体的制备和对杀虫剂抗性的初步检测

Preparation of polyclonal antibody against bovine trypsin and the preliminary detection of the insecticide resistance in mosquitoes

目的制备抗牛胰蛋白酶(TRY)多克隆抗体检测杀虫剂抗性,为建立蚊媒菊酯类杀虫剂抗性的现场检测方法奠定基础。方法用牛TRY配成200mg/mL的储备液,与弗氏完全佐剂等体积混合均匀后,按照每只BALB/c小鼠20μg的量背部皮下多点注射,每隔15d加强免疫1次。25d后尾部采血,用ELISA方法测定抗体滴度。45d后眼球采血分离血清,标记分装冷藏备用。用此抗体Western blot研究抗性和敏感品系淡色库蚊成蚊粗提蛋白,观察显色结果。结果所得多克隆抗体小鼠抗牛TRY抗血清滴度达到1:10000,ELISA重复性稳定。在抗TRY抗血清组抗性品系出现约28kD大小的显色带。结论成功制备小鼠抗TRY多克隆IgG抗血清,其检测蚊虫抗性是可行的,值得进一步研究。

To prepare the polyclonal antibody against bovine trypsin （TRY） and its use to detect the insecticide resistance in mosquitoes, the bovine trypsin equally mixed with complete Freund＇s adjuvant （CFA） was used to immunize BALB/c mice with multi-point subcutaneous injection of antigen on back of mice at a dosage of 20μg per mouse, and mice were re-immunized 15 days later. Twenty five days after the last immunization, blood was taken from tail vein, and serun antibody titer was determined by ELISA assay, and 45 days after the last imminization blood was taken post-bulbarily for the investigation of insecticide resistance and the crude proteins from the sensitive strains of adult mosquitoes by Western blotting. It was demonstrated that the titer of the polyclonal antibody obtained was attained up to 1 ： 10 000, and a tape of protein about 28 kDa on the NC membrane was observed in the insectcide resistant strains ,when the mouse anti-TRY antibody was used as the first antibody in the Western blot assay. This protein was coincident with the protein of NYD-Tr. These results indicate that the polyclonal mouse anti-TRY antibody thus prepared can be used to detect the insecticide resistance in mosquitoes