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
发布时间：2019-06-04 12:30
【摘要】：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%。
[Abstract]:Mulberry wrinkled leaf virus (mulberry crinkle leaf virus,MCLV) is a new mulberry virus isolated and identified from susceptible mulberry in recent years, which belongs to Geminiviridae. The expression of MCLV coat protein gene (cp) contains more rare codon of Escherichia coli (Escherichia mori) BL21 (DE3), which limits its expression in Escherichia coli. The sequence of MCLV cp gene was optimized by synthetic DNA, and 40 rare codon of Escherichia coli were transformed into synonymous preference codon. The optimized prokaryotic expression vector p GEX4T-MCLV CP of MCLV cp gene was constructed and transformed into Escherichia coli BL21 (DE3) strain. It was induced by 0.1 mmol/L IPTG and identified by SDS-PAGE electrophoresis. The fusion protein GST-MCLV CP was highly expressed in Escherichia coli BL21 (DE3). The polyclonal antibody of MCLV CP was prepared by immunizing New Zealand white rabbits with the fusion protein GST-MCLV CP, which was purified and purified by SDS-PAGE, and the titer of the antiserum was determined to be 1:4 096 by indirect ELISA assay. Western blot detected that it could react specifically with MCLV. The indirect ELISA detection of MCLV positive mulberry leaf samples was carried out with the prepared antiserum, and the detection rate was only 25%. However, the detection rate of MCLV was significantly increased by boiling the crude juice of mulberry leaf samples, and the detection rate was 83. 3%.
【作者单位】： 江苏科技大学生物技术学院;农业部蚕桑遗传改良重点实验室中国农业科学院蚕业研究所;
【基金】：江苏省自然科学基金项目(No.BK20151320)
【分类号】：S888.71
，
本文编号：2492724
[Abstract]:Mulberry wrinkled leaf virus (mulberry crinkle leaf virus,MCLV) is a new mulberry virus isolated and identified from susceptible mulberry in recent years, which belongs to Geminiviridae. The expression of MCLV coat protein gene (cp) contains more rare codon of Escherichia coli (Escherichia mori) BL21 (DE3), which limits its expression in Escherichia coli. The sequence of MCLV cp gene was optimized by synthetic DNA, and 40 rare codon of Escherichia coli were transformed into synonymous preference codon. The optimized prokaryotic expression vector p GEX4T-MCLV CP of MCLV cp gene was constructed and transformed into Escherichia coli BL21 (DE3) strain. It was induced by 0.1 mmol/L IPTG and identified by SDS-PAGE electrophoresis. The fusion protein GST-MCLV CP was highly expressed in Escherichia coli BL21 (DE3). The polyclonal antibody of MCLV CP was prepared by immunizing New Zealand white rabbits with the fusion protein GST-MCLV CP, which was purified and purified by SDS-PAGE, and the titer of the antiserum was determined to be 1:4 096 by indirect ELISA assay. Western blot detected that it could react specifically with MCLV. The indirect ELISA detection of MCLV positive mulberry leaf samples was carried out with the prepared antiserum, and the detection rate was only 25%. However, the detection rate of MCLV was significantly increased by boiling the crude juice of mulberry leaf samples, and the detection rate was 83. 3%.
【作者单位】： 江苏科技大学生物技术学院;农业部蚕桑遗传改良重点实验室中国农业科学院蚕业研究所;
【基金】：江苏省自然科学基金项目(No.BK20151320)
【分类号】：S888.71
，
本文编号：2492724
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