Cloning And Expression Of Ubiquitin Gene Of Helicoverpa Armigera Single-nucleocapsid Nucleopolyhedrovirus And Its Antiserum Preparation

Ubiquitin(Ubi) is a highly conserved 76 amino acid protein present in all eukaryotic organisms either as a free molecule or covalently attached to lysines in a wide variety of proteins. The ubiquitin-proteasome pathway(UPP) has important roles in selective protein degradation, especially short-lived function proteins, denaturalized proteins, products of oncogenes, and some regulative factors, etc. So the ubiquitin pathway plays an essential role in DNA repair, progression through the cell cycle, signal transduction, transcriptional regulation, the nuclear transport process, receptor control by endocytosis, the processing of antigens in the immune system, pathological alterations and programmed cell death.Ubiquitin-related proteins and ubiquitined vims proteins have been found in some plant viruses and animal viruses. Host Ubi molecules have been found covalently linked to some plant viruses; A lipid modified Ubi is packaged into particles of African swine fever vims, vaccinia vims and Herpes simplex vims. The infected cell protein 0 of HSV-1 can act as a unimolecular E3 Ubi ligase and contains two E3 ligase sites specific for different E2 Ubi-conjugating enzymes; A putative causal link between human immunodeficiency vims type 1 budding and Ubi was established by showing that depletion of the intracellular pool of free Ubi inhibits the vims budding. Baculovimses and entomopoxvirus are the viruses which have been reported so far encoding homologs of ubiquitin. All these viral Ubi genes encodes an N-terminal Ubi sequence and 1-256 amino acids C-terminal peptides. Most of the residues known to be essential for Ubi function have been conserved in the viral variant. But the function of these homologs is unknown, and the research of the function is underway.The ubiquitin gene of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) was amplified by PCR.Nucleotide analysis showedthat it was 252bp,encoded 83 amino acids with a predicted size of 9.24kDa. The PCR product containing ubiquitin gene was inserted into pET-28a(+) expressive vector. The Ubi-His-tag fusion protein was expressed in E.coli BL21(DE3). The highest expression quantity reaches to 20%. The fusion protein was identified by Western-blot with His-tag antibody. The expression condition including the content of IPTG and the inducing time were optimized to achieve an optional expression. The best expression time is 1 hour, and when the density of IPTG is l.Ommol/L, the fusion protein expression quantity has reached in a big way. The fusion protein was purified using the Ni-NTA resin column. SDS-PAGE analysis showed that the purity may reach above 90%.The antibody anti the HaSNPV ubiquitin was preparated in rabbit with purified ubiquitin fusion protein,and the further study on the function of HaSNPV UBI is underway.