大豆花叶病毒CP基因原核表达与抗血清制备

Prokaryotic Expression of SMV CP Gene and Preparation of the Antibody Against CP

根据已报道的大豆花叶病毒(SMV)序列设计引物,扩增SMV的外壳蛋白(CP)基因,序列同源性分析结果表明,与目前已报道的SMV不同分离物CP基因核苷酸序列同源性为85.0%～96.8%,相应推测的氨基酸序列同源性为87.9%～98.9%。将CP基因插入原核表达载体pSBET后在大肠杆菌BL21(DE3)PlysS中诱导表达。通过12%SDS-PAGE和5%～20%梯度SDS-PAGE两次制备电泳纯化诱导产物,免疫家兔获得抗CP血清,Western blot分析对CP具有高度特异性。硫酸铵沉淀法与Protein A-Red Sepharose亲和层析相结合提取IgG,获得效价达1∶3800的一抗,可用于田间SMV病样检测。

Specific primer was designed to amplify SMV CP gene. The CP gene was then inserted into pSBET and expressed in Escheriehia coli BL21 （DE3） plys S strain. It shared 85,0% - 96.8% nucleotide acids identities and 87.9% - 98.9% amino acids identities with the sequences of CP genes isolates reported in the world. The object protein was purified by 12% SDS-PAGE firstly and subsequently 5% - 20% gradient SDS-PAGE. The antiserum against the CP was raised in rabbit and its specificity was confirmed by Western blot analysis. IgG against SMV-CP was purified by ammonium sulfate sedimentation and protein A-Red Sepharose affinity chromatography and antiserum with value of 1 ： 3 800 was obtained. The lgG prepared in this study could be suitable for SMV detection.