桑树皱叶病毒外壳蛋白基因的原核表达和抗体制备及应用

Prokaryotic Expression, Antibody Preparation and Application of Coat Protein Gene of Mulberry Crinkle Leaf Virus

桑树皱叶病毒(mulberry crinkle leaf virus,MCLV)是近年从感病桑树中分离鉴定的一种新的桑树病毒,该病毒属于双生病毒科。由于MCLV外壳蛋白基因(cp)序列中含有较多大肠埃希菌(Escherichia mori)BL21(DE3)的稀有密码子而限制了其在大肠埃希菌中的表达。通过人工合成DNA的方法对MCLV cp基因序列进行优化,将其中40个大肠埃希菌稀有密码子变换成同义偏爱密码子,构建优化后的MCLV cp基因的原核表达载体p GEX4T-MCLV CP并转化大肠埃希菌BL21(DE3)菌株,经0.1 mmol/L IPTG诱导及SDS-PAGE电泳鉴定,实现了融合蛋白GST-MCLV CP在大肠埃希菌BL21(DE3)中高效表达。通过SDS-PAGE纯化后切胶回收原核表达的融合蛋白GST-MCLV CP,以其为抗原免疫新西兰大白兔制备MCLV CP的多克隆抗体,经间接ELISA法测定该抗血清的效价为1∶4 096,Western blot检测其能与MCLV发生特异性反应。用制备的抗血清对MCLV感染阳性的桑叶样品进行间接ELISA检测,其检出率仅为25%,但将桑叶样品的粗汁液煮沸处理后能显著提高MCLV的检出率,检出率达83.3%。

Mulberry crinkle leaf virus（MCLV）,which was identified from the diseased mulberry in recent year,is a novel member of the family Geminiviridae.The coat protein gene（cp）of MCLV is expressed poorly in prokaryotic expression system because it contains high proportion of rare codons of Escherichia coli BL21（DE3）.In this study,cp gene of MCLV was optimized via artificial DNA synthesis,in which a total of 40 rare codons were substituted with synonymous codons preferred by E.coli BL21（DE3）.The optimized MCLV cp gene was cloned for construction of recombinant plasmid p GEX4T-MCLV CP.SDS-PAGE assay indicated that the fusion protein,GST-MCLV CP,was over-expressed in E.coli BL21（DE3）after induction with 0.1 mmol/L IPTG.GST-MCLV CP fusion protein expressed in E.coli was purified and recovered from SDS-PAGE and used as antigen to generate anti-MCLV polyclonal antiserum in New Zealand white rabbit.The prepared antiserum titer was 1∶4 096 measured by indirect enzyme-linked immunosorbent assay（ELISA）.Analysis by Western blotting indicated that the prepared antibodies could specifically bind MCLV.When the prepared antiserum was used in examining MCLV-infected mulberry leaf samples through indirect ELISA,the detection rate of MCLV was only 25%.However,after the crude sap of MCLV-infected mulberry leaf samples was boiling-treated,the detection rate was increased to 83.3%.