The Development Of Fluorescent Probe Of Phycobiliprotein/streptavidin And Application In Early Diagnosis Of Liver Cancer

Phycobiliprotein is a kind of specific protein which can capture light in algae.The phycobilin coupled with subunits has specific absorption spectrum and fluorescence emission spectrum.Meanwhile,Streptavidin binds with biotin in high strength and specificity which is also widely used inthe field of immunodetection.We build the phycobiliprotein/streptavidin（Streptavidin-phycobiliprotein）bifunctional molecules based on the excellent features of phycobiliprotein and streptavidin,.It has a excellent fluorescence properity of phycobiliprotein,as well as an efficient and specific targeting characteristic when binding with biotin.The main contributions are as follows:We used the technology of overlapping PCR fusing the phycobiliprotein gene with the streptavidin gene and constructed the fusion sequence of sa-cpcA based on the P5 vector（pCDFDuet-cpcA-cpcE/F-hox1-pcyA）preserved in the laboratory,finally getting the recombinant vector of pCDFDuet-sa-cpcA-cpcE/F-hox1-pcyA.Based upon this,we replaced the key gene cluster of phycocyanobilin hox1-pcyA by the key gene cluster of phycoerythrin hox1-pebS,constructedtherecombinantexpressionvectorof pCDFDuet-sa-cpcA-cpcE/F-hox1-pebS which can express the SA-PCA-PEB.Then we opitimized the condition of protein’s expression systematically.Eventually we get the best conditions for the expression of recombinant proteins:Using the TB medium in37℃,and when the OD₆₀₀ is 1.3,adding 2 mm lactose to induce protein expression.The induced temperature is decreased to 18℃and continue to culture for 22 h.The final yield of recombinant protein is 60 mg/L.We purified the protein by Ni²⁺affinity chromatography from the supernatant.The recombinant protein has a high purity above 95%.The anaLysis of the properties of recombinant proteins’spectroscopy and biotin binding activity showed that the maximum absorption wavelength of SA-PCA-PCB is at 624 nm and fluorescence emission wavelength is 646 nm.The fluorescence quantum yield is 0.10 and the biotin binding activity is 2.2 U.The maximum absorption wavelength of SA-PCA-PEB is at 556 nm and fluorescence emission wavelength is 568 nm.The fluorescence quantum yield is 0.41 and the biotin binding activity is 4.1 U.In order to keep the stability of its fluorescence properties and biotin binding ability,we explored the storage conditions of the recombinant protein.According to the study,the optimized condition is:in PBS buffer（pH 7.0）at the temperature of 4℃contained 750 mM NaCl,and 1 mM EDTA.We compared three different detection methods including direct method,indirect method and double-antibody sandwich ELISA to observe their effects on the detection sensitivity and stability.The results show that,compared to the direct method and indirect method,double sandwich ELISA method had the good correlation between concentration and intensity with stronger specificity.Then we optimized the condition of double sandwich ELISA,the optimized condition of ELISA detection is captured antibody at 4℃overnight,the concentration of captured antibody and biotinylation detected antibody is 5μg/mL,concentration of fluorescent probes is 10μg/mL.According to the opitimized condition,the concentration of SA-RPE probes for detection of AFP from 0 to 50ng/mL had a good linear relationship and the minimum detection limit is 0.19ng/mL.We used two kinds of Phycobiliprotein/streptavidin probes（SA-PCA-PCB and SA-PCA-PEB）which are constructed as above to detect two kinds of markers of liver cancer（AFP and CEA）.The results showed that as for those using the probe of SA-PCA-PCB and SA-PCA-PEB,AFP and CEA had a good linear relationship within 0 to 50 ng/mL,the minimum detection limit of AFP is 1.01 ng/mL and 0.25 ng/mL,and the detection limit to CEA is 1.12ng/mL and 0.28ng/mL.This result shows that the probe has reached the detection standard of liver cancer and it has a good potential applications of clinical detection.