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当前位置: 首页 > 文章 > 棉铃虫核型多角体病毒几丁质酶的原核表达及其协同增效作用 西北农林科技大学学报(自然科学版) 2011,39 (2) 87-91
Position: Home > Articles > Expression of HearNPV chitinase gene in E.coliand its enhancement on HearNPV virulence Journal of Northwest A & F University(Natural Science Edition) 2011,39 (2) 87-91

棉铃虫核型多角体病毒几丁质酶的原核表达及其协同增效作用

作  者：
项林平;刘小芸;田强强;刘强;吕婷婷
单  位：
植保资源与病虫害治理教育部重点实验室西北农林科技大学陕西杨凌;西北农林科技大学林学院
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
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(Chi A),利用表达产物进行病毒增效作用研究,以明确杆状病毒Chi A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Chi A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-Chi A和pET-32a-Chi A。重组载体转化相应菌株并进行诱导表达,发现pMAL-p2x-Chi A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-Chi A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-Chi A在BL21(DE3)中的表达产物作为增效蛋白,当表达的可溶性HearNPV Chi A的添加水平为1.0μg/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 Chi A,对HearNPV具有明显的增效作用。
译  名：
Expression of HearNPV chitinase gene in E.coliand its enhancement on HearNPV virulence
作  者：
XIANG Lin-ping1,LIU Xiao-yun1,TIAN Qiang-qiang2,LIU Qiang2,Lü Ting-ting1,WANG Dun1 (1 Key Laboratory of Plant Protection Resources and Pest Management,Ministry of Education,Yangling,Shaanxi 712100,China;2 College of Forestry,Northwest A&F University,Yangling,Shaanxi 712100,China)
关键词：
HearNPV;chitinase;prokaryotic expression;virulence enhancement
摘  要：
【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.

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