RESUMO
Human arrest defective 1(hARD1) is an acetyltransferase catalyzing the N-terminal acetylation of proteins after translation. The high expression of hARD1 could be an indicator of the breast cancer. In current study, we produced an anti-hARD lp monoclonal antibody that could specifically recognize ARD1 in breast cancer tissues by using the immunohistochemical assay. The full-length His-tag hARD1 protein (1-235 aa) was over-expressed in Escherichia coli, and purified recombinant protein was injected into Balb/c mice to perform immunization procedure. Eight stable positive monoclonal cell lines were isolated. ELISA results demonstrated that all light chains of antibodies were kappa, and the heavy chains displayed three subtypes IgG1, IgG2a and IgG2b, respectively. A monoclonal antibody, which could specifically recognize hARD1 protein in breast cancer tissues, was identified by screening different cancer tissues using antibody-specificity method. Further, the specificity of the antibody was confirmed by Western blotting analysis. Our study would facilitate breast cancer diagnosis by using this ARD1 monoclonal antibody in clinic. Also, this antibody could be used as an important tool for further investigating the role of ARD1 in tumorigenesis.
Assuntos
Animais , Feminino , Humanos , Camundongos , Acetiltransferases , Genética , Alergia e Imunologia , Anticorpos Monoclonais , Genética , Alergia e Imunologia , Biomarcadores Tumorais , Alergia e Imunologia , Neoplasias da Mama , Alergia e Imunologia , Metabolismo , Patologia , Escherichia coli , Genética , Metabolismo , Imunização , Camundongos Endogâmicos BALB C , Acetiltransferase N-Terminal A , Acetiltransferase N-Terminal E , Proteínas Recombinantes , Genética , Alergia e ImunologiaRESUMO
Human arrest defective 1 (hARD1) is an acetyltransferase; its physiological significance remains unclear. To explore the relationship between ARD1 protein and tumors, we detected the hARD1 protein in tumor tissues in vivo. We cloned hARD1 gene from Hela cell and construct recombinant plasmid pET28b-hARD1. The recombinant plasmid was transformed into E. coli BL21 (DE3)plysS. hARD1 protein was expressed by inducing with IPTG(1 mmol/L) and purified up to 95% through Ni2+ chelation affinity chromatography. We used the purified hARD1 protein as antigen immunized the Balb/c mice and obtained the hARD1 specific polyclonal antiserum. Through immunohistochemical analysis of different tumor tissues in vivo, we found that hARD1 expressed at high frequency in breast cancer, prostate cancer and lung cancer, especially, hARD1 expression frequency in breast cancer was up to 70%, which is higher than in the other tumors. These results indicate that the high expression level of hARD1 could be an indicator of the breast cancer. This new finding would be a foundation to further explore the relationship between breast tumor and hARD1.
Assuntos
Animais , Feminino , Humanos , Masculino , Camundongos , Acetiltransferases , Genética , Alergia e Imunologia , Sequência de Aminoácidos , Anticorpos , Sangue , Alergia e Imunologia , Sequência de Bases , Biomarcadores Tumorais , Neoplasias da Mama , Metabolismo , Patologia , Linhagem Celular Tumoral , Clonagem Molecular , Escherichia coli , Genética , Metabolismo , Soros Imunes , Imunização , Imuno-Histoquímica , Neoplasias Pulmonares , Metabolismo , Patologia , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Acetiltransferase N-Terminal A , Acetiltransferase N-Terminal E , Neoplasias da Próstata , Metabolismo , Patologia , Proteínas Recombinantes , Genética , Alergia e ImunologiaRESUMO
A serum inhibited gene Si1(GenBank acession number:AY050169) was previously cloned and identified by differential expression of genes in U251 cells.For the further study of biological function of Si1, prediction procedure was performed to predict its subcelluar-localization.Relative experiments were carried out at the same time.The expression of EGFP/Si1 recombinant in HeLa cells showed Si1 protein located in nuclear which corroborated the prediction results of PsortⅡ, Proloc, Cello version2, Subnuclear compartments prediction system, NUCLEO and NUCPRED.According to the PredictNLS prediction, twelve different fragments of EGFP/Si1 recombinants were constructed to identify precise NLS regulation sequence.Findings proved that the real NLS regulation sequence was not the same as the software predicted(1 206 bp～1 239 bp on Si1 ORF), but located on 1 395 bp～1 594 bp of Si1.A tumor relatived mutation/EGFP recombinant localization result showed though the mutation site(1 639bp on Si1 ORF) does not located in NLS regulation sequence, it did affect wildtype Si1 protein divert to nuclear and may affect its natural function in cell, perhaps it is the main reason for highly mutation rate of Si1 in tumor.
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Objective To study on the relations between endometrial carcinoma and Infections of HSVⅡ.Methods Using to skill of PCR-SSCP to test 30 cases of endometrial carcinoma,others 15 cases of the hyperplastic endometrial and 15 cases of the normal of endometrial were infected HSVⅡ.Results Positive rates of endometrial carcinoma that was infected of HSVⅡ were 50%(15/30),Positive rates of the hyperplastic endometrial that was infected of HSVⅡ were 6.67%(1/15),15 cases of the normal of endometrials were not infected.Compared with the endometrial carcinoma and the hyperplastic endometrial and the normal of endometrial,P
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The loci of cDNA sequences for valid diagnosis have been identified through the selection of the genome of SARS coronaries. The gene chips for diagnosing such virus have been developed, based on our own-developed technology for manufacturing and application of gene chips. The diagnoses given by such gene chips were consistent well with the reports of clinical laboratories (94.29%) and the sensitivity reached 10(-2)/ml virus molecules. This method is well suited for the clinical use in SARS coronaries diagnosis.
Assuntos
Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Sensibilidade e Especificidade , Síndrome Respiratória Aguda Grave , VirologiaRESUMO
Objective:To construct a phage display antibody library of special antigen responding to serum starved U251 cell,from which the serum responding gene and protein would be gotten.Methods:U251 cells were cultured in serum-absent midium for 48 h.Its protein was extracted and used to immunize BALB/c mice.Total RNA of the spleenocytes of immunized mice was extracted.VH and VL were amplified by RT-PCR and were linked into ScFv(Single chain fragment of variation) with a linker.ScFv was recombined to pCANTAB5E vector and was transformed to TG1 strain.Results:The library capacity was up to 3?106 cfu/L.A positive clone was identified from 8 random clones of this library.Conclusion:The special ScFv phage library is constructed successfully.It is the basis for screening special antibodies which can recognize serum responding protein.