[Cloning and expression of a novel ubiquitin fusion gene Uba256].
Spodoptera litura nucleopolyhedrovirus (SpltMNPV) Uba256 gene is the only Ub-gp37 fusion gene in the genome of insect viruses. With the specific primers designed for Uba256 gene that was reported recently, the coding regions of Uba256, N-terminal ubiquitin and C-terminal GP37 that lacking the signal sequence, were amplified from SpltMNPV genomic DNA by PCR. The Uba256 coding region was expressed using the expression vector pBV220, and a band of 38 kD was detected with Western blot analysis, indicating that ubiquitin-GP37 fusion protein did not undergo post-transcriptional processing in E. coli. The ubiquitin and GP37 coding region were highly expressed, respectively, using pQE30 expression vector. Antibody to the purified ubiquitin reacted not only to the recombinant ubiquitin but also to bovine ubiquitin, and an antibody to the purified GP37 also reacted to the recombinant GP37. The results indicated that the purified ubiquitin and GP37 retained their antigenicity. Western blot analysis results of SpltMNPV-infected Sl-zsu-1 cells revealed that the intact Uba256 was processed in this insect cell line to yield free ubiquitin and GP37 protein.
Li
Zhao-Fei
ZF
State Key Laboratory for Biocontrol, Institute of Entomology, Zhongshan University, Guangzhou 510275, China. Ls12@zsu.edu.cn
Li
Chong-Bi
CB
Tang
Mu-Jing
MJ
Yin
Chong
C
Gong
Ying-Xue
YX
Pang
Yi
Y
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D007074:Immunoglobulin G / Q000097:blood; D008297:Male; D017924:Nucleopolyhedroviruses / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology / Q000821:virology*; D014452:Ubiquitins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins