DNA Shuffling of HPV16E7 and Construction of pcDNA3.1-HPV16E7sh Eukaryotic Expression Recombinant
|School||Hebei Medical University|
|Course||Dermatology and Venereology|
|Keywords||Cervical Cancer DNA shuffling HPV16E7 Recombinant plasmid HPV therapeutic vaccine|
Objective HPV16 and HPV18 are considered the leading cause of cervical cancer in women, especially high-risk HPV E6, E7 genes were confirmed to be transforming gene, stably expressed in relevant organizations, with strong antigenicity, and not in normal tissues With the two proteins. Therefore, E6 and E7 proteins become HPV16-related cervical cancer and precancerous lesions of the ideal therapeutic vaccine target antigen. Study using DNA shuffling reorganization HPV16E7 gene construct HPV16E7 gene mutant library, and successfully constructed the eukaryotic expression plasmid pcDNA3.1-HPV16E7sh, expectations screened out higher immunogenicity HPV16E7, to build a new HPV therapeutic vaccine provides an important theoretical basis and experimental basis. Method 1. DNA template preparation reorganization reorganization: We first extract samples of tissue DNA, tissue DNA extracts were stored at -20 ℃. Then using conventional PCR method using primers designed HPV16E7 detect DNA extraction step on the tissue fluid, screened positive samples, PCR products 1% agarose gel electrophoresis using gel extraction kit HPV16E7 gene fragment was dissolved in pure water stored at -20 ℃. DNase Ⅰ digestion: 30μl enzyme in the reaction system, adding purified HPV16E7, digestion buffer, 15 ℃ for 5 minutes, then add 0.05U DNase Ⅰ, 15 ℃ reaction 2 minutes, 90 ℃ 10 minutes to terminate. Digestion products were 2% agarose gel electrophoresis, excised with a small DNA fragment of about 50bp fragment recovered. No primer PCR: the 50μl PCR reaction system, adding 10μl purified digested small fragments, 10 × PCR buffer, dNTPs, Taq enzyme, water make up 50μl. Reaction conditions: 94 ℃ denaturation for 3 minutes, 94 ℃ 30 秒, 52 ℃ 30 秒, 72 ℃ 35 seconds, 40 cycles, 72 ℃ for 10 minutes. A primer PCR: PCR product taking no primer 1:100 dilution, 50μl PCR reaction was added to the primer, the upper and lower primers, Taq enzyme, 5μl of diluted PCR product no primer, 10 × PCR buffer, dNTPs. Reaction conditions: 94 ℃ denaturation for 3 minutes, 94 ℃ 45 秒, 55 ℃ 1 分钟, 72 ℃ 1 min, 20 cycles, 72 ℃ for 10 minutes. Electrophoresis, if the strip is unclear, the reaction product of this step desirable 10μl, a primer for the second round of PCR, the reaction conditions as above. PCR products were cut plastic recycling approximately 300bp fragment. 2. PcDNA3.1-HPV16E7sh and screening plasmid DNA purification of PCR products amplified product and the plasmid vector pcDNA3.1 restriction endonuclease cleavage reaction 0.5ml sterile centrifuge tube for the 50μl enzyme reaction system by adding HPV16E7DNA (500ng/μl) 32μl, BamH Ⅰ enzyme, EcoR Ⅰ enzyme, 1 × TangoTM, mix gently, 37 ℃ water bath for 2 hours to digest. After digestion solution were purified directly from the digested fragments, the same way double digested pcDNA3.1. Ligation reaction: The ligation reaction system: pcDNA3.1 digested products (100ng/μl) 1.5μl, HPV16E7 digestion product (70ng/μl) 0.5μl, T4 DNA ligase (3u/μl) 0.2μl ultrapure water make up 10μl, 45 ℃ for 5 minutes, to allow re-annealing cohesive ends melting. The mixture was cooled to 0 ℃. Reaction conditions: 15 ℃ water bath for 12 hours. Preparation of CaCl2 competent E. coli DH5α cells and recombinant plasmids and plasmid screening and identification methods using conventional recombinant plasmid, while doing two controls: control group 1: with the same volume of sterile distilled water instead of DNA solution. Control group 2: the same volume of sterile double distilled water instead of the DNA solution, the coated plate coated on only take 5μl bacteria on LB plates without antibiotics. Each ligation reaction liquid taken into 100μl sterile glass rod uniformly coated on the filter medium, 37 ℃ incubated for half an hour or more. Inverted plate and cultured at 37 ℃ for 12-16 hours out flat, put at 4 ℃ for several hours, so that the color completely. The recombinant plasmid was digested: white picked with a sterile toothpick a single colony was inoculated in 5ml LB Amp 50μg/ml containing liquid medium, 37 ℃ with shaking for 12 hours. Rapid extraction kit using a plasmid plasmid double digestion of the plasmid recovered. 1 first with tissue DNA extraction kit extraction 10 specimens of the DNA, to obtain the large fragment tissue DNA, then add HPV16E7-specific primers using conventional PCR screening and purified to obtain about 300bp of about HPV16E7 clip to purified 300bp fragment Substrate with DNase Ⅰ enzyme fragmentation of the fragment, to obtain about 50bp so a DNA fragment digested small fragment step without primer PCR, to obtain a uniform distribution strip electrophoresis, no significant concentration of strips, take the step product diluted by adding restriction sites with primers amplified a 300bp fragment of 300bp with the original template of HPV16E7 restructuring agreement, we got reorganized HPV16E7, namely HPV16E7sh. 2 Use the reconstituted HPV16E7 carrier pcDNA3.1, constructed plasmid pcDNA3.1-HPV16E7sh after the reorganization. First, the success of the pcDNA3.1 vector HPV16E7 and enzymes BamH Ⅰ and EcoR Ⅰ double enzyme digestion. Then the action of DNA ligase and pcDNA3.1 HPV16E7 connection electrophoresis and recovered large fragment of the connection. Previously prepared competent E. coli DH5α cells were transformed with the pcDNA3.1-HPV16E7 competent E. coli DH5α cells, and we picked white colonies were cultured for 12 hours after a single plasmid extraction kit using the extracted plasmid DNA, and agarose gel electrophoresis bands, in order to further identify whether the plasmid was constructed successfully, we use enzymes BamH Ⅰ and EcoR Ⅰ double digestion enzymes extracted recombinant plasmid obtained 5Kb about fragments and 300Bp fragments illustrate HPV16E7sh after DNA shuffling has been successfully transferred to pcDNA3. a vector, the recombinant eukaryotic expression plasmid pcDNA3.1-HPV16E7sh constructed successfully. Conclusion The use of DNA shuffling 1, under the right conditions, can be in the independent evolution of sequence comparison HPV16E7 gene sequence rearrangement HPV16E7 gene construct mutant library, which may include new, higher immunogenicity E7 gene for further research E7 gene in cervical cancer, genital warts and other diseases in the role of the foundation. 2 restructured HPV16E7 genes and the recombinant plasmid vector pcDNA3.1 and pcDNA3.1-HPV16E7sh successfully constructed, in order to further establish highly immunogenic HPV16E7 gene screening model provides the premise, not to further build the foundation HPV therapeutic vaccine.