Cervical cancer therapeutic vaccine based on recombinant attenuated Listeria monocytogenes
A Listeria monocytogenes and therapeutic vaccine technology, applied in the biological field, can solve problems such as defects and low immunogenicity, and achieve the effect of good tolerance and low production cost
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Embodiment 1
[0066] Example 1. Preparation of vaccine bacteria LmΔactAplcB-HPV
[0067] (1) HPV16E6E7 gene fragment synthesis
[0068] Query HPV 16 E6 protein and E7 protein genes from NCBI, use online software to analyze and predict their dominant T cell antigen epitopes, select and combine them into fusion antigen epitope peptide genes, and then optimize according to Listeria codons, A HindⅢ restriction site was added upstream, and an XhoI restriction site was added downstream. Finally, the DNA sequence was directly obtained by synthesis (obtained in the form of the cloning plasmid pUC57-E6E7 provided by the company), and the fragment length was 866bp (such as SEQ ID NO.1 shown).
[0069] (2) Construction of targeting plasmid
[0070] ① Construction of intermediate plasmid pCW203-E6E7
[0071] Plasmids pUC57-E6E7 and pCW203 were extracted according to the instructions of the plasmids, and eluted with 30 μL Elution Buffer.
[0072] pUC57-E6E7 and pCW203 were double-digested with HindⅢ...
Embodiment 2
[0096] Example 2: Evaluation of the therapeutic effect of vaccine strains on cervical cancer
[0097] (1) TC-1 tumor cell culture and establishment of cervical cancer model
[0098] Use RPMI1640 Medium containing 10% fetal bovine serum, 1% penicillin, and streptomycin at 37°C in 5% CO 2 Cultivate TC-1 cells in an incubator until the density reaches about 80%, rinse the cells with Hank’s solution for 2 to 3 times, digest with trypsin and resuspend with PBS for counting, and prepare the cells to a concentration of 1×10 6 100 μl of cell suspension was subcutaneously injected into the right abdomen of 6-8 week-old C57BL / 6 female mice to establish a mouse HPV-infected tumor model, and a macroscopic tumor was formed in about 7 days. Tumor volume according to the formula 1 / 2×(a×b 2 ) calculation (a, b are the longest diameter and shortest diameter of the tumor, respectively). Statistical analysis of data was performed using SPSS software. The t-test was used to compare the means ...
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