Prokaryotic Expression of Human Cytomegalovirus pp65 Gene Fragment and Identification of Expressed Product

Objective To construct the prokaryotic expression vector for human cytomegalovirus(HCMV) pp65 gene fragment and test for the reactogenicity of express product. Methods Amplify the nucleotides 1087-1515 of HCMV pp65 gene by PCR using plasmid pGEM-T-pp65 containing the full-length of HCMVpp65 gene as template, identify by restriction analysis and insert into vector pET-21a(+). The constructed recombinant plasmid pET-21a(+)-pp65 was transformed to E. coli DH5α, and positive clones were screened and identified by PCR, from which recombinant plasmid was extracted and identified by sequencing, then transformed to E. coli BL21(DE3) for expression under induction of IPTG. Purify the expressed product by affinity chromatography and identify by Western blot. Results Restriction analysis and sequencing proved that recombinant plasmid pET-21a(+)-pp65 was constructed correctly and the expression level of target protein reached a peak value after induction with 1. 0 mmol / L IPTG for 5 h. The expressed fusion protein mainly existed in a form of inclusion body, and showed good reactogenicity after purification. Conclusion The prokaryotic expression vector for HCMV pp65 was successfully constructed and expressed in E. coli.