Expression of non-structural protein NS3 gene of Bombyx mori densovirus (China isolate).
The invertebrate parvovirus Bombyx mori Densonucleosis Virus type 3 (China isolate), named BmDNV-3, is a kind of bidensovirus. It is a new type of virus with unique replication mechanisms. To investigate the effects of the NS3 gene during viral DNA replication, a pair of primers was designed for amplifying NS3 gene of Bombyx mori densovirus (China isolate). Gene NS3 amplified was cloned into a prokaryotic expression vector pET-30a and the donor plasmid pFastBacHTe, respectively. The NS3 protein was expressed in Escherichia coli BL21. The pFastBacHTe-NS3 was transformed to E. coli DH10Bac. The recombinant bacmid baculoviruses (rBacmid-EGFP-NS3) isolated from the white colonies were transfected into BmN-4 cells using a transfection reagent. BmN-4 cells were infected with recombinant virus to express fusion proteins. The expression of fusion protein around 30 kDa in E. coli BL21 was identified by SDS-PAGE, Western blotting, and mass spectrometry. The expressed NS3 protein by B. mori nucleopolyhedrovirus bacmid system was confirmed by Western blotting using an anti-NS3 polyclonal antibody. And about 45 kDa protein was found. The expressed fusion protein was smaller than the expected size of EGFP-NS3, 55 kDa. Western blotting analysis indicated that EGFP-NS3 protein was expressed in infected larvae with smaller molecular size.
Yin
Huijuan
H
Institute of Life Sciences, Jiangsu University, 301# Xuefu Road, Zhenjiang 212013, China.
Yao
Qin
Q
Guo
Zhongjian
Z
Bao
Fang
F
Yu
Wei
W
Li
Jun
J
Chen
Keping
K
D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx / Q000254:growth & development / Q000821:virology*; D002460:Cell Line; D002681:China; D004274:DNA, Recombinant / Q000235:genetics; D017990:Densovirus / Q000235:genetics* / Q000302:isolation & purification; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics; D007814:Larva / Q000254:growth & development / Q000821:virology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics*
D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins