Development Of A Novel Gaussia-luciferase-based System For The Detection Of Antibody

Gaussia luciferase (GLuc) is novel luciferase which is purificatied from Gaussiaprinceps.GLuc could use coelenterazine as substrate to excite475nm fluorescence.Aswe know, GLuc is the smallest but the brightest luciferase among the knownluciferases such as Firefly Luciferase, Renilla Luciferase,Copepod Luciferase ect.SoGLuc will have a bright future in the application of detection.GB virus C also named HGV was a novel virus which was isolated from aNon-A-E hepatitis patient in1995.As the virus was discovered from a hepatitis patientwithout any known etiology, It was considered to be responsible for hepatitis.However, evidences from epidemiological investigations of this virus show thatthis virus is also widespread in healthy people.For example, about2%-5%of healthyindividuals in America are infected by GBV-C, but none of them has any symptoms ofhepatitis.Recently, an amount of investigations have confirm that this virus isreplicating in PBMC cells rather than liver cells.But until now,we still don’t know theexact replication site of this virus. And there is no any exact disease that associatedwith GBV-C.However,a lot of researchers has confirmed that GBV-C can inhibitatthe replication of HIV.Further more,some research has proved that the protein E2ofGBV-C could inhibitat the replication of HIV in many ways.And the proteinNS3,NS5a and anti-E2Ab also can inhibitat the replication of HIV.HIV,which was found in America in1981,has infected more30million pople andlead to the death of more than12million.Unfortunately,until now there is still no anyeffictive treatments for the terrible disease.As to some special features of HIV,thevaccine against it also has a uncertain future.Until now, ELISA is still the key method for diagnosis of these two viruses. As toGBV-C, the antibody to the protein E2is the marker of GBV-C serum positive.However, E2is an envelop protein of GBV-C. there are3glycosylation sites on E2.And some researchers has confirmed that it can’t react with anti-E2Ab in humanserum if E2is expressed in prokaryotic cells,it is because that the anti-E2Ab onlyrecognise with the conformational epitope of E2.However,if E2is expressed inprokaryotic cells,it can’t be modified efficiently and it may not have the rightconformational epitopes.In order to get the right conformational epitopes,E2isalways expressed in eukaryotic cell.But purification of E2from eukaryotic cell is noteasy,and this will cost a lot of time and money,which may block the development ofdiagnosis of these two viruses.In this work, we combine Gluc with E2of GBV-C or P24of HIV.As to Gluc issecreted from cell to supernatant, so E2of GBV-C will be glycosylated in this process,but P24of HIV will not be glycosylated since it has no glycosylation sites.And wecan also use the supernatant of the cell culture to diagnosis of these two virusesdirectly without purificating them from the cells.We use Immunomagnetic beads with proteinA which can be used to adsorbedIgG.And we mix serum,the GLu-L-AF-E2or GLu-L-P24and the Immunomagneticbeads together to diagnose these antibodies.In this work,we use this method to detectGBV-C positive serum and HIV positive serum successfully.In summary,we havedeveloped a novel method based on GLuc to detect anti-GBV-C-E2Ab and anti-P24Ab.Comparing with conventional ELISA,This method is easy to establish because itdoes not need to purified antigens from the cells which will also save us a lot of timeto control some novel pathogens.