棉铃虫单核衣壳核型多角体病毒HaSNPV orf101的原核表达及抗体制备

[目的]摸索棉铃虫单核衣壳核型多角体病毒(HaSNPV)orf101编码蛋白原核表达条件并制备其多克隆抗体。[方法]根据HaS-NPVorf101序列,设计引物,引入适当的酶切位点,利用PCR扩增出基因片段并将其克隆至pGEM-TEasy载体,然后亚克隆至原核表达载体pGEX-KG,检测在不同条件下融合蛋白的表达产量及其折叠性质。原核表达产物经SDS-PAGE分离纯化,免疫新西兰大白兔制备多克隆抗体。[结果]在IPTG诱导的条件下,融合蛋白GST-HA101可在大肠杆菌BL21中以包涵体形式高效表达,表达产物的大小为55.8 kDa。制备的多克隆抗体经1∶8 000倍稀释后用于Western Blot分析,获得特异性显色信号。[结论]原核表达蛋白及其多克隆抗体的获得为深入研究Ha101的基因功能打下了基础。

Prokaryotic Expression of orf101 from HaSNPV and the Antibody Preparation

[Objective] This study aimed to seek the optimum expression conditions of HaSNPV orf101 coding protein in prokaryotic cells,and to prepare the anti-HA101 multiclonal antibodies.[Method] The orf101 was amplified by PCR from the genome DNA of HaSNPV.The PCR products was cloned into pGEM-T Easy vector,then it was subcloned into the GST-fused expression vector pGEX-KG.The expressed yield and folded property of the GST fusion protein were detected in different conditions.The protein was purified by SDS-PAGE.New Zealand white rabbit was immunized with the purified protein.[Result] After IPTG induction,the E.coli BL21 containing recombinant plasmids expressed a considerable fusion protein of GST-HA101 in the form of inclusion body.The molecular weight of the fusion protein was 55.8 kDa,which was in agreement with the anticipation.Western blot analysis using the multiclonal antibodies with 1∶8 000 diluted showed a specific reaction to GST-HA101 fusion protein expressed in E.coli BL21.[Conclusion] The prokaryotic expression protein and its antibodies made it possible to analyze the function of HA101 in infected insect cells in the future.