Expression of Recombinant Bovine Prion Protein PrP27-30 in CHO-K1 Cells

[Objective] To investigate the possibilities of expressing bovine PrP27-30 gene in CHO-K1 cells. [Method] The purified PCR products of PrP27-30 were digested and ligated to the pCI-neo vector to yield pCI-neo-PrP27-30 that was used as an expression vector. Then CHO-K1 cells were transfected by pCI-neo-PrP27-30, and stable expression clone cells were screened by methotrexate (MTX) at a concentration of 0.1 and 1.0 umol/L. The transient expression was detected by indirect immunofluorescence assay and western blot. [ Result] After drug selection with MTX, the expression of PrP27-30 gene was detected in CHO-K1 cells. [Conclusion] Recombinant protein PrP27-30 expressed in CHO-K1 cells has better immunoreactivity and can be used to study secondary structure and regulation mechanism of pathological isoform of prion protein (prpC).

Expression of Recombinant Protein Bovine Prion pCIp264 in COS-7 Cells and Its Detection

Bovine spongiform encephalopathy （BSE） is thought to be caused by prions initially derived from sheep scrapie, and epidemiological studies suggest that new viriant form of CJD manifest in Great Britain may be caused by BSE prions. PrP^Sc is thought to pathogenic factor of transmissible spongiform encephalopathy （TSE）, which invariably involve a post-translational modification process of PrPc encoded by the host euchromosome PrP gene during the period it converted into the pathogenic form （PrP^Sc）, PrP is nomal cellular protein which has been found in both neuronal and nonneuronal tissues. Since the crucial infectious event in protein-transmitted diseases is an induced misfolding of prion proteins （PrP^c） catalyzed by already misfolded PrP^Sc, it is of high importance that such collisions are enhanced by two-dimensional diffusion in cell membranes is of high importance compared to three-dimensional diffusion in solution. The level of PrP mRNA in brain is higher than other tissue, but purification of PrPc from rodent has been difficult. To understand the formation of PrP^Sc, it seemed useful to develop a system for produced a large quantities of PrPc since there is no nature source of PrP^c. The pCI-neo mammalian expression vector contains the neomycin phosphtransferase gene which serves as a marker for the selection of stable transfected cells with G418. COS-7 cells constitutively express simian viruse 40 （SV40） T-antigen and support replication of expression plasmids containing the SV40 origin of replication, amplifying the introduced expression cassettes, now become important routine of expression a large number of heterologous gene products. In this paper, the authors used pCI-neo vector to construct a recombnant pCIp264 （cotains mPrP, N-signalpeptide and C-GPI anchor） plasmid to express it in the COS-7 cells and meanwhile detect the expression fusion using IN-ELISA, IN-IFA and western blot, and obtain some approximative nature PrPc.

Development and characterization of monoclonal antibodies against human aryl hydrocarbon receptor

Aryl hydrocarbon receptor（Ah R）, a ligand-dependent nuclear receptor, is involved in a diverse spectrum of biological and toxicological effects. Due to the lack of three dimensional（3D）crystal or nuclear magnetic resonance structure, the mechanisms of these complex effects of AhR remain to be unclear. Also, commercial monoclonal antibodies（mA bs） against human AhR protein（h Ah R）, as alternative immunological tools, are very limited. Thus, in order to provide more tools for further studies on h Ah R, we prepared two m Abs（1D6 and 4A6） against h Ah R. The two newly generated m Abs specifically bound to amino acids 484–508（located in transcription activation domain） and amino acids 201–215（located in Per-ARNT-Sim domain）of h Ah R, respectively. These epitopes were new as compared with those of commercial m Abs.The m Abs were also characterized by enzyme-linked immunosorbent assay, western blot,immunoprecipitation and indirect immunofluorescence assay in different cell lines. The results showed that the two m Abs could recognize the linearized AhR s in six different human cell lines and a rat hepatoma cell line, as well as the h Ah R with native conformations. We concluded that the newly generated m Abs could be employed in AhR-based bioassays for analysis of environmental contaminants, and held great potential for further revealing the spatial structure of AhR and its biological functions in future studies.