Construction And Expression Of Single Chain Variable Antibody Fragment With Hepatitis B Surface Antigen

Objective:We construct a recombinant plasmid containing hepatitis B surface antigen sc Fv gene,which can express the single chain antibody of hepatitis B surface antigen in eukaryotic cells,in order to explore whether it has the activity and the ability to specifically combine with human hepatitis B surface antigen.Methods:1.Construction of the recombinant plasmid.The plasmid containing the single chain antibody gene of hepatitis B surface antigen was synthesized,and the single chain antibody gene of hepatitis B surface antigen was obtained by PCR.It was inserted into the eukaryotic expression vector p Fuse-m Ig G2-Fc,then we got the recombinant plasmid named p Fuse-m Ig G2-Fc-Sc Fv.2.Identification of the recombinant plasmid.The recombinant plasmid was digested and sequenced.3.Collection of the target protein.The recombinant plasmid correctly sequenced was transfected into 293 F cells.The target protein can be expressed and secreted into the cell culture supernatant.We got the target protein via collecting the cell culture supernatant.And then we use a Protein G agarose affinity chromatography and a super filter to purify and concentrate the cell culture supernatant respectively to obtain the target protein with higher purity and concentration.4.Detection of the target protein purity.The assay was performed by SDS-PAGE gel stained by Coomassie brilliant blue and Western blotting.5.Test of the target protein activity and specific binding capacity.We detected the target protein activity and its specificially binding ability to human hepatitis B surface antigen by a native-PAGE gel stained by Coomassie brilliant blue,ELISA,immunohistochemistry and immunofluorescence.Results:1.After the digestion of the recombinant plasmid,two theoretical bands were obtained by agarose gel electrophoresis,and the sequencing results showed that the inserted sequence was correct.2.The target protein with relative molecular weight of about 50 KD and high purity was obtained.3.The results of ELISA,immunohistochemistry and immunofluorescence showed that the target protein had specifically binding ability to human hepatitis B surface antigen.Conclusions:1.The recombinant plasmid was successfully constructed.2.The single chain antibody of hepatitis B surface antigen was successfully expressed in eukaryotic cells.3.The specifically binding ability of the single chain antibody of hepatitis B surface antigen to human hepatitis B surface antigen was identified.