家蚕质多角体病毒RDRP的免疫电镜定位研究

Study on the location of the RNA-dependent RNA polymerase gene from Bombyx mori cytoplasmic polyhedrosis virus

以原核表达BmCPV RDRP重组蛋白为抗原制备的兔抗血清为一抗,直径15nm山羊抗兔IgG 胶体金为二抗。应用3%多聚甲醛 0 1%戊二醛混和固定液、K4M低温包埋剂和紫外光聚合制备的样品进行免疫电镜观察,结果感染BmCPV(C)病蚕的中肠柱状细胞中,游离和病毒多角体中BmCPV的病毒粒子均能结合胶体金颗粒,平均标记率为25%左右,认为BmCPV(C) RDRP基因编码的蛋白应为BmCPV病毒的结构蛋白,位于病毒的衣壳上。其中一抗的使用浓度为1∶200,二抗的使用浓度为1∶40,可以获得较好的免疫标记效果和较少的非特异性标记。

The first antibody was rabbit antibody prepared using recombinant protein and the second antibody was 15 nm colloidal gold labeled goat anti rabbit IgG. The third instar silkworms were infected by BmCPV in the midgut reer part and then immobilized by 3% polyformaldehyde-0 1% glutaraldehyde mixture, low-temperature embedded by K_4M before it was ultraviolet polymerized and prepared as antigen plate using ultrathin sectioning and immuno marked by 1∶200 first antibody and 1∶40 second antibody. Colloidal gold bound mostly to the virion that dispersedly located in virus generation matrix in the pillar cell of the midgut of silkworms and that located in polyhedron with a higher marking ratio while the average marking ration is about 25%.