Clone Of HPV18L1 Gene From Human Cervical Carcinoma Tissues In GuangDong And Its Expression In Pichia Pastoris

ObjectiveTo investigate the structure specificity of human papillomavirus(HPV)type 18 in Guangdong area and to construct its Pichia pastories secretion type expression vector.Then transfer the reconstructed expression vector into P.pastoris GS115,to induce its expression and optimize the conditions of HPV18 L1 expression in GS115,Subsequently,the expression of the gene in P.pastories was evaluated.Methods1.To amplify the HPV18 L1 gene from human cervical carcinoma tissues in Guangdong area by PCR,and then cloned the HPV18 L1 gene into P.pastories expression vector.The HPV18 L1 gene was sequenced and analyzed.2.The recombinant vector pPICZαB-HPV18 L1 was transformed into P.pastories GS115 by electroporation and chemical conversion,the electroporation conditions were optimized. Positive recombinants were screened through phenotype and Zeocin resistance. Recombinant yeast expression strains were analysised by PCR.3.Methanol with a final concentration of 0.5%(V/V) was added to fermentation broth to induce HPV18 L1 protein induction.The expressed protein in P.pastories was detected by SDS-PAGE and Western blot methods.Results1.HPV18 L1 gene was amplified successfully from human cervical carcinoma tissues in Guangdong area,There are 4 nucleotide differences between Guangdong strain and Germanic standard strain,the two sequences are found with similarities of 99.8%,3 nucleotide mutations lead to the changes of amino acid.2.Recombinant vector pPICZαB-HPV18 L1 was constructed.The vector was confirmed by restriction endonuclease digestion and sequencing methods. 3.Successfully transfected the recombinant vectors into the P.pastories,and obtained the best electroporation conditions.4.The expression of HPV18 L1 protein:a.At the temperature of 30℃,methanol with a final concentration of 0.5%(V/V) was added to induce the GS115/pPICZαB-HPV18 L1 to express the HPV18 L1 protein.The result of SDS-PAGE indicated that the aimed protein can be detected in fermentation broth,b.The optimal induction conditions were found to be:30℃for induction temperature,0.5%(V/V) methanol,and 72h for induction time. Expression level of HPV18L1 was found to be satisfactory with 105mg/mL in P.pastories, and the product counted for about 70%of the total proteins in the expression system.Conclusion1.There are nucleotide differences of HPV18 L1 gene among Germanic standard strain, Chinese other region strains and Guangdong strain.2.Guangdong HPV18 L1 P.pastories expression vector was constructed.The optimal electroporation conditions were explored.3.The HPV18 L1 protein was expressed in P.pastories,and high level expression was obtained in the optimized conditions.