ABSTRACT
BACKGROUND:RACK1 is strongly associated with the occurrence and development of oral squamous cel carcinoma. However, the occurrence and development of tumor do not depend on a gene or protein, but a long-term complex process of a network structure of multiple genes and multiple molecules, multi-step, multi-stage joint action. Synergism between tumor genes promotes the formation and development of tumor cel s. Therefore, we cannot limit on a single gene or protein to discover the action mechanism of oral squamous cel carcinoma, but should pay attention on signaling network path related to differential protein or gene, investigate the alterations in related protein or gene expression in the whole signaling pathway, and analyze the action mechanism of the interaction of these molecules. OBJECTIVE:To screen differential genes related to oral squamous cel carcinoma, construct an interaction network through bioinformatics using STRING database, and provide clues for future tests. METHODS:In accordance with our previous classic proteomics results and microarray results of oral squamous cel carcinoma, genes with consistent expression and big differences were selected as differential genes. The differential genes were inputted into the database of STRING to find the possible relationship among the protein subunits and to construct network structure of their interaction. RESULTS AND CONCLUSION:The 19 differential proteins of oral squamous cel carcinoma construct a complicated net work, and the differential proteins interact through these networks. GNB2L1-encoded RACK1 is a node protein and interacts with other differential proteins via WD40 repeated protein (number COG2319) andβ-G protein subunit (number KOG0279). WD40 repeated protein (number COG2319) interacts with 5 differential proteins directly and constructs 10 interacting pathways.β-G protein subunit (number KOG0279) interacts with 8 differential proteins directly, which has 11 interacting pathways. We make a network structure picture based on the interaction of these 19 differential genes by the analysis of the STRING database. The results show that the two subunits of RACK1 protein have direct interaction with 8 differential proteins and have 18 interaction pathways on the picture. As a result, RACK1 is the core protein of the network, suggesting RACK1 is the key node protein in oral squamous cel carcinoma.
ABSTRACT
Objective To prepare a high‐titer rabbit specific serum antibody against Kaposi′s sarcoma associated herpesvirus (KSHV) ORF65 capsid protein and identify the specificity of serum antibody .Methods Artificial synthetic peptide of ORF65 pro‐tein was emulsified with Freund adjuvant .4 rabbits were immunized with the prepared antigen by subcutaneous injection at various sites of skin of back and jaw once every two weeks .Immunization was carried out in total 4 times .The serum of the immunized rab‐bits was collected at a week after the last immunization .The titer of rabbit anti‐serum was assayed by ELISA .Specificity of the rab‐bit anti‐serum was analyzed by immunofluorescence assay and Western blot .Results The immunized rabbits produced high‐titer se‐rum antibody after total immunization .The highest titer of anti‐serum against ORF65 protein peptide was 1∶12 800 .The results of Immunofluorescence assay showed that antibody was binded in plasma of BCBL‐1 cell mostly ,which was consistent with the expres‐sion location of ORF65 in BCBL‐1 cell .Subsequently ,the data of Western blot revealed a specific band about 21 kD which accorded with the size of ORF65 protein .Meanwhile ,the expression of ORF65 in TPA treated BCBL‐1 cells was higher than the control cells ,which was consistent with the expression characteristics of lytic protein .Conclusion High‐titer specific rabbit serum antibody against KSHV capsid ORF65 antigen could be successfully prepared by rabbits immunization with ORF65 protein peptide .The pre‐pared antibody could be revealed immune reaction specificity with KSHV ORF65 protein .