RESUMO
OBJECTIVE: To develop attenuated Salmonella which harboring enterovirus 71 (EV71) VP1 gene. METHODS: The plasmid which expressed VP1 protein of EV71 was constructed by gene recombination. Cellular expression was assessed by Western Blot analysis. The recombinant plasmid was then transformed into attenuated Salmonella SL7207. RESULTS: EV71 VP1 gene sequence was inserted into a eukaryotic expression plasmid VR1012. VP1 protein was detected by Western Blot analysis in the culture supernatant. And the attenuated Salmonella harbored the plasmid stable. CONCLUSION: The plasmid was constructed successfully and it can express effectively in vitro. The bacteria which harboring the plasmid were constructed successfully. This has provided a basis for further research of an oral EV71 vaccine.
Assuntos
Proteínas do Capsídeo/genética , Enterovirus Humano A/genética , Expressão Gênica , Vetores Genéticos/genética , Salmonella/genética , Proteínas do Capsídeo/metabolismo , Engenharia Genética , Vetores Genéticos/metabolismo , Salmonella/metabolismoRESUMO
OBJECTIVE: To develop a system to rescue virus by intracellular expression of T7 RNA Polymerase. METHODS: The gene of T7 RNA Polymerase was amplified and cloned to VR1012 by molecular biological technology. The expression plasmid VR-1a was then identified. VR-1a and EV71 infectious plasmid were co-transfected in Vero cell. CPE was observed and viral gene viral antigen were detected. RESULTS: The gene of T7 RNA Polymerase was successfully cloned into vector VR1012. Vero cell developed to CPE after being transfected VR-1a and EV71 infectious plasmid. EV71 gene was amplified by RT-PCR from the culture. EV71 antigen was also detected by ELISA. CONCLUSION: The method can be used to rescue virus. It could apply to immunologic research of EV71 DNA vaccine.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Engenharia Genética/métodos , Vetores Genéticos/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Animais , Chlorocebus aethiops , Enterovirus Humano A/genética , Enterovirus Humano A/fisiologia , Expressão Gênica , Vetores Genéticos/metabolismo , Células HeLa , Humanos , Plasmídeos/genética , Plasmídeos/metabolismo , Transfecção , Células Vero , Replicação ViralRESUMO
OBJECTIVE: To construct the recombinant plasmid containing S1 gene of new type of reovirus, and to study the expression of protein sigma1 in Vero cells. METHODS: The recombinant plasmid, named pC-S, was constructed by cloning S1 gene into vector pCAGGS/MCS. Then Vero cells were transfected with pC-S and collected at 24, 48, 72 h post transfection followed by SDS-PAGE and Western-Blot assay. RESULTS: Results both SDS-PAGE and Western-Blot assay indicated that sigma1 protein could be expressed well and the highest expression level was 72 h post transfection. CONCLUSIONS: Sigma1 protein could be expressed well in Vero cells by transfected with recombinant plasmid containing S1 gene, and could give some implications for subsequent research on virus-host interactions.
