DNA Shuffling Of HPV16E7 And Construction Of PcDNA3.1-HPV16E7sh Eukaryotic Expression Recombinant

ObjectiveHPV16 and HPV18 is considered the main pathogen of cervical cancer for women, especially high-risk HPV E6, E7 genes were confirmed to be transforming gene, in the relevant organizations in stable expression, are highly antigenic and, does not exist in normal tissues these two proteins. Therefore, E6 and E7 proteins have become HPV16-related cervical cancer and precancerous lesions of the ideal therapeutic vaccine target antigen. This study uses DNA shuffling technology restrict HPV16E7 gene, constructed HPV16E7 gene mutant library, and successfully constructed the eukaryotic expression plasmid pcDNA3.1-HPV16E7sh, expectations from selected higher immunogenicity HPV16E7, for building new type of HPV therapeutic vaccines provide an important theoretical basis and experimental evidence.Methods1 DNA shuffling of HPV16E7:Preparation of the restructuring of the template: We first of all extracted samples organizations DNA, organizations extract DNA preservation of -20℃. And with conventional PCR method, using primers designed HPV16E7, the step-by-step organizations Detect DNA extract selected positive samples, PCR product of 1% agarose gel electrophoresis followed by gel extraction kit recovery HPV16E7 gene fragments, soluble in pure water -20℃to preserve the reserve. DNaseⅠdigestion: digestion in 30μl reaction system, by adding purified HPV16E7, digestion buffer, 15℃heat 5 minutes, then add 0.05U DNaseⅠ, 15℃reaction of 2 minutes, 90℃10 minutes to terminate. Digestion products were 2% agarose gel electrophoresis, cut around 50bp fragment with DNA recovery of small fragments. No primer PCR: in the 50μl PCR reaction system, by adding 10μl purified after digestion of the small fragment, 10×PCR buffer, dNTPs, Taq enzyme, water filled 50μl. Reaction conditions: 94℃predegeneration 3 minutes, 94℃30 seconds, 52℃30 seconds, 72℃35 seconds, 40 cycles, 72℃extension for 10 minutes. Has primer PCR: to take no-PCR product 1:100 dilution, 50μl PCR reaction system add primers, upper and lower primers, Taq enzyme, 5μl of the non-diluted PCR product of primers, 10×PCR buffer, dNTPs. Reaction conditions: 94℃predegeneration 3 minutes,, 94℃45 seconds, 55℃1 minutes, 72℃1 minute, 20 cycles, 72℃extension for 10 minutes. Electrophoresis observation that if bands unclear, the desirability of this step the reaction product of 10μl, have a second round primer PCR, reaction conditions Ibid. Gel-cut amplification products of about 300bp fragment.2 plasmid pcDNA3.1-HPV16E7sh a Construction and Screening:PCR amplification products of purified DNA and plasmid vector pcDNA3.1 of restriction endonuclease digestion reaction in a 0.5ml centrifuge tube sterilization carried out in 50μl enzyme reaction system, add HPV16E7DNA (500ng/μl) 32μl , BamHⅠenzyme, EcoRⅠenzyme, 1×TangoTM, mixing gently, 37℃water bath for 2 hours to digest. Upon completion of digestion, respectively, directly from the solution after digestion of purified fragments, the same way double-digested pcDNA3.1. Connect with the response: Connect the reaction system: pcDNA3.1 digested product (100ng/μl) 1.5μl, HPV16E7 digestion product (70ng/μl) 0.5μl, T4 DNA ligase (3u/μl) 0.2μl complement ultrapure water 10μl, 45℃heat 5 minutes, so that re-annealing of the sticky-side-chain solution. The mixture cooled to 0℃. Reaction conditions: 15℃water bath for 12 hours. Recombinant plasmid transformation, bolting and verification: Construct recombinant plasmid with routine methods of plasmid construction. At the same time to set up two control group: control group 1: the same volume of sterile double distilled water in place of DNA solution. Control group 2: the same volume of sterile double distilled water instead of DNA solution, but when painted plate 5μl bacilli, only coated in antibiotic-free LB plate. Each stock solution to connect the reaction conversion from 100μl with sterile glass rod uniformly coated on the filter medium, 37℃under cultivation more than half an hour. Inverted plate at 37℃to continue to foster a 12-16 hour flat out put it in a 4℃for several hours, so that the color completely. Enzyme digestion of recombinant plasmid: picked with sterile toothpicks and white single colony inoculated into the Amp 50μg/ml containing 5ml LB liquid medium, 37℃under the oscillation train 12 hours a day. Products extracted by plasmid quick extraction kit were digested by double enzymes in order to identification.Results1 First of all tissue DNA extraction kit extract 10 samples of DNA, has been the organization of large fragment of DNA, then add HPV16E7 specific primer, using conventional PCR screening and purified from about HPV16E7 fragment about 300bp to 300bp fragment purified as substrate with DNaseⅠenzyme fragments of the fragment of about 50bp around the DNA fragment will be digested in small fragments without step primer PCR, have been a uniformly distributed strip electrophoresis map, no significant concentration of bands, check on the step diluted product add restriction sites with primers amplified a 300bp fragment, and the restructuring of the original template 300bp of HPV16E7 consistent, we have been restructured HPV16E7, namely HPV16E7sh.The use of restructured HPV16E7 with vector pcDNA3.1, constructed after the restructuring of the plasmid pcDNA3.1-HPV16E7sh. First of all, success will HPV16E7 and pcDNA3.1 vector enzyme BamHⅠand EcoRⅠdualenzyme digestion. And in the role of DNA ligase and pcDNA3.1 under the HPV16E7 connection, electrophoresis and recovery of large fragments connected. Preparation of the feelings of pre-state E. coli DH5αcells transformed with pcDNA3.1-HPV16E7 competence E. coli DH5αcells, and then we picked a single colony of white culture 12 hours after the use of plasmid extraction kit extraction of recombinant plasmid DNA, and agar Observation of sugar gel electrophoresis bands, Plasmid in order to further identify whether the build is successful, we used enzyme BamHⅠand EcoRⅠenzyme double digestion of recombinant plasmid extraction, have been around 5KB and 300Bp fragment fragment, indicating that DNA formed after the reorganization has been successfully transferred HPV16E7sh into the pcDNA3.1 vector, the recombinant eukaryotic expression plasmid pcDNA3.1-HPV16E7sh was successfully constructed.Conclusions1 Restructuring of the use of DNA technology, in suitable conditions, can be in the autonomous evolution of HPV16E7 gene sequence comparison, sequence rearrangements, build HPV16E7 gene mutant library, which may include a higher immunogenicity of a new type of E7 genes, for further study of E7 gene in cervical cancer, condyloma and other diseases the role of laying the foundation2 Restructured HPV16E7 gene and with the vector pcDNA3.1 recombinant plasmid pcDNA3.1-HPV16E7sh successfully constructed, in order to further the establishment of high immunogenicity HPV16E7 gene selection model provides the premise is not to further build HPV therapeutic vaccine to lay the foundation.