Expression Of Recombinant Rubella Virus E1 Protein And Its Epitope In Escherichia Coli

ObjectivesTo research efficient recombinant antigens for serological assays for rubella virus infection, full-length rubella virus E1 gene was synthesized with overlapping PCR-Restriction enzyme ligation method and major E1 epitope gene was amplified by PCR; Full-length E1 gene and E1 epitope gene were constructed into prokaryotic vectors for expression of recombinant antigen, respectively.MethodsBioinformatic analysis of E1 gene was done and the E1 sequence containing Escherichia coli preferred codons was designed. Two restriction enzymes were used to cut the full-length sequence of E1 to generate three fragments. Which were then amplified, respectively. With overlapping PCR using multiple pairs of oligonucleotide primers, a full-length of E1 sequence was assembled via restriction enzyme ligation. The full-length of E1 sequence was cloned into expression vector pET32a. The recombinant plasmid pET32-RV E1 was confirmed by PCR, restriction enzyme digestion and DNA sequencing. The gene of epitope,195-305 amino acid residues of the rubella virus E1 glycoprotein, was amplified from new synthesized E1 sequence, and cloned into expression vector pET32a to construct recombinant plasmid pET32-E1epitope-The expression of the two recombinant proteins was induced by IPTG, and analyzed by SDS-PAGE and Western blotting.Results1. The three fragments were amplified, and verified by DNA sequencing. The full-length sequence was inserted into plasmid pET32a which was confirmed as the designed one. The recombinant protein was expressed with IPTG inducement and identified by western blotting.2. The desired epitope gene fragment was amplified with PCR and confirmed with DNA sequencing. The sequence was cloned into plasmid pET32a, which was confirmed as the desired with PCR, restriction enzyme digestion and DNA sequencing. Western blotting showed the expression of recombinant protein with IPTG inducement. The recombinant protein of high level was expressed at 6 hours after induced with 0.1 mmol/L IPTG.ConclusionsThe rubella virus E1 gene sequence containing Escherichia coli preferred codons was synthesized with overlapping PCR-Restriction enzyme ligation method, and the plasmid pET32-RV E1 was constructed. The plasmid pET32-E1epitope for E1 epitope gene was constructed too. The recombinant full-length El protein and E1 epitope was expressed.