Objective The cDNA expression library,which was constructed with human colorectal cancer cell HRT-18,was screened with the phage antibody CH209 in order to find novel antigen genes of colorectal carcinoma.Methods Total RNA was extracted from cell HRT-18 and mRNA was isolated from total RNA and then double strand cDNA was synthesized by SMART technique.cDNA was ligated into ? TripIEX2 vector and then was packaged into ? phage in vitro.The primary library was titrated and the percentage of recombinant clones were determined.The length of cDNA inserts was tested for ligation efficiency.The library was screened with phage fusion antibody CH209 and the sequences of the reacted clones were determined.Results The primary library consisted of 6.5?10~(6)pfu/mL,and the percentage of recombinant clones was 97%.The length of cDNA inserts was 0.5-2.0kb.The titer of the amplified cDNA library was 1.1?10~9 pfu/mL.Ten positive clones were obtained and derived from ten different genes.Five of these genes were high homologous to genes known in GenBank,and there were also three genes with low homology to genes known in GenBank.The remainder two genes might be novel genes by matching in GenBank with BLAST software.Conclusion The quality of the constructed cDNA library from human CRC cell HRT-18 is excellent.Ten positive clones were obtained and two of them may be novel genes.

Objective To identify potential candidate genes related to the cancerous phenotype by analyzing databases publicly available. Methods Using a data-mining tool called Digital Differential Display (DDD) from the Cancer Gene Anatomy Project database, ESTs from 17 different tumor types were analyzed for differential expression. Results We obtained 130 up-regulated and 159 down-regulated genes, most of which are related to cytoskeleton, ribosomal subunit, substance metabolism, cell cycle, signal conduction, transcription and translation. These genes appear most frequently on chromosome 12 but rarely on chromosome 21 and Y. Conclusion In silico identification is a high-throughput screening strategy. Our study may lay a foundation for identification of future caner markers and provide a new thought for screening strategy of cancer markers.

Tumor markers refer to the specific substances that exist in tumor cells themselves or are secreted by tumor cells. They can reflect the existence and growth of the tumor. The serum tumor biomarkers have been widely applied in tumor detection,but the detection of these markers is based on the tumor antigens,and thus has many inadequacies in tumor screening and diagnosis. In this paper,we reviewed autoantibodies as tumor biomarkers against self-antigens in vivo. This method is to examine the tumor autoantibodies by using the tumor antigens,and its specificity and sensitivity are superior to the traditional examination methods. Using autoantibodies to detect tumors would provide a new method for tumor screening and diagnosis.

Objective To construct a cDNA library from human nasopharyngeal carcinoma cell HNE 2.Methods The total RNA was separated from human NPC(nasopharyngeal carcinoma)cell HNE 2 and the mRNA was isolated from the total RNA by MagneSphere technique,then the first-strand cDNA was synthesized with oligo(dT) primer containing sfiI site while the double-strand cDNA was amplified through LD-PCR(Long-distance PCR) by SMART technique.The double-strand cDNA was digested by sfiI(IA &IB)restriction enzyme before cDNA size fractionation ,the double-strand cDNA fractionated was ligated into the ?TripIEx2 vector and then was packaged in vitro.Results The unamplified human NPC cell HNE 2 cDNA library consists of 0 78?10 6 independent clones,and the percentage of recombinant clones was more than 96%.The titer of the amplified cDNA library was 1 02?10 9 pfu/ml and the average insert of the recombinants was 1 2kb.Conclusions The quality of the constructed human NPC cell HNE 2 cDNA library is excellent and helpful to screen NPC specific-antigen.

Objective To obtain high pure myocardiac Troponin T from myocardiac tissue. Methods After the myocardiac tissues were homogenized and purified with high salt solution, the Sephacryl S-300HR column chromatography was employed to separate the cTnT from cardiac proteins, and identified them with SDS-PAGE and Western blot. Results The crude cTn was purified by column chromatography, and there appeared three peaks. The second peak was single component, which could react with mouse anti-human Troponin T antibody, and its molecular weight was about 34kD. Conclusions In this experiment, high pure cTnT was obtained, which could pave a way for further research.

According to the immune network hypothesis proposed by Jeme, certain anti-idiotypic antibodies (Ab2) express three dimensional shapes which resemble the structure of natural antigens. Here we propose a study of active immunotherapy by Ab2 for patients with Nasopharyngeal Cancer (NPC) . Two Ab2,designated 2H4 and 5D3,against two Abl (FC_2 and HNL5)that recognize NPC associated antigen were generated. They could substitute NPC antigen to induce humoral and cellular immune response against NPC cells in syngeneic mice. Nineteen patients with NPC at stage IV were chosen for active immunotherapy. They were treated with Alum-precipitated 2H4 or 5D3 accompanying radiotherapy. Nine patients with radiotherapy alone were as control. Both anti-anti-idiotypic antibodies (Ab3) and anti-NPC antibodies (Abl') were increased and human anti-mouse antibodies (HAMA) occurred in nineteen patients of the experimental group; whereas the levels of Abl' did not rise in control group.Serum IL-2, IFN-? and TNF-? level were increased in most patients in experimental group. While in the control group, there was no difference of cytokine level between pretherapy and post- therapy.

