| Objectives To construct the EGFP (enhanced green fluorescent protein) expressing vector of NP (nucleocapsid protein) gene of HV (hantavirus) Junan amplifying isolate JUN05. To express the NP of HV together with EGFP, and to observe the expressing density, location and distribution of the fused protein through fluorescence microscopy and immunohistochemistry, to lay foundation for the development of new type diagnosis reagent and further research on the structure and function of NP.Methods The NP gene was amplified from cloning vector constructed formerly, by PCR after primers were designed according to the gene sequence of nucleocapsid protein of Junan amplifying isolate JUN05 and MCS sequence of expressing vector pEGFP-N1. The products of PCR and pEGFP-N1 were digested by Bgl â…¡ and Pst â… respectively and ligated together. The NP gene was located downstream of the EGFP gene, and they were fused in codon frame. The clones carrying NP gene were selected. After PCR, double enzyme digestion, and DNA sequencing, the expressing clone was identified. The recombinant plasmid was transfected into BHK21 cells with lipofectamine and the expression was observed under fluorescence microscope and assayed by immunohistochemistry after 24 hours.Results 1. The expression vector of HV NP gene was constructed and enzyme digestion, PCR, and DNA sequencing showed the clone was constructed successfully.2. The sequencing result showed that the NP gene was recombinated into thevector ahead of the EGFP gene, and the two genes of NP and EGFP were fused in codon frame. Compared with the sequence of NP gene of former clone vector, no mutation caused by PCR amplification was observed. And the stopping codon of NP gene was removed, which guaranteed the fused genes could be expressed successfully. The introduction of kozak sequence improved the level of expression.3. The fluorescence microscopy showed that the NP was expressed effectively in the cytoplasm of BHK21 cells. The strong green fluorescence was distributed in the cytoplasm, the aim of observing the NP by EGFP was achieved. We also obtained the positive signal though immunohischemistry.Conclusions The expressing vector pEGFP-NP, which expresses the HV NP gene of Junan amplifying isolate JUN05 together with EGFP gene, is constructed successfully. Analyses of nucleotide showed that the two genes of NP and EGFP were fused in codon frame. The NP gene was located at the downstream of CMV promoter, and kozak sequence was added into the NP gene. This lays foundation for high-level expression. The fused gene of NP-EGFP was expressed at a high level in BHK21 cells, and bright green fluorescence was observed. The fused protein possesses both character of NP and EGFP, so the EGFP can be served as a reporter gene to observe the expression and location of NP. This research also lays foundation for the further study of selecting the stably transfected cell lines, and substantive production and purification of this protein, which can be used in the HV experimental diagnosis. |
