Change of the Antigenecity of Human Papillomavirus Type 16 E7 Oncoprotein according to Phosphorylation / 대한암학회지

PURPOSE: It was suggested that immunogenic region of E7 proteins of human papillo- mavirus (HPV) type 16 encompass casein kinase (CK) II phosphorylation site and the resulting negative charge may affect the various biologic function of E7 protein. This study was undertaken to analyze the change of antigenic characteristics of HPV type 16, E7 oncoprotein according to phosphorylation. MATERIALS AND METHODS: We produced two monoclonal antibodies (VD6 and IB10) which showed different reactivities to E7 proteins expressed from bacteria or extracted from CaSki cell. These reaction were analyzed by Western blotting. Also the antigenic sites estimation of these antibodies using nested deletion sets was done. On the basis of above experiments, we performed in vitro phosphorylation assay using CK II and its specific inhibitor, DRB (5, 6-dichloro-l-beta-D-ribofuranosylbenzimidazole), to analyze the IB10 reactivity to E7 oncoproteins according to phosphorylation. RESULTS: In Westem blot analysis, VD6 and IB10 antibodies reacted strongly to bacterially expressed E7 protein. But using E7 extracted from CaSki cell, VD6 reacted to 2.0 kDa E7 protein whereas IB10 showed weak reactivity. The antigenic sites estimation of these antibodies showed that antigenic site of VD6 was located in amino terminal region and that of IB10 in the middle portion in the range of approximate amino acid 25-45. The antigenic site of IB10 might contain the possible phosphorylation sites (Ser-31, 32) in E7. Considering this, the different reactivities of IB10 to E7 proteins expressed in bacteria and extracted from CaSki cell might be due to phosphorylation. In in vitro phosphorylation assay using CK II, the phosphorylation of E7 increased according to reaction time. And this phosphorylation reduced the reactivity of IB10 to E7 protein whereas the reactivity of VD6 did not change. Also the reactivity of IB10 to E7 protein increased in a dose dependent manner with CK II specific inhibitor, DRB treated CaSki cell extracts. CONCLUSION: These result showed the antigenecity is affected by the degree of phosphorylation of E7 protein.

Cloning and expression of the human MMP-1 gene fragment and antigenecity analysis of its expression products / 中华传染病杂志

Objective To construct the recombinant plasmid of human MMP 1 clone and study its antigenecity of MMP 1 fusion protein. Methods The total RNA was extracted from human liver and used as a template for reverse transcription. After PCR amplification, a 1 432 bp fragment was obtained and cloned into T vector. After digested with restriction enzyme, the target fragment was subcloned into plasmid pMAL c2x. The recombinant plasmid was transferred into JM109 which can express a fusion protein. We analyze the protein with SDS PAGE and Western blot. Results We obtained human MMP 1 gene and its recombinant plasmid clone. The expressed protein can be recognized by MMP 1 polyclonal antibody in Western blot.Conclusions We obtained the MMP 1 protein with antigenicity. The fusion protein can be used to prepare polyclonal antibody against MMP 1.

Study on Histopathologic Changes of Suckling Rats Inoculated with Hantaan Virus

Hantaan and related viruses have been implicated as causative agents for a diverse group of human diseases known collectively as "hemorrhagic fevers with renal syndrome" (HFRS). Outbred SD rats obtained within 24 hours after birth were inoculated by intracerebral (the first group) or intramuscular routes (the second group) with 10(9.5)/ml DL50 of Hantaan seed virus suspension in 0.02 ml and 0.1 ml, respectively. Brain, lung, liver, kidney and spleen were used for virus antigen detection by immunofluorecence and histopathologic examination. In the first group, immunofluorescent intensity of virus antigen was increased in all organs (especially brain) and persisted until time of death(day 9). The histopathologic changes were relatively mild in brain and spleen and unremarkable in liver, lung and kidney. In the second group, immunofluorescent intensity of virus antigen was markedly increased in brain until time of death(day 17), but decreased in other organs. The histopathologic findings, such as meningoencephalitis, interstitial pneumonitis with focal hemorrhage, and lymphoid hyperplasia of splenic white pulp were much prominent compared to the first group. However, those of liver and kidney were unremarkable. The chronology of virologic and pathologic findings in Hantaan-infected suckling rats suggests a possible immune-mediated mechanism in disease pathogenesis.

Prokaryotic expression of fusion gene A?-HBcAg and analysis of the immunoreactivity and immunogenicity of the expression protein / 西安交通大学学报(医学版)

Objective To study the prokaryotic expression of fusion gene A?-HBcAg and analyze the immunoreactivity and immunogenicity of expression protein. Methods Recombinant plasmid pBV220/A?-HBcAg was transformed into E.coli DH5?, and expressed by temperature inducing. The bacteria were split by ultrasonic wave. The expression of the fusion protein was studied by SDS-PAGE and Coomassie brilliant blue staining. The immunoreactivity of the fusion protein was determined using ELISA. After immunized intraperitoneally with the fusion protein, 5 Balb/c mice's sera titers of anti- A? and anti-HBc were evaluated by ELISA. Results Fusion protein was in sediment of the split bacteria as inclusion bodies and its expression level was 5% of the total sediment protein. The fusion protein had both immunoreactivity of A? and HBcAg. The titers of anti-A? and anti-HBc were very low after 3 times of immunization. After immunization for 5 times, the titers reached 1∶800 and 1∶3 200 for anti-A? and anti-HBc, respectively. Conclusion Recombinant gene A?-HBcAg can be expressed in E.coli DH5? and the expression protein has certain immunoreactivity and immunogenicity. It indicates that further work should be done to enhance the expression level of fusion gene A?-HBcAg and improve the immunogenicity of the fusion protein.