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期 刊 ：植物保护学报 2008年

关键词：粘虫颗粒体病毒；苏云金杆菌；δ-内毒素；酶解活化；

Keywords:Psedualetia unipuncta granulovirus, Bacillus thuringiensis, δ-endotoxin, degradation,

摘 要 ：粘虫颗粒体病毒(Psedualetia unipuncta granulovirus,PuGV-Ps)对苏云金杆菌(Bacillus thuring-iensis,Bt)具有增效作用.为明确其增效机制,采用SDS-PAGE方法研究PuGV-Ps对Bt δ-内毒素的降解活化作用.结果表明,碱性条件下晶体蛋白和PuGV-Ps共同孵育,130kD的δ-内毒素被进一步酶解为分子量为110、87、61、47 kD等多种不同的肽链,其酶解活化程度随缓冲液pH的升高不断加深,在pH值10.7的0.1 mol/L Na₂CO₃缓冲液中,δ-内毒素完全降解,并产生具有一定抗蛋白酶继续降解的分子量为47、60和61 kD的活性片段.用氨苯磺胺偶氮酪蛋白为底物测定PuGV-Ps中总蛋白酶活性的结果表明,PuGV-Ps在pH值为7.38～10.38时均具有蛋白酶活性,且蛋白酶活性随pH升高而显著提高.4种蛋白酶抑制剂均可抑制PuGV-Ps的蛋白酶活性,且以STI的抑制作用最强.SDS-PAGE试验同样显示了STI可以抑制PuGV-Ps对δ-内毒素的酶解活化.

Abstract:Psedualetia unipuncta granulovirus (PuGV-Ps) can enhance the toxicity of Bacillus thuringiensis (Bt）to lepidopterous pests. In this study, the effect of PuGV-Ps on the degradation of δ-endotoxin from Bt was demonstrated. SDS-PAGE analysis showed that a large amount of activated toxin proteins were yielded from δ-endotoxin of Bt incubated with PuGV-Ps under alkaline condition. δ-endotoxin with molecular weights about 130kD was degraded into 47kD to 110kD toxic protein fragments. pH value influenced the degradation effect greatly, which elevated along with the increase of pH value. In buffer of 0.1mol/L Na₂CO₃, pH value 10.7, δ-endotoxin was completely cleaved into 47, 60 and 61kD activated toxins and resisted further degradation. The amount of PuGV-Ps and the time of incubation also influenced the degree of degradation. Total proteolysis of PuGV-Ps was measured by using azocasein under different pH conditions from value 7.38 to 10.38, and the activity of proteolysis was improved following the rising of pH value. All the four kinds of inhibitors influenced the activity of PuGV-Ps proteolysis, among them soybean trypsin inhibitor (STI) made the greatest effect. STI also inhibited the degradation of δ-endotoxin by PuGV-Ps.

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