Synthesis of rubella virus E1 gene and construction of plasmid vector

Aim:Rubella virus E1 gene was synthesized with overlapping PCR-Restriction Enzyme ligation method. Methods: Bioinformatics 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 plamid pET32a.The recombinant plasmid was confirmed by PCR,restriction enzyme digestion and DNA sequencing.Results: The three fragments were separately synthesized,and identified by DNA sequencing.The full-length sequence was inserted into plasmid pET32a successfully,which was confirmed as the one designed.Conclusion:The rubella virus E1 gene sequence containing Escherichia coli preferred-codons was synthesized with overlapping PCR-Restriction enzyme ligation method,and the plasmid pET32-RV rE1 for the synthetic E1 gene was constructed.