Cloning And Secreting Expression Of Human Papillomavirus 16 L1 Gene From Guangdong Area

[Objective]①To clone the human papillomavirus(HPV) type 16 L1 gene in Guangdong area and to investigate its structure specificity;to construct its Pichia pastories secretion type expression vector.②To optimize the HPV16 L1 gene,which was amplified from human cervical carcinoma tissues in Guangdong area,accordine to the codon bias of Pichia pastories and to evaluate the expression of the optimized gene in Pichia pastories.[Methods]①To amplify the HPV16 L1 gene from human cervical carcinoma tissues in Guangdong area by PCR,and then cloned the HPV16L1 gene into Pichia pastories expression vectors.The HPV16 L1 gene were sequenced and analyzed.②To optimize the HPV16L1 gene,accordine to the codon bias of Pichia pastories,then sequencing to evaluate.②The recombinant vectors pPICZαC-HPV16L1 and the gene modified recombinant vectors pPICZαC-HPV16L1m were transformed into Pichia GS115 by electroporation.Positive recombinant were screened through phenotype and Zeocin resistance.Recombinant yeast expression strains were analysised by PCR.④Add 100%methanol to a final concentration of 0.5%(v/v) methanol to maintain induction.Detected the expression of HPV16L1 protein in Pichia pastories by SDS-PAGE and Western blot.[Results]①Successfully amplified the HPV16L1 gene from human cervical carcinoma tissues in Guangdong area.There were 16 nucleotide differences between Guangdong strain and Germanic standard strain,the sequences were 98.99%homology,subsequently changed 8 amino acids it coded.There were 9 nucleotide differences between Guangdong strain and Chinese standard strain,the sequences were 99.18%homology,subsequently changed 4 amino acids it coded.②The hydrophobicity prediction and predicted antigen determinants results of Guangdong strain are different from that of Germanic standard strain.③Successfully optimized the HPV16 L1 gene.After sequenced,all of the 12 sites which need to be mutated were optimized.④The codon optimized recombinant plasmid pPICZαC-HPV16 L1m were constructed.⑤The expression of HPV16 L1 protein:a.Under the condition of 30℃,adding 0.5%(v/v) methanol to induce the GS115/pPICZαC-HPV16 L1 and GS115/pPICZαC-HPV16 L1m to express the HPV16 L1 protein.The result of SDS-PAGE indicated that the aimed protein band can be detected and the expression level of codon optimized GS115/pPICZαC-HPV16L1m was higher than that of GS115/pPICZαC-HPV16L1.b.The optimal induction condition were found to be:30℃for induction temperature,0.5%(v/v)methanol, and 72 hours for induction time.Expression of HPV16L1 was found to be satisfactory (107mg/L)in Pichia pastories,and the product counted for about 45%of the total proteins in the expression system.[Conclusion]①There are nucleotide differences of HPV16 L1 gene among Germanic standard strain, Chinese standard strain and Guangdong strain.②The codon optimized recombinant plasmid pPICZαC-HPV16 L1m were constructed.③The HPV16 L1 protein was expressed in Pichia pastories.And the expression level of codon optimized GS115/pPICZαC-HPV16L1m was higher than that of GS115/pPICZαC-HPV16 L1.