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目的利用活体电穿孔法将携带有棉铃虫精氨酸激酶(HarmAK)基因的表达质粒导入昆明小白鼠制备抗体。方法在构建重组质粒pcDNA3-HarmAK并测序验证序列的正确性后,利用活体电穿孔辅助核酸免疫的方法将重组质粒pcDNA3-HarmAK导入到小鼠体内,免疫5次后,用ELISA检测确定抗体的效价,并对抗体的特异性进行Western blot法检测。结果小鼠抗HarmAK血清的效价达到1∶40 000,Western blot法检测的结果显示此抗血清能与融合蛋白His-HarmAK和棉铃虫总蛋白中的AK蛋白特异性结合。结论采用活体电穿孔辅助核酸免疫的方法制备了高滴度和特异性的小鼠抗HarmAK的抗体。
OBJECTIVE To introduce the expression plasmid carrying the HarmAK gene of Helicoverpa armigera into Kunming mice by in vivo electroporation. Methods The recombinant plasmid pcDNA3-HarmAK was constructed and sequenced. After the recombinant plasmid pcDNA3-HarmAK was introduced into mice, the recombinant plasmid pcDNA3-HarmAK was introduced into the mouse and the antibody was determined by ELISA Price, and the specificity of the antibody Western blot test. Results The titer of mouse anti-HarmAK serum reached 1:40 000. The result of Western blot showed that this antiserum could specifically bind to the AK protein in His-HarmAK and Helicoverpa armigera proteins. Conclusion The mouse anti-HarmAK antibody with high titer and specificity was prepared by in vivo electroporation-assisted nucleic acid immunization.