Construction, Expression Of Fusion Protein A-enhanced Green Fluorescent Protein (PA-EGFP) And Its Application To Immunoassay

The aim of exploitation and application of new immunoindicators is to enhance the sensitivity specificity and to make the assay process easier and quicker. Genetic engineering is an important technique in biotechnology. Through recombinant DNA technology, the genetic conjugation between labels and binding proteins retains the bifunctional effects of the two proteins. There is no need to purify indicator and antibody,and it can avoid complex and low efficiency chemical crosslinking between protein and enzyme. In this research, we constructed a recombinant fusion protein of protein A (PA) with enhanced green fluorescent protein (EGFP) as a signal protein. The fluorescence can be detected by observing the green light emission without any additional substrates or cofactors. Protein A has the ability to selectively bind to immunoglobulins G(IgG) of many mammalian, via Fc region of IgG. The fusion protein could be applied to immunoassay, as a new alternative reagent.The EGFP gene was amplified by PCR from plasmid pT1EGFP-N1. The amplified fragment was digested with EcoR I and Pst I, and then subcloned into the plasmid pEZZ18 which was digested with the same restriction endonucleases, finally we got the plasmid pEZZ-EGFP. The vector pEZZ-EGFP contains the protein A signal sequence and two synthetic "Z" domains, the protein can be expressed as fusions with the "ZZ" peptide and secreted into the liquid cultures under the direction of the Protein A signal sequence. Expression is controlled by the lacUV5 and Protein A promoters and is not inducible. Without any additional substrates or cofactors, fluorescence can be observed. When the recombinant strains incubated on the plate(Amp+) ,it can emit green fluorescence under the UV light. Thus,we can select the recombinant rapidly and simply on plates containing Amp.The purified PA-EGFP migrated at approximately 44kD in SDS-PAGE, and this corresponds to the theoretical molecular weight. The measurement of fluorescence properties and competitive ELISA indicated the plasmid pEZZ-EGFP correctly expressed in E.coli. PA-EGFP retains the bifunctional effects of protein A and EGFP. PA-EGFP has an absorbance/excitation peak at 489 nm, and has a peak emission at 511 nm. The excitation and fluorescence emission spectra of PA-EGFP are similar to what were reported. The linear relationship of the fluorescenceintensity-concentration standard curve of the purified PA-EGFP is rather good. So we can assess the level of excretion of PA-EGFP into the liquid cultures easily and quickly just by detecting the fluorescenceintensity of the supernatant. PA-EGFP secreted into culture medium with 1% glycine, 1% Triton X-100 is 6-fold higher than that without the two chemicals. The final yield of PA-EGFP is estimated to be 6.44 mg/liter.When PA-EGFP applied to Dot-ELISA,HRP/DAB system is more sensitive than PA-EGFP detection. Sensitivity in this experiment was 50ng IgG for fluorescence detection, compared to 6.25ng IgG for HRP/DAB detection. But PA-EGFP detection is rapid and simple and does not need any additional substrates or cofactors. We used PA-EGFP as the substituted second-antibody to detect actin in rat's neurocyte. We saw the green fluorescence which was the formation reticularis marked by PA-EGFP. This is a good way to observe the actin in rat's neurocyte.