Abstract: This work aims to construct eukaryotic expression vector of the extracellular domain of gE gene for generating stable soluble gE protein in HEK293F cells,and to obtain specific monoclonal antibody of gE by hybridoma screening. Bioinformatics was used to analyze the gE gene sequence,and the expression vectors pcDNA3.4-gE-6×His and pcDNA3.4-gE-mFc were constructed and then transfected into HEK293F cells via transient transfer method for the expression and further purification. Western blot and SDS-PAGE were used to identify and compare the fusion protein gE-6×Hia and gE-mFc. Pseudorabies inactivated whole virus was used to immunize the mice,hybridoma fusion cells were obtained by electrofusion,positive hybridoma cell lines stably and specifically binding to gE protein were screened by gE-mFc protein and IFA. As results,the pcDNA3.4-gE-6×His and pcDNA3.4-gE-mFc expression vectors were successfully constructed and the gE-6×His and gE-mouse Fc soluble proteins were obtained after expression and purification. The results of SDS-PAGE and Western blot showed that gE-mFc had a higher expression level and better stability than gE-6×His,and the purity reached 85% after only one purification. Further,9 stably and specifically hybridoma positive cell lines were screened using gE-mFc protein. In sum,this is the first time that stable and soluble gE protein is expressed by mammalian cell expression system,and by which gE specific monoclonal hybridoma cell lines are screened,laying a fine material foundation for the development of pseudorabies diagnostic reagents.

Key words: pseudorabies virus, gE, eukaryotic expression, monoclonal antibody