BACULOVIRUS-MEDIATED EXPRESSION OF A MANDUCA-SEXTA CHITINASE GENE - PROPERTIES OF THE RECOMBINANT PROTEIN
BACULOVIRUS-MEDIATED EXPRESSION OF A MANDUCA-SEXTA CHITINASE GENE - PROPERTIES OF THE RECOMBINANT PROTEIN
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DOI：
10.1016/0965-1748(94)00070-x
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发表时间：
1995-02-01
影响因子：
3.8
通讯作者：
KRAMER, KJ
中科院分区：
文献类型：
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作者：
GOPALAKRISHNAN, B;MUTHUKRISHNAN, S;KRAMER, KJ
我们构建了一个重组的非封闭杆状病毒，苜蓿银纹夜蛾核型多角体病毒（AcMNPV），含有一个1.8 kb的DNA片段，从烟草天蛾（烟草天蛾）几丁质酶cDNA的多角体蛋白基因启动子的控制下。当草地贪夜蛾（秋粘虫）细胞（SF 9）感染这种重组病毒，具有85 kDa的表观分子量的蛋白质分泌到培养基中。该蛋白水解甲壳素并与抗M. Sexta蜕皮液几丁质酶衣霉素处理感染的SF 9细胞和随后的蛋白质印迹分析表明，分泌的酶是一种糖蛋白。GC-MS分析表明，糖蛋白的质量约占25%。重组几丁质酶和蜕皮液酶是无法区分的N-末端测序，聚丙烯酰胺凝胶电泳和碳水化合物分析，表明重组蛋白是相似的，如果不是相同的，蜕皮液酶。重组几丁质酶在SF 9、SF 21和粉纹夜蛾（Hi-5）细胞系中的表达量分析表明，表达量大小顺序为Hi-5 > SF 21> SF 9。注射几丁质酶后，血淋巴中有几丁质酶积累。sexta和S.用重组病毒感染草地贪夜蛾幼虫。S.用重组病毒感染的草地贪夜蛾四龄幼虫比用野生型病毒感染的昆虫短约20小时。这些结果支持昆虫几丁质酶具有增强昆虫病原菌杀虫活性的潜力的假设。
We constructed a recombinant nonoccluded baculovirus, Autographa californica nuclear polyhedrosis virus (AcMNPV), containing a 1.8 kb DNA fragment from a Manduca sexta (tobacco hornworm) chitinase cDNA under the control of the polyhedrin gene promoter. When Spodoptera frugiperda (fall armyworm) cells (SF9) were infected with this recombinant virus, a protein with an apparent molecular weight of 85 kDa was secreted into the culture medium. This protein hydrolyzed chitin and cross-reacted with a polyclonal antibody to M. sexta molting fluid chitinase. Tunicamycin treatment of infected SF9 cells and subsequent western blot analysis indicated that the secreted enzyme was a glycoprotein. GC-MS analysis revealed that carbohydrate accounted for approximately 25% of the mass of glycoprotein. The recombinant chitinase and the molting fluid enzyme were indistinguishable by N-terminal sequencing, polyacrylamide gel electrophoresis and carbohydrate analysis, indicating that the recombinant protein was similar, if not identical, to the molting fluid enzyme. Analysis of the expression level of recombinant chitinase in SF9, SF21 and Trichoplusia al (Hi-5) cell lines showed that the yields were in the order Hi-5 > SF21 > SF9. Chitinase accumulated in hemolymph after injection of fourth instar M. sexta and S. frugiperda larvae with recombinant virus. The median time for mortality of S. frugiperda fourth instar larvae infected with the recombinant virus was approximately 20 h shorter than that for insects infected with a wild type virus. The results support the hypothesis that insect chitinase has potential to enhance the insecticidal activity of entomopathogens.