[Autophagy inhibitor 3-MA decreases the production and release of infectious enterovirus 71 particles].

OBJECTIVE: To determine whether or not enterovirus 71 (enteroviurs 71, EV71) may induce autophagy and affect the production and release of EV71 after the treatment of autophagy inhibitor. METHODS: Western blots were performed to examine the conversion of LC3-I to LC3- II and the degradation of P62 after the RD-A cells were infected with EV71. CCID50 was determined by checking the virus titer in the supernatant of cells that treated with autophagy inhibitor 3-MA. RESULTS: EV71 infection enhances the type conversion of LC3 and degradation of P62. The infectious virus particles were decreased after the treatment of 3-MA. CONCLUSION: EV71 infection could induce cell autophagy and the autophagy might contribute to the production and release of infectious EV71 particles.

[Expression and biological function of ULBP4 in Rosetta-gamiTMB(DE3) and mAb preparation].

AIM: To express human ULBP4 in Rosetta-gami(TM) B(DE3) and to prepare monoclonal antibody against ULBP4 for the research of gamma deltaT cells recognition mechanism. METHODS: DNA fragments of ULBP4 were derived from HO-8910 RNA by reverse-transcriptase polymerase chain reaction(RT-PCR). The fragments encoding the former 225 amino acids of ULBP4 were cloned into Histag fusion protein expression vector pET22b(+). The C-Histag fusion ULBP4(225a) protein was expressed in inclusion body and purified step by step according to manufactory's protocol and renatured in bag filter. It's functional effect on NK cells was evaluated by NKG2D binding assay and IFN-gamma secretion experiments. Prokaryotic expressed human ULBP4 was used as an antigen to prepare monoclonal antibodies by means of the B lymphocyte hybridoma technique. RESULTS: ULBP4 recombinant protein can stimulate NK cells to secrete IFN-gamma. Through PEG fusion and screening by limited dilution, we obtained four strains of hybridoma cells secreting anti-ULBP4 antibodies. CONCLUSION: The fusion protein was expressed successfully and functional. At the same time, the anti-ULBP4 mAbs were prepared successfully. Both of them provide a platform for further research.

[Inhibition of intracellular Ca(2+)-chelating agent on Hela cell apoptosis induced by HSV-1].

AIM: To explore the variation of intracellular free-Ca (2+) concentration during Hela cell apoptosis induced by HSV-1 and inhibition of Ca(2+)-chelating agent on the apoptosis. METHODS: Apoptosis of the Hela cells infected by HSV-1 was observed under scanning electron microscope (SEM) and transmission electron microscope (TEM), and the variation of intracellular free-Ca (2+) concentration, labeled with fluorescent probe was observed under fluorescence-microscope at a series of timepoints. RESULTS: The Hela cells infected by HSV-1 showed typical apoptotic morphological changes. Free-Ca (2+) concentration increased and reached peak at the 12 hours after HSV-1 infection. Characteristic morphological features of apoptosis occurred at the 24 hours after infection. This apoptosis could be inhibited by intracellular Ca(2+)-chelating agent. CONCLUSION: Intracellular free-Ca(2+) might play an important role in Hela cell apoptosis induced by HSV-1, which provides useful clues for clinical treatment of associated diseases.