| Aflatoxin (Aspergillus flavus toxin) are compounds with a similar type of structure, among which aflatoxin B1 (AFB1) is the most toxic. Though there are some conventional detection methods such as biological method, HPLC and TLC, due to the limitations of each method, application of immunological method is gradually attracting researchers'interest. Since immunological detection needs to use the enzyme, while the enzyme itself is unstable, and will lead to false positive or false negative results. In order to overcome the instability of the results, a fusion protein with green fluorescent antibody and single chain fragment variable was conducted, through the intensity of the fluorescence, we can get a stable result. And finally the research laid good basis for immunological detection of AFB1.Green fluorescent protein (GFP) was originally isolated from the jellyfish(Jellyfish aequorea). Due to its fluorescence stability, no toxicity, versatile, easy to build carrier, and many other advantages, GFP, as a reporter gene, has been widely used in detection, tracking and positioning of target cells. The single chain fragment variable (ScFv) gene against aflatoxin was previously selected by our research team. On the basis of the above ScFv and GFP gene,8 different types of plasmids were constructed and then expressed in E.coli. The fusion protein from the E.coli with double activity of antibody and fluorescent was finally selected.Firstly, we constructed the GFP-linker-ScFv and ScFv-linker-GFP, and the recombinant gene was cloned into plasmids pET22b and pET32a to construct the recombinant plasmids pET22b-ScFv-GFP, pET22b-GFP-ScFv, pET32a-ScFv-GFP, pET32a-GFP-ScFv. We transformed the recombinant plasmids into host bacteria E.coli BL21(DE3), and the recombinant bacterias were harvested after 20h fermentation and broken, the target protein were extracted, separated and purificated.The activity of the fusion proteins were detected by enzyme-linked immunosorbent assay (ELISA)and fluorescence microscopying, respectively, the results showed that the antibody titer of the expression product from BL21(DE3) /pET22b-ScFv-GFP and BL21(DE3)/pET32a-ScFv-GFP were 0.930 and 0.918 respectively, while there were no obvious fluorescence signals. Expression product of BL21(DE3)/pET22b-GFP-ScFv and BL21(DE3)/pET32a-GFP-ScFv had strong green fluorescence signals, but the recombinant protein lost the activity of bonding with the antigen. SDS-PAGE results showed that the recombinant plasmids pET22b-ScFv-GFP, pET22b-GFP-ScFv, pET32a-ScFv-GFP and pET32a-GFP-ScFv expressed in host bacteria would be degraded.The single VH has been proved to have the ability to bind antigen by our previous research. On the basis of that, recombinant plasmids pET22b-VH-GFP, pET22b-GFP-VH, pET32a-VH-GFP, pET32a-GFP-VH were constructed, and then they there transformed into E.coli BL21(DE3).After inducing and expression, the products were detected by ELISA and fluorescence microscopy, the titer of the fusion protein from BL21(DE3)/pET22b-VH-GFP was 0.915, while no fluorescence signal was produced. The expression product from BL21(DE3)/pET22b-GFP-VH had a strong fluorescence activity, but the titer was just 0.223. It was concluded that pET32a-VH-GFP, pET32a-GFP-VH could be expressed in E.coli BL21(DE3) better, and the target protein had dual activity, the antibody liter was 0.920 and 0.506, the fusion protein can transmit certain intensity of bright under the excitation of 488nm, the expression yields were 145μg/L,131μg/L, respectively.In conclusion, this research showed that the recombinant strains of BL21(DE3)/pET32a-VH-GFP and BL21(DE3)/pET32a-GFP-VH could express the fusion protein with dual activities. If the fusion protein binds with AFB1, a certain intensity of fluorescence will be emitted, and we can get the quanlitative of AFB1 through the fluoresence without the enzyme, and this will overcome the instability of the detecting result. The specific equipment such as fluorescence spectrophotometer could be used to get the quatitative of AFB1. Our research laid a good foundation for application of the flourescent single-chain antibody in AFB1 detection.
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