| 曾智，王梅，肖虎松，钟日超.球孢白僵菌CDEP2基因原核表达、蛋白纯化及多克隆抗血清制备[J].广东农业科学,2013,40(16):131-133 |
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球孢白僵菌CDEP2基因原核表达、蛋白纯化及多克隆抗血清制备 |
| Prokaryotic expression, protein purification and polyclonal antiserum preparation of Beauveria bassiana CDEP2 Gene |
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| DOI： |
| 中文关键词: 类枯草杆菌蛋白酶 原核表达曰融合蛋白 多克隆抗血清 |
| 英文关键词: subtilisin-like protease prokaryotic expression fusion protein polyclonal antiserum |
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| 中文摘要: |
| 类枯草杆菌蛋白酶是昆虫致病真菌重要的毒力因子，利用合成的特异性引物通过RT-PCR 扩增了球孢白僵菌类枯草杆菌蛋白酶CDEP2 基因。将其与质粒pET28a连接，构建重组原核表达载体pET28-CDEP2，转化大肠杆菌BL21（DE3），并用IPTG进行诱导表达。SDS-PAGE检测His-CDEP2 融合蛋白以包涵体方式大量表达并对其进行纯化。用SDS-PAGE 分离纯化的CDEP2蛋白免疫家兔，制备了多克隆抗血清。Western blotting 免疫印迹检测分析表明，该抗血清对CDEP2蛋白有特异性识别能力，可用于对CDEP2蛋白功能的进一步研究。 |
| 英文摘要: |
| Subtilisin-like protease was the most important factor for the insecticidal activity of entomopathogenic fungi. Using the specificity primers, a cDNA encoding the subtilisin-like protease CDEP2 gene was amplified from Beauveria bassiana by RT-PCR. The recombination prokaryotic expression plasmid pET28-CDEP2 was constructed by subcloning the maturation CDEP2 protein gene encoding sequence into the vector pET28a. It was transformed into E.coli BL21 (DE3) and induced to express by IPTG. SDS-PAGE results showed that the His-CDEP2 fusion protein was high express in the form of inclusion body and purified by affinity chromatography. The rabbits were immuned by the purified CDEP2 protein to produce polyclonal antiserum. Western blotting analysis indicated that the antiserum could react with the corresponding protein, and was suitable to be used for further analysis of CDEP2 protein. |
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