Biosynthesis Of Phycobiliprotein Fluorescent Probe And Its Application In Immunofluorescence Assay

Phycobiliproteins,which are protein complex composed of multiple subunits,are light-harvesting antennae found in cyanobacteria,red algae and cryptomonas.Phycobiliproteins covalently bind to one or more algal phycocyanins,which have intense fluorescence and can be used as fluorescent probes for immunofluorescence assay.Compared with radioimmunoassay,enzyme-linked immunosorbent assay and other immuno-labeling techniques,immunofluorescence technology has high sensitivity and safety.It is widely used in environmental monitoring,food inspection and medical examination,and other fields.The phycobiliprotein fluorescent probe has become a hotspot for its high fluorescence intensity,stable property,and safe reliability.As one of phycobiliproteins,the allophycocyanin has excellent fluorescent properties.The allophycocyanin monomer has low molecular weight and is easily cross-linked with other biomacromolecules,so it could be applied as a fluorescent label.There are two main sources of phycobiliproteins,natural extraction and heterologous expression.When extraction and purification process of natural allophycocyanin is complex,the advantages of genetic engineering are that the obtained protein has simple structure,is easy to purify and artificial modification,and has the spectral properties similar to natural phycobiliproteins.In this study,the expression vector containing the fusion gene of streptavidin and allophycocyanin alpha subunit(SLA)and the gene of lyase(cpcS)was co-transformed into Escherichia coli BL21(DE3)with the expression vector containing phycoerythrin synthase gene(Ho1,PebS)or the expression vector containing phycocyanobilin synthase gene(Ho1,PcyA).The recombinant SLA covalently bound PCB(denoted as SLA-PEB)or PEB(denoted as SLA-PCB)were biosynthesized during induction.The expression and activity of SLA-PEB and SLA-PCB were analyzed.The molecular weight of the recombinant proteins was 34.5 kDa and the purity was above 95%.SLA-PEB had an absorption maximum at 550 nm,an emission maximum at 562 nm,extinction coefficient of 402400 M-1·cm-1,quantum yield of 0.91,photobleaching rate of 1.5 × 10-3 min-1,biotin-binding activity of 16.4 U,and chromophorylation rate of 34.2%.SLA-PCB had an absorption maximum at 614 nm,an emission maximum at 633 nm,extinction coefficient of 422400 M-1·cm-1,quantum yield of 0.33,photobleaching rate of 1.7 × 10-3 min-1,biotin-binding activity of 6.9 U,and chromophorylation rate of 21.1%.In addition,the stability of recombinant proteins was analyzed.The inhibitor of EDTA and NaN3 could improve the stability of recombinant proteins.The fluorescence intensity of SLA-PEB and SLA-PCB decreased by less than 10% after stored at 4 ℃ for 80 days,and decreased by about 20.8% and 26.4%,respectively,after stored at 37 ℃ for 7 days.Because of low chromophorylation rate of recombinant proteins biosynthesized in E.coli,an in vitro chromophore attachment experiment was designed.PEB,PCB and CpcS were biosynthesized and purified respectively.The purified recombinant proteins with low chromophorylation rate and bilins were recombined under the catalysis of lyase in vitro.The purified proteins further chromophorylated in vitro were purified,and the spectra were analyzed.The results showed that the chromophorylation rate of SLA-PEB was not significantly improved,and the chromophorylation rate of SLA-PCB increased from 21.1% to 86.5%.The solid-phase immunofluorescence assay for AFP,CEA and NSE was established using recombinant allophycocyanin as fluorescent probes,with commercial SA-PE as control.Analytical performance was evaluated normatively.The linear range of AFP,CEA and NSE was 0.02-50 ng/m L,0.05-200 ng/mL,and 0.1-1000 ng/mL,respectively.The detection range was consistent for SA-PE,SLA-PEB and SLA-PCB used as probes.The detection limit of AFP and CEA was less than 1 ng/m L,indicating that the method has high sensitivity.The intra-assay precision of AFP,CEA and NSE were less than 10%,and the inter-assay precision of AFP,CEA and NSE were less than 15%,indicating that the method has good repeatability.All the components of immunofluorescence assay reagent were placed under 37 ℃ for 3 days and 6 days.Compared with components without treatment,the standard curve of AFP maintained good linearity.The limit of detection increased by less than 10%.The intra-assay precision were less than 10%,while the inter-assay precision were less than 15%.The above data showed that the detection method has good stability.The specificity test showed that the specific deviation of AFP was less than 10%,indicating that the method has good specificity.The specificity test showed that AFP,CEA and NSE antibodies had a strong detection signal with the related antigen,and there was no obvious signal with unrelated antigen,indicating that the detection method has good specificity.In this study,the recombinant allophycocyanin fluorescent probes biosynthesized in E.coli were achieved.High purity proteins were obtained by efficient extraction and purification,and the fluorescence properties of the fluorescent probes were improved by in vitro chromophore attachment,that laid the foundation for the preparation of recombinant phycobiliprotein fluorescent probes.In addition,the method of AFP,CEA,NSE solid phase immunofluorescence assay was established,which laid the foundation for its clinical application.