Construction and evaluation of recombinant adenovirus serotype 4 expressing RBD of SARS-CoV-2 Omicron variant
Objective To construct and identify a recombinant adenovirus type 4 expressing spike protein receptor binding domain(RBD)of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)omicron variant.Methods Enhanced green fluorescent protein and RBD dual cis-trans expression cassette was constructed by overlap extension PCR and seamless cloning.The intermediate plasmid pBR322-Ad4-ccdB was generated from ccdB fragment obtained by PCR and pBR322-Ad4 using homologous recombineering technique.After digestion with PacI and SpeI restriction enzymes,the vector fragment was recovered and homologously recombined with the dual cis-trans expression cassette in E.coli GB08Red to acquire the recombinant plasmid pBR322-Ad4-RBD.After linearisation with AsiSI,the recombinant pBR322-Ad4-RBD was transfected into HEK293T cells to generate recombinant Ad4-RBD.Transcription of the RBD gene was detected by RT-PCR and the expression of the RBD protein in transfected cells was assessed by western blot.Results The titer of the recombinant adenovirus Ad4-RBD was 5×108 PFU/mL and the expression of exogenous gene in infected cells was stable.Conclusions The recombinant adenovirus expressing the RBD protein of the SARS CoV-2 Omicron variant was constructed successfully.This may provide a scientific and effective basis for the vaccine development for SARS-CoV-2 Omicron variant.
Severe acute respiratory coronavirus 2 Omicron variantRBD proteinRcombinant adenovirus type 4
NETL
NSTL
万方数据
