We investigated the mechanism of human aspartyl beta-hydroxylase (HAAH) in early diagnosis of tumors. The encoding gene of HAAH was cloned from the hepatic carcinoma by RT-PCR and expressed as a fused protein in the prokaryotic vector pBV-IL1. The expressed HAAH was purified by Ni(2+)-NTA purification column and the purified protein was then used to immunize Balb/c mice. Three hybridoma cell lines (respectively designated H3/E10, E4/F12 and G4/D8) stably expressing the monoclonal antibody specific to HAAH fusion protein were obtained. The specificity and sensitivity of the monoclonal antibody were assessed by indirect enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Finally, the monoclonal antibody expressed by H3/E10 cell line was used to detect the expression of HAAH in several tumor cell lines by indirect immuno-fluorescence, and the specific fluorescence was observed. In conclusion, this study successfully constructed the recombinant prokaryotic vector pBV-IL1-HAAH and prepared HAAH-specific monoclonal antibody for further study of the structure and function of the protein. The result may also lay solid foundation for the research of the molecular mechanism of HAAH in early diagnosis of tumors.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Cell Line, Tumor ; Cloning, Molecular ; Genetic Vectors ; genetics ; Humans ; Hybridomas ; metabolism ; Immunization ; Liver Neoplasms ; pathology ; Mice ; Mice, Inbred BALB C ; Mixed Function Oxygenases ; biosynthesis ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology

Objective To summarize the latest studies on the biological effects of microgravity or simulated microgravity on microorganisms in the world. Literature resource and selection The related published articles and reviews in the field. Literature quotation Thirty-eight references were cited. Literature synthesis Studies showed that microgravity environment of space could induce the variations of microorganisms on their physiological, biochemical and genetic characteristics.Some microorganism populations, those are originally unharmful to human body or environment, show variations on virulence, pathogenicity, sensitivity to antibiotics, etc. Conclusion Studies on the effects of microgravity on microorganisms have important theoretical and practical significance on clarifying the survival and mutation mechanism of microorganisms in microgravity, establishing the future's space controlled ecological life support system, monitoring the safety of microorganisms in the space and rationally utilizing microgravity resources.

Objective To summarize the latest studies on the biological effects of microgravity or simulated microgravity on microorganisms in the world. Literature resource and selection The related published articles and reviews in the field. Literature quotation Thirty-eight references were cited. Literature synthesis Studies showed that microgravity environment of space could induce the variations of microorganisms on their physiological, biochemical and genetic characteristics.Some microorganism populations, those are originally unharmful to human body or environment, show variations on virulence, pathogenicity, sensitivity to antibiotics, etc. Conclusion Studies on the effects of microgravity on microorganisms have important theoretical and practical significance on clarifying the survival and mutation mechanism of microorganisms in microgravity, establishing the future's space controlled ecological life support system, monitoring the safety of microorganisms in the space and rationally utilizing microgravity resources.

We constructed the recombinant adenovirus expressing the glycoprotein G2 of Hantaan virus. Firstly we obtained coding gene fragment of G2 by PCR, and subsequently inserted the gene of interest into the Adenoviral pShuttle vector pAd5-CMV. Then we co-transfected the recombinant pShuttle vector and adenovirus skeleton plasmid into HEK293 cells by Calcium phosphate precipitation method. After the recombinant adenovirus were packaged and amplified in HEK293 cells, we observed the expression of reporter gene eGFP by fluorescent microscopy, and we obtained the recombinant adenovirus containing Hantaan virus glycoprotein G2. The recombinant adenoviruses were used to infect Hela cells and the expressed protein was detected by Indirect Immuno-fluorescence and Western blotting. The construct was confirmed at several levels: first restriction enzyme analysis demonstrated that the recombinant adenovirus vector was constructed correctly, second RT-PCR showed that the G2 gene could transcribe correctly in Hela cells. Then Fluorescent microscopy proved the expression of eGFP in the infected Hela cells. Finally, Indirect Immune-fluorescence and Western-blot confirmed the expression of interested protein identified by anti-G2 monoclonal antibody. In conclusions, this study successfully constructed the recombinant adenovirus containing Hantaan virus envelope glycoprotein G2, meanwhile obtained the G2 protein, it may lay solid foundation for the structure study of G2 protein and the new vaccine of Hantaan virus.
Adenoviridae ; genetics ; metabolism ; Genetic Vectors ; Green Fluorescent Proteins ; biosynthesis ; genetics ; HEK293 Cells ; HeLa Cells ; Humans ; Kidney ; cytology ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection ; Viral Envelope Proteins ; biosynthesis ; genetics