Font Size: a A A

Based On The Preparation Of Anti-human Gp73 Monoclonal Antibody Epitopes

Posted on:2011-12-31Degree:MasterType:Thesis
Country:ChinaCandidate:X N YaoFull Text:PDF
GTID:2190360308974901Subject:Biochemistry and Molecular Biology
Abstract/Summary:PDF Full Text Request
Hepatocellular carcinoma (HCC) is one of the most common malignant tumor in China, and alpha-fetoprotein (AFP) is the most commonly used serum marker for HCC diagnosis currently. AFP has a sensitivity of 39~64% and a specificity of 76~69% in diagnosis of HCC. Such a sensitivity and a specificity could not meet the requirement of HCC diagnosis, so it is urgent to find ideal biomarkers for HCC diagnosis.Golgi protein 73(GP73) is a typeⅡtransmembrane glucoprotein, and is closely related with the occurrence and development of hepatitis, cirrhosis, HCC and other liver diseases. GP73 has a sensitivity of 65% and a specificity of 90% in detecting HCC, but fucosylated GP73 has a sensitivity of 90% and a specificity of 100% in HCC diagnosis. Evidently, GP73 is a good marker candidate for HCC diagnosis, while the core fucosylated GP73 will be more valuable in early diagnosis of HCC. The purpose of this study is to prepare specific, high affinity monoclonal antibodies to GP73 for early diagnosis of HCC.The whole native protein antigen or synthetic peptide is required for traditional method of antibody preparation, but the whole native protein is not always available, and the protein purification is laborious and time-consumed, while the synthesis of peptide through chemical methods is of high cost, and the synthetic peptides should be coupled to carrier protein for animal immunization. In this study, the specific B cell epitopes were selected and displayed on the surface of T7 phages, then the recombinant T7 phages were used to immunize the animals to prepare the monoclonal antibody(McAb) against the B cell epitope, from which the McAbs specific for the corresponding whole native protein were selected.?Firstly, the B cell epitopes should be selected. Because of absence of the information about the advanced structure of GP73, it is impossible to select B cell epitopes based on its three dimensional structure. Generally, the N-terminus and the C-terminus of the protein are accessible, they are good B cell epitope candidates. Serum GP73 (sGP73) is the product of GP73 cleaved by Furin between aa55 and aa56, it has different N terminus from GP73. The N-terminal and C-terminal peptides (GP73N and GP73C) of sGP73 all have good hydrophilicity and accessibility, and are unique in the human protein library, so they were selected as candidate epitopes. Two kinds of the recombinant T7 phages, which displayed GP73N and GP73C epitopes respectively, were constructed, and were used as the alternative antigens of sGP73. GP73N and GP73C epitopes were also cloned into pGEX-4T-2 vector to prepare the GST-epitope fusion protein for the antibody screening. The high purity of the GST-GP73N and GST-GP73C fusion protein were obtained by affinity chromatography. In addition, GST-GP73 and GST-GP73 56-195 were also constructed for the use in the subsequent McAb identification.The recombinant phage T7-GP73N and T7-GP73C were amplified and extracted, and then were used to immunize Balb/c mice. When the serum antibody titers meet the fusion requirement, the spleen cells of mice were fused with SP2/0 cells. After HAT medium selection, ELISA screening, and subcloning by limited dilution, three hybridoma cell lines, which stably secret anti-GP73N antibodies, were established, the were designated GP73N1, GP73N4 and GP73N8. ELISA was used to analyze their specificity and affinity. The results demonstrated that GP73N1 had a high affinity, but a low specificity, GP73N4 had a good specificity, but low affinity, GP73N8 had good affinity and specificity. Then GP73N8 was systematically identified subsequently.ELISA results showed that GP73N8 specifically recognized GP73N peptide, but did not react with other eight proteins; Western blot analysis indicated that GP73N8 also specifically bind GP73 proteins in cell lysates, no reaction were found with other cell proteins. This demonstrated that GP73N8 had a high specificity, and could recognize not only the recombinant GP73 protein, but also the native GP73.GP73N8 was found to be of IgG1 subclass analyzed by Sigma IsoQ5 antibody test strip.The affinity constant K of GP73N8 was determined by indirect ELISA, it was 3.36×107 L/mol.Immunofluorescence experiments exhibited that GP73 protein mainly accumulated around the nucleus in HepG2 cells, this consisted with the fact that GP73 is Golgi protein.The expression of GP73 in different cell lines was analyzed by Western blot. The results exhibited that the selected HCC cell lines all expressed GP73 protein. In non-HCC cell lines, Raji and GLC-82 cells lines highly expressed GP73, Hela cell line expressed GP73 at low level, but no expression of GP73 was found in Lovo and MCF-7 cell lines. This means that GP73 is not specifically expressed in HCC cells.A method for detecting the core fucosylated GP73 in serum was established using McAb GP73N8 and lentil lectin by bio-chip technology. But the results were not good, it requires further optimization. The expression status of GP73 in HCC tissues was assayed using McAb GP73N8. 45 cases of HCC tissues, 5 cases of cholangiocarcinoma tissues and 10 cases of normal liver tissues were detected. The results hinted that the expression of GP73 gradually increased with the development of HCC, while no expression was found in normal liver and cholangiocarcinoma tissues.In summary, an alternative antigen strategy was adopted in this work. The recombinant T7 phage, which displayed the epitope of GP73 antigen, was used as alternative antigen to prepare the McAb to GP73, and the McAb specifically recognizing GP73 protein was successfully selected. It could be used in ELISA, Western blot, immunofluorescence and immunohistochemistry. Preparation of the antibody specific for GP73 will be benefit to develop the kit of HCC diagnosis based on detecting serum GP73.
Keywords/Search Tags:epitope, phage display, GP73, monoclonal antibody (McAb)
PDF Full Text Request
Related items