Expression, Purification Of Zta-P54Fusion Protein In Escherichia Coli Of Epstein-barr Virus And Research The Detection Methods

EB virus is closely related to occurrence of nasopharyngeal carcinoma,the virusis integrated into the host cell chromosome in the form of circular DNA.As NPClocated relatively deep, early lesions in clinical routine medical examinations are noteasy to find, and diagnostic imaging such as CT is hard to distinguish between theearly nasopharyngeal carcinoma and other nasopharyngeal hyperplasticlesions.Therefore, less traumatic, low cost, fast detection of serological diagnosticmethods are currently hot and difficult study of early diagnosis of nasopharyngealcarcinoma.In view of this, we proposed the establishment of detection of EB virusantibody in serum of patients through the protein antigen of the BZLF1-BMRF1fusion gene of nasopharyngeal carcinoma,thus Providing technical methods for earlydiagnosis and screening of nasopharyngeal carcinoma.In this study,according to the pGEX5T-BZLF1-BMRF1plasmid sequencingresults,we designed primers using premer5.0and obtained target gene fragment fromthe plasmid containing EB virus antigen gene sequences through PCR method.Andthen,we connect the target gene and plasmid pET32a to construct the recombinantplasmid pET32a/BZLF1-BMRF1.The plasmid expressed in Escherichia ColiBL21(DE3) by IPTG induction.We used SDS-PAGE to analysis proteinsolubility,western blotting to detect the immunogenicity of the expressed protein,andpurified the recombinant protein by optimized ammonium sulfate precipitation, ionexchange chromatography and affinity chromatography.Purified Zta-P54fusionprotein was labeled by Horseradish peroxidase, chessboard method was used todetermine the working concentration of recombinant antigen package andenzyme-labeled antibody,preliminary establishing a new ELISA method of screeningand diagnosis of nasopharyngeal carcinoma,and its specificity, precision, and clinicaleffect was analyzed.Results showed that:1) we successfully constructed therecombinant plasmid PET32a/BZLF1-BMRF1and achieved the soluble expression ofZta-P54fusion protein;2) We have established the optimization and purification procedures of the Antigen, purity was up to96.5%;3) ELISA indirect detectionmethod of Nasopharyngeal carcinoma was established primarily based on expressedprotein,this method is highly in line with the clinical diagnosis,which is expected tobe used in mass screening for the early auxiliary diagnosis and nasopharyngealcarcinoma (NPC), at the same time,we can providing the reference for theestablishment of specific serological diagnosis of nasopharyngeal carcinoma (NPC)method.