Expression of Trichoderma viride endoglucanase III in the larvae of silkworm, Bombyx mori L. and characteristic analysis of the recombinant protein.
Endoglucanase is a part of cellulase which hydrolyzes cellulose into glucose. In this study, we cloned endoglucanase III (EG III) gene from Trichoderma viride strain AS 3.3711 using a PCR-based exon splicing method, and expressed EG III recombinant protein in both silkworm BmN cell line and silkworm larvae with an improved Bac-to-Bac/BmNPV mutant baculovirus expression system, which lacks the chiA and v-cath genes of Bombyx mori nucleopolyhedrovirus (BmNPV). The result showed that around 45 kDa protein was visualized in BmN cells at 48 h after the second generation recombinant mBacmid/BmNPV/EG III baculovirus infection. The enzymes from recombinant baculoviruses infected silkworms exhibited significant maximum enzyme activity at the environmental condition of pH 8.0 and temperature 50°C, and increased 20.94 and 19.13% compared with that from blank mBacmid/BmNPV baculoviruses infected silkworms and normal silkworms, respectively. It was stable at pH range from 5.0 to 9.0 and at temperature range from 40 to 60°C. It provided a possibility to generate transgenic silkworms expressing bio-active cellulase, which can catabolize dietary fibers more efficiently, and it might be of great significance for sericulture industry.
Key Laboratory of Animal Epidemic Etiology and Immunological Prevention of Ministry of Agriculture, Zhejiang University, Hangzhou, 310029, People's Republic of China.
Xing-hua
XH
Li
Peng
P
Zhang
Mei-xian
MX
Wang
Fang
F
Zhou
Firdose Ahmad
FA
Malik
Hua-jun
HJ
Yang
Roy
R
Bhaskar
Jia-biao
JB
Hu
Chun-guang
CG
Sun
Yun-gen
YG
Miao
D000818:Animals; D016367:Baculoviridae / Q000502:physiology; D015153:Blotting, Western; D012831:Bombyx / Q000378:metabolism*; D002460:Cell Line; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D005091:Exons / Q000235:genetics; D015870:Gene Expression; D007438:Introns / Q000235:genetics; D007814:Larva / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism*; D014242:Trichoderma / Q000201:enzymology*
D011994:Recombinant Proteins; D002480:Cellulase