| 摘要: |
| 【目的】在大肠杆菌中表达棉铃虫单粒包埋型核型多角体病毒（HearNPV）几丁质酶（ChiA），利用表达产物进行病毒增效作用研究，以明确杆状病毒ChiA对HearNPV的协同增效作用。【方法】用PCR方法扩增出不含N端信号肽编码序列的ChiA基因片段,分别克隆至原核表达载体pMAL-p2x和pET-32a中,构建重组表达载体，并分别在大肠杆菌TB1和BL21(DE3)中进行融合表达，测定融合蛋白对HearNPV的增效作用。【结果】克隆的基因片段经过测序后连接表达载体，成功构建了重组表达载体pMAL-p2x-ChiA和pET-32a-ChiA。重组载体转化相应菌株并进行诱导表达，发现pMAL-p2x-ChiA在大肠杆菌TB1中的表达量占细菌总蛋白的15.8%，其中20.0%为可溶性表达；pET-32a-ChiA在BL21(DE3)中的表达量占细菌总蛋白的26.8%，其中30.2%为可溶性表达。选用pET-32a-ChiA在BL21(DE3)中的表达产物作为增效蛋白，当表达的可溶性HearNPV ChiA的添加水平为1.0 μg/mL时，其能够显著提高病毒毒力，棉铃虫的死亡率较对照提高36.6%。【结论】 利用大肠杆菌表达的可溶性HearNPV ChiA，对HearNPV具有明显的增效作用。 |
| 关键词: 棉铃虫核型多角体病毒 几丁质酶 原核表达 增效作用 |
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| 基金项目:国家自然科学基金项目(30872033);教育部新世纪优秀人才支持计划项目（NCET-06-0867） |
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| Expression of HearNPV chitinase gene in E.coli and it enhancement on HearNPV virulence |
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XIANG Linping1, LIU Xiaoyun1, TIAN Qiangqiang,et al2
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1.Key Laboratory of Plant Protection Resources and Pest Management Ministry of Education;2.College of Forestry, Northwest A＆F University
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| Abstract: |
| 【Objective】The study was conducted to investigate the virulence enhancement on Helicoverpa armigera single nucleopolyhedrovirus (HearNPV) by baculovirus chitinase (ChiA).The HearNPV chitinase gene was cloned and expressed in E.coli TB1 and BL21(DE3),respectively.And the bioassay was carried out to evaluate virulence enhancement.【Method】The PCR product of the chitinase gene was cloned into the prokaryotic expression vector pMAL-p2x and the pET-32a,and then expressed in E.coli strains TB1 and BL21(DE3),respectively.The BL21(DE3) products were used for bioassay.【Result】The chitinase (ChiA) was cloned and confirmed by sequencing and then inserted into expression vectors resulting in pMAL-p2x-ChiA and pET-32a-ChiA.The pMAL-p2x-ChiA was expressed with a yield of 15.8% of the total cellular proteins in TB1 while the pET-32a-ChiA was expressed with a higher yield of 26.8% in BL21(DE3).And the soluble products were 20.0% and 30.2% to the fusion proteins expressed in TB1 and BL21(DE3) respectively.The BL21(DE3) product was fed to the third instar larvae of the H.armigera for bioassay of virulence enhancement.The bioassay results showed that the mortality was 36.6% higher than the control virus at a combination of 1.0 μg/mL of soluble ChiA to HearNPV.【Conclusion】The virulence of HearNPV could be enhanced by soluble ChiA expressed in E.coli significantly. |
| Key words: HearNPV chitinase prokaryotic expression virulence enhancement |
