| In humans, the HER-2/neu proto-oncogene is frequently amplified or overexpressed in many types of cancers, including breast, ovary, lung, stomach, and oral cancers, suggesting that HER-2/neu overexpression plays a critical role in the growth of tumors and the development of malignancies. In addition, HER-2/neu overexpression represents a pivotal biological marker that can help to determine more accurately the poor prognosis for individual patients. HER-2/neu oncogenic protein has been identified as a suitable target for tumor vaccine, which would generate an active immune response directed against established tumors. The HER-2/neu oncoprotein extracellular part consists of two cysteine-rich domains and two receptor ligand domains (RLD), which are responsible for the formation of both homo- and heterodimers. In the study, HER-2/neu RLD protein was expressed, purified and mannosylated, which will lay foundation for the research of the new type of mannosylated tumor vaccine.In this study, four subjects were investigated:1 Bioinformatic analysis of HER-2/neu RLD proteinThe biological characters of HER-2/neu RLD protein, including antigenic specificity, CTL peptide epitopes, hydrophilic/ hydrophobic nature and isoelectric point, were analysed by Blast program and computer algorithms, such as BIMAS, SYFPEITHI and Goldkey software.2 Soluble expression and purification of HER-2/neu RLD2HER-2/neu RLD2 cDNA was amplified by PCR, cloned into the prokaryotic expression vector containing thioredoxin A (TrxA) gene and expressed in soluble form as TrxA-RLD2 fusion protein. The co-expression of TrxA and RLD2 was realized by inserting a tandem sequence of stop and start codon between TrxA and RLD2 cDNA. Western-blotting analysis showed that the expressed protein could react with the specific antibody against proto-oncoprotein HER-2/neu. With the applicationof DEAE-Sepharose FF column and cobalt-based affinity resins, the purity of RLD2 obtained was over 90%. The purified protein was further subject to mass spectrography molecular weight monitoring, and the molecular weight of RLD2 protein was identical to the expectation. By using TrxA expression system, the soluble expression of RLD2 protein was obtained with high efficiency, which will make it possible to further study of protein mannosylation.3 Expression, purification and renaturation of HER-2/neu RLD proteinThe two pieces of HER-2/neu RLD cDNA were amplified by PCR respectively, and linked up by using the same cleavage site Kpn I , so that the genetic fragment of cysteine-rich domain between RLDs was deleted. The ligation product was inserted into prokaryotic expression vector pET-28a(+), and expressed as the fusion protein with high level in E.coli. Western-blotting analysis showed that the expression product was recognized by the specific antibody against proto-oncoprotein HER-2/neu, which indicated that the antigen specificity of the fusion protein was retained. SDS-PAGE showed that the fusion protein existed in the form of insoluble inclusion body. After denaturation, purification and renaturation, the fusion protein containing two pieces of HER-2/neu RLD proteins was obtained in soluble form, which will be helpful to the study of the vaccine for the tumors overexpressing HER-2/neu oncoprotein.4 Mannosylation of HER-2/neu RLD2The purified HER-2/neu RLD2 protein was mannosylated by chemical method in vitro. RLD2 neoglycoprotein was identified by MALDI-TOF-MS. With the application of resorcinol-sulfuric acid method, the chemical compounds attached to RLD2 protein were certified to be the sugar molecule. The synthesis of RLD2 neoglycoprotein lays foundation for the study of antigen uptake and presentation mediated by mannose receptor and tumor vaccine.
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