Construction And Expression Of HPV16 L1\E7 L2\E7 Recombinant Vaccine In Pichia Pastoris And The Immunogenicity Preliminary Study Of HPV16 L1/E7 Vaccine

Cervical cancer is one of the important reasons of cancer death in women. Many studies have shown that about 90% of cervical cancer tissue could be detected in the HPV16 subtype, which is closely related to squamous cell carcinoma, and 90% of cervical cancer was squamous cell carcinoma. Therefore, high-risk HPV16 is the main reason for women's cervical cancer.Pichia pastoris expression system, which is now widely used, is an efficient eukaryotic system in expressing the heterologous protein. The expression system has virtues of good stability, high expression, high secretion, easy industrialization and low cost, as well as glycosylation and post-translated modification.The present study was done as follows:①Cloning of HPV16 L1/E7_ L2/E7 gene and construction of plasmid pPIC9k-L1/E7 and pPIC9k-L2/E7;②Transforming the constructed expression plasmid pPIC9k-L1/E7 and pPIC9k-L2/E7 into P·pastoris via electroporation. Afterward, HPV16-L1/E7 and HPV16-L2/E7 were secreted from the secrecting expressing strain with high efficience and stability by the P·pastoris;③Studied the large-scale fermentation process of HPV16L1/E7 in the New Brunswick Scientific bioflow 5000 fermentor;④Researched the immunogenicity and anti-tumor effect of the vaccine.The results of the research were as follows:1. Cloning of HPV16L1 gene and construction of cloning plasmid pPIC9k-Ll/E7 and pPIC9k-L2/E7The DNA sequences encoding HPV16L1, L2 and E7 were obtained by PCR using specific cloning primers from human genome. Then the purified PCR production was inserted into pPIC9k vector. The results showed that the recombinant cloning vector pPIC9k-L1/E7 and pPIC9k-L2/E7 were constructed correctly after being identified by endonuclease digestation assay and DNA sequencing.2. Screening and identification of stable and efficient secretion and expression in P·pastoris(1) Competent P·pastoris preparation and electroporation (In accordance with the Pichia expression manual)After identification by endonuclease digestation assay and sequencing, the expression plasmids pPIC9k-L1/E7 and pPIC9k-L2/E7 were linearized, and transformed into P·pastoris via electroporation.(2) Confirmed the growing shape of transformants on MD/MM plate, and then screened His+Muts transformants. The multiple copy integration of recombinants was screened on the MM plate with 2.0 mg/ml G418. The genomic DNA of the transformed yeasts was extracted and characterized with PCR using the expression primers.Expression and identification of HPV16L1/E7 and HPV16L2/E7:positive yeast clones were tested by PCR, and then the fusion protein was induced with 0.5% methanol in BMMY media. The results showed that only pPIC9k-L1/E7 fusion protein was successfully expressed with strong immunogenicity, but pPIC9k-L2/E7 fusion protein had not been expressed successfully. Test showed that the expression yield of fusion protein was 50ng/ul.Many factors would affect the expression of foreign protein in P·pastoris, especially pH, methanol concentration, and inducing time, so this study carried out optimization experiments from these three aspects. The results showed that the foreign proteins would be secreted most significantly in P·pastoris at pH of 6.0, with inducing for 60 hours and methanol concentration of 0.5%.3. The study of immunogenicity and anti-tumor aspect of the fusion protein(1) In this study, in order to determine the immunogenicity of the HPV16-L1/E7 fusion protein, C57BL/6 mice were immunized with the fusion protein, and then the humoral and cellular immune responses were detected. The results showed that the protein vaccine had a strong immunogenicity, and produced high-titer antiserum that could maintain a long time, which showed that the fusion protein could induce humoral immunity in mice. The spleen lympHocytes could yield lympHocyte proliferation against HPV16-L1/E7 protein. (2) In preventive tests, protein vaccine was injected into C57BL/6 mice, and then the mice were tumor-born. The results showed that immunized group had slow-growing and smaller tumors and the average weight was 2.08 g; the control group had rapid-growing and bigger tumors and the average weight was 2.99 g.(3) In therapeutic trial, C57BL/6 mice were tumor-born firstly, and then protein vaccine was injected into C57BL/6 mice till tumors grew to a certain size. Results showed that the immunized group had the obvious effect in inhibition of tumor development, and the average tumor weight was 2.57 g, but the PBS group had no inhibition and the average weight of tumor was 3.03 g.In short, this HPV16 L1/E7 protein vaccine had a satisfactory prevention and treatment effect to cervical cancer, which established significant foundation for the treatment of cervical cancer and related drug research.