Expression And Purification Of HPV16, 18 E6E7 Fusion Protein In E.coli

ObjectiveThe prokaryotic expression vector pET28a-HPV16-E6E7 and pET28a-HPV18-E6E7 were constructed for the fusion protein of HPV16 and HPV18 E6E7.Expressed the fusion proteins in E.coli,purified and tested its physical and chemical properties.The project provided a possible basis for the preparation of HPV detection reagent in vitro and the development of HPV therapeutic vaccine.Methods1.Accodering to the genomic information of HPV16 and 18 published in Genbank,N₀01526.4?HPV16?and NC₀01357.1?HPV18?,removed the termination codon TAA of E6 gene and inserted a flexible amino acid?Gly4Ser?3between E6 and E7 genes to connect them.2.Designed the primers with restriction sites?BamH ?/Xho ??and protective bases according to the genetic information of HPV16/18 E6 and E7 genes.The primers between E6 and E7 genes included the sequence of flexible amino acids.3.Amplified the HPV16/18 E6 and E7 genes under the action of taq enzyme,by using the extracted genome as a template;then,amplified HPV16 and 18 fusion gene E6E7 using the amplified E6,E7 fragment as a template;recovered the gene fragment of E6E7.4.Constructed the recombinant expression plasmids pET28a-HPV16-E6E7 and pET28a-HPV18-E6E7 by inserted the E6E7 fusion gene into the plasmid pET28 a with connection and conversion experiment;identified the recombinantplasmid DNA by colony PCR,agarose gel electrophoresis,nucleic acid sequence alignment,etc.5.Transfered the recombinant plasmid into BL21,and induced the expression of the corresponding fusion protein at 28 ? and IPTG 0.5 mmol/L.6.Identified the molecular weight and purity of the fusion protein by SDS-PAGE and Western blot.7.Purified the fusion proteins HPV16-E6E7 and HPV18-E6E7 by Ni_NTA.8.Calculated the concentration of fusion protein by BCA.Results1.HPV16/18 E6 and E7 gene fragments and HPV16 and 18 E6E7 fusion gene were successfully obtained.2.Prokaryotic expression plasmids pET28a-HPV16-E6E7 and pET28a-HPV18-E6E7 were successfully constructed.The size,direction,insertion site and reading frame of the target gene fragments inserted in recombinant plasmids were confirmed by double digestion and nucleic acid sequencing technique.3.The fusion proteins HPV16-E6E7 and HPV18-E6E7 were highly expressed under the conditions of 28 ? and 0.5 mmol/L IPTG.4.The physical and chemical properties of the fusion protein were analyzed by SDS-PAGE.The apparent molecular weights of the fusion proteins HPV16-E6E7,HPV18-E6E7 with six histidine labels were about 37 KD and 39 KD,which were in accordance with the expectation.5.Western blot analysis showed that the fusion proteins HPV16-E6E7 and HPV18-E6E7 is the target proteins.6.Two fusion proteins were purified by nickel column affinity chromatography,and the purified target proteins were obtained.7.The concentration of fusion protein HPV16-E6E7 was 2.15 mg/mL,and the concentration of fusion protein HPV18-E6E7 was 1.30 mg/mL,measuered by method BCA.ConclusionThe recombinant prokaryotic expression vectors pET28a-HPV16-E6E7 and pET28a-HPV18-E6E7 were successfully constructed.The fusion proteins HPV16-E6E7 and HPV18-E6E7 were highly prokaryotic expression,and purified proteins were obtained.The target protein laid the foundation for further development of HPV in vitro detection reagents and HPV therapeutic vaccines.