苹果褪绿叶斑病毒外壳蛋白基因的原核表达及抗血清制备

【目的】为了制备苹果褪绿叶斑病毒(Apple chlorotic leaf spot virus,ACLSV)的抗血清,【方法】将ACLSV外壳蛋白(Coat protein,CP)基因克隆到原核表达载体pEHISTEV和pEHISEGFPTEV,转化大肠杆菌Rosetta,切胶回收诱导表达的CP融合蛋白免疫新西兰长耳兔,制备ACLSV的抗血清。【结果】利用原核表达载体pEHISTEV和pE-HISEGFPTEV在Rosetta中分别表达出了分子质量为25 ku和55 ku的ACLSV CP融合蛋白。切胶回收25 ku的融合蛋白免疫新西兰长耳兔。经ELISA测定,所制备的抗血清效价为1∶1 024,Western blot及组织印迹分析表明抗血清能与ACLSV CP发生特异性的免疫反应。【结论】该血清特异性强,效价高,可用于苹果叶片、果实和枝条等样品中A-CLSV的检测。

Preparation of antiserum to Apple chlorotic leaf spot virus with coat protein expressed in E.coli

【Objective】The objective of this study is to obtain antibody to the CP of Apple chlorotic leaf spot virus(ACLSV) using coat protein(CP) prepared in E.coil.【Method】The CP gene of ACLSV was cloned to prokaryotic expression vectors pEHISTEV and pEHISEGFPTEV,and transformed into cells of E.coil Rosetta,and expressed fusion proteins after induction with IPTG.Antiserum was prepared with fusion protein extracted from the gel.【Result】The fusion protein of 25 ku and 55 ku were expressed from vectors pEHISTEV and pEHISEGFPTEV,respectively.The 25 ku fusion protein of ACLSV CP were used as antigen to prepare antiserum.ELISA results indicated that the titer of resultant antiserum was 1: 1024.The antiserum showed strong positive reaction with coat protein of ACLSV in Western blot and tissue printing assays.【Conclusion】The antiserum prepared in this research had high titer and specificity and can be used in the detection of ACLSV from leaf,fruit and branch samples of apple trees.