Objective To compare the in vitro antitumor immune responses induced by bivalent bispecific anti-idiotype antibody G22-I50 and monovalent anti-idiotype antibody G22 and I50, and explore its possible mechanism. Methods Proteins G22-I50, G22, and I50 were induced and identified by Western blot and ELISA. Peripheral blood monoclear cells (PBMC) were isolated and stimulated with G22-I50, G22, and I50 anti-idiotype antibodies, respectively. MTT assay and LDH release test were employed to examine the proliferation and cytotoxicity of the PBMC. The levels of IFN-γ, IL-2, and IL-4 in the supernatant were detected by ELISA and changes of T lymphocyte subsets were determined by flow cytometry. Results Western blot showed that G22-I50, G22, and I50 had specific binding capabilities to FC2 (Ab1). The activities of G22-I50, G22, and I50 had recovered and these proteins could be used in the in vitro study. The proliferation and cytotoxicity of the PBMC stimulated with G22-I50 were significantly higher than those stimulated with G22 or I50, The level of IFN-γ and IL-2 in the culture supernatant of the PBMC stimulated with G22-I50 was higher than that in the G22 or I50 group, but the level of IL-4 did not increase.Compared with the G22 or I50 group, the proportion of CD4+ and CD8+ T cells and CD4+/CD8+ ratio significantly increased, and the proportion of CD4+CD25+ T cells significantly decreased in the PBMC stimulated with G22-I50. Conclusion G22-I50 has more potent immunogenicity and would enhance specific antitumor effect which might relate to improving PBMC proliferation, inducing the secretion of Th1 type cytokines, activating CD8+T cells, and suppressing the expression of CD4+CD25+ T cells.

Objective To obtain I50 anti-idiotype antibody and identify its activity in vitro.Methods I50 anti-idiotype (Id) antibody gene was amplified from the template of fuse 5-I50 by PCR to construct a prokaryotic expression vector pET25b-I50. The expression of pET25b-I50 in E. coli BL21(DE3) was induced by isopropylthio-β-D-galactopyranoside (IPTG) and was confirmed by SDS-PAGE and Western blot with Ab1(FC2) monoclonal antibody and an anti-hexahistidine tag antibody. The method of dialysis refolding was used to restore the activity of I50 anti-Id antibody, which was measured by Dot-ELISA and lymphocyte proliferation assay. Results The recombinant vector was successfully constructed and the recombinant protein was successfully expressed and purified with 90% purity. The relative molecular weight of the expressed protein was 15 kD, which was in accordance with expectation. The activity of I50 anti-Id antibody could be restored and could promote the proliferation of lymphocyte in a dose-dependent manner. Conclusion These results suggested that I50 anti-Id protein vaccine is likely an option in the therapy against nasopharyngeal carcinoma in vivo.

Objective To construct human phage antibody library against hepatoma carcinoma.Methods Peripheral blood mononuclear cells(PBMCs) of patients with liver cancer were sensitized in vitro and transformed by Epstein-Barr virus(EBV).The genes of light chain and Fd of antibodies were amplified by RT-PCR.Fab genes were cloned into vector pComb3 and transformed into E.coli XLI-Blue by electroporation to construct the Fab-displaying phage antibody library.Results ELISA detection showed that 4 liver cancer patients' B cells transformed by EBV could produce specific antibodies to hepatoma carcinoma cell.Totally 13 types of light chain genes and 28 types of Fd genes were obtained by RT-PCR.The capacity of the primary phage library was 1.7?107pfu/mL.The percentage of recombinant clones was about 100%.Conclusion A human phage antibody library has been constructed successfully by means of EBV transformation technique.

Objective:To obtain the full length cDNA sequences of hepatoma associated gene HTA, analyze its alternative splicing, detect the expression pattern of 2 HTA gene transcripts in different hepatic cell lines, and to establish a base for further study of HTA gene function in hepatocellular carcinoma (HCC) occurence and development. Methods:The full length cDNA of HTA gene was cloned by rapid amplification of cDNA 3' ends (3'-RACE), rapid ampliifcation of cDNA 5' ends (5'-RACE) and DNA sequencing. The gene structure and alternative splicing were analysed. Northern blot assay was performed to detect the expression pattern of 2 HTA gene transcripts in different hepatic cell lines. Results:The full length of HTA gene was 1414 bp, composed of 3 exons and 2 introns, and the second intron could be retained in mRNA. Northern blot assay showed that 2 transcripts of HTA mRNA(1.4 kb and 1.7 kb) could express in the HCC cell lines HepG2 and QGY-7703, but not in the non-malignant cell line L-02 and HUVEC. The expression level of 1.4 kb transcript was much higher than 1.7 kb one. Conclusion:This study successfully has obtained the full length cDNA of HTA gene, and analysed the gene sequence and alternative splicing, 2 transcripts of HTA mRNA specifically expressed in HCC cell lines. As a hepatoma associated gene, HTA deserves further investigation.