Cloning,recombinant Expression And Fluorescence Function Of The Cyanobacteriochrome SPI1085g2 From Spirulina Subsalsa

Heavy metal pollution has threat to human life and the environment seriously.To establish a sensitive and selective method become particularly important.Fluorescent proteins exhibit an important value applied in biosensors,biomarkers,and fluorescence imaging.Recently,biliproteins derived from cyanobacteriochromes attracted a wide attention due to their small molecular weight,high fluorescence quantum yield and spectral diversity.Therefore,it is feasible to use fluorescent protein in biosensor technology to detect heavy metal ions.In this study,the gene SPI1085g2 encoding the CBCRs was amplified from S.subsalsa FACHB351 genomic DNA by the polymerase chain reaction（PCR）,and constructed into p ETDuet-1 by molecular cloning.The recombinant plasmid p ETDuet-SPI1085g2 was co-expressed with PEB in E.coli BL21（DE3）.The biliprotein PEB-SPI1085g2 exhibited the maximum absorption peak at 523 nm and the maximum fluorescence emission peak at 555 nm,with fluorescence quantum yield nearly close to 1.This means it is an excellent fluorescent biomaterial.In order to study the physical and chemical characteristics of PEB-SPI1085g2,we researched its fluorescence stability.The p H stability showed that PEB-SPI1085g2 had good stability under neutral or alkaline conditions（p H≥6）,and fluorescence quenching occurs under strong acid conditions（p H≤4）,its stability of p H is obviously better than that of YFP;The results of thermal stability showed that the fluorescence intensity of PEB-SPI1085g2 was stable in the range of 25℃to 60℃,when the temperature is higher than 60℃,the fluorescence intensity decreases obviously.The photostability experiment showed that when the sample was bleached with blue light（450s）,the fluorescence of PEB-SPI1085g2 lost about 20%at the initial stage（800s）,and then the fluorescence intensity（within 1800 s）tended to be unchanged.The salty stability showed that when the salty concentration up to 5 mol/L,the fluorescence intensity of PEB-SPI1085g2 was still 80%of original example,compared with 20%of the original YFP fluorescence intensity in the salty concentration of 2 mol/L,which indicateed that PEB-SPI1085g2 had high salty stability and was suitable to apply to biosensors in high salty environment.In order to develop a new biosensor for the detection of heavy metals,we tested the response of PEB-SPI1085g2 to metal ions The results showed that PEB-SPI1085g2 below 10μmol/L had a specific response to Cu²⁺.Besides,Cu²⁺concentration and fluorescence intensity showed a dose-effect relationship,which laid a foundation for the development of Cu²⁺biosensor based on the biliprotein PEB-SPI1085g2.On the other hand,SPI1085g2 and green fluorescent protein（GFP）or cyanobacteria photosensitive pigment（All2699g1）were fused and pigmented with PEB in E.coli to construct fusion biliprotein PEB-GFP::GGS::SPI1085g2 and PEB-All2699g1::GGS::SPI1085g2.The results showed that PEB-GFP::GGS::SPI1085g2exhibited the absorption peak at 398、520 nm and the fluorescence emission peak at 509、544 nm,while PEB-All2699g1::GGS::SPI1085g2 exhibited the absorption peak at 523、576 nm and the fluorescence emission peak at 555、590 nm,which laid a foundation for the further construction of fluorescence resonance energy transfer pairs based on SPI1085g2 to facilitate the development of new biosensors for the detection of environmental pollutants.