Construction Of HPV16 E6 And IL-28B In Eukaryotic Expression Vector And Study On The Anti-cervical Cancer Effect Of Glycolysis Inhibitor

Part? Construction of HPV16 E6 and IL-28 B genes in Eukaryotic expression VectorObjective: The E6 and IL-28 B gene sequences of human papillomavirus 16 were constructed into eukaryotic expression vector Pichia pastoris by cloning technique.The protein was further screened and optimized and induced by methanol to provide the basis for immunotherapy of cervical cancer.Methods: The cDNA sequences of E6 and IL-28 B were found in geneback library.The cDNA sequences of E6 and IL-28 B constructed in prokaryotic expression vector in the early stage of laboratory were consistent with those of gene library.When Ppic-9k-his plasmid was purchased,the company provided the sequence consistent with the results of gene pool alignment,selected EcoR ? and NoT ? restriction sites,and designed primers with primer software.The cDNA sequences of E6 and IL-28 B were constructed into eukaryotic expression vector Ppic-9k-his by PCR and other molecular cloning techniques.GS115 host bacteria were transformed and high copy bacteria were screened by G418 antibiotics,and the expression conditions were optimized.Results: The Pichia pastoris expression vectors Ppic9k-his-E6 and Ppic9k-his-IL-28 B,were successfully constructed by molecular cloning technique.the high copy transformants were screened by G418,and the induced expression conditions were optimized.The expression of exogenous genes IL-28 B and E6 in Pichia pastoris receptor cells were explored.The induction conditions need to be further improved,which is prepared for the further study of immunotherapy and therapeutic vaccine for cervical cancer.Conclusion: The recombinant plasmids of Ppic9k-his-E6 and Ppic9k-his-IL-28 B were successfully constructed and multi-cuff shellfish were screened out.Part II Study on the anti-cervical cancer effect of glycolysis inhibitor Objective: The inhibitory effects of glycolysis inhibitors 3-BrPA,2-DG and clonidamine on cervical cancer TC-1 cells were observed in vitro.The glycolysis inhibitors with obvious inhibitory effect were screened out and combined with cisplatin.In order to provide experimental basis for further study of glycolysis inhibitor against cervical cancer,the author observe the therapeutic effect and lactic acid level of TC-1 cervical cancer xenografts in mice,Methods:(1)To culture mouse TC-1 cervical cancer cells,(2)to evaluate the inhibitory effect of glycolysis inhibitor on cervical cancer TC-1 cells by CCK8,scratch test and plate cloning test,(3)to establish the subcutaneous transplantation tumor model of C57 mice,(3)to establish the subcutaneous transplantation tumor model of C57 mice,and(3)to establish the subcutaneous transplantation tumor model of C57 mice.(4)the diameter of the tumor to be transplanted subcutaneously was up to.3mm,the experimental mice were randomly divided into 6 groups with random number table.The transplanted tumor size was observed and the tumor inhibition rate was calculated in 3-BrPA,Clonidamine,3 + BrPA + Cisplatin,Chloridamine + Cisplatin,Cisplatin and PBS groups.(5)after 20 days,the serum of the experimental mice in each group was centrifuged by eyeball blood,and the transplanted tumor size was observed and the tumor inhibition rate was calculated.after 20 days,the serum of the experimental mice in each group was centrifuged by eyeball blood.The changes of lactic acid in serum were detected by enzyme-linked immunosorbent assay(Elisa).Results :(1)In the proliferation test,it was found that the growth inhibition rate of cervical cancer TC-1 cells increased with the increase of drug concentration after treated with3-BrPA,2-DG and clonidamine for 24 h,48 h and 72 h,respectively.Microscopic observation showed that compared with the blank control group,the number of 3-BrPA,2-DG and Lonidamine was less,the number of cells in the combined cisplatin group was less than that in the cisplatin group,and the morphology of 3-BrPA,2-DG and Lonidamine groups changed.It becomes smaller and smaller,and the nucleus shrinks.(2)in the scratch experiment,24 h mean mobility cisplatin(4.35 ±0.69%)< 3-BrPA(16.26% ±0.018%)<2-DG(18% ±0.57%)< Lonidamine(21.26% ±2.08%)< PBS(28.81 ±1.1%)(P < 0.05);48 h mean mobility cisplatin(10.23% ± 0.7%)< 3-BrPA(26.95% ± 1.32%)< 2-DG(30.68% ±1.10%)<Lonidamine(36.18% ±3.70%)< PBS(39.80 ±2.1%)(P < 0.05);(3)in the plate cloning experiment,(3)in plate cloning test,cisplatin(7.6 ± 0.37%)<3-BrPA(17.13% ±0.59%)< 2-DG(23.04 ±0.82%)<Lonidamine(33.62±0.26%)< PBS(57.4% ± 0.27%)(P < 0.05).(4)the subcutaneous transplantation tumor model of experimental mice was successfully established.The tumor inhibition rate was 3-BrPA +cisplatin(91.30 ± 1.24%)> 2-DG + cisplatin(86.50 ± 0.38%)> cisplatin(78.36 ±1.28%)> 3-BrPA(71.27 ±0.93%)> 2-DG(65.44 ±1.74%)> PBS(0)(P < 0.05).In the experiment of lactic acid content detection,it was found that there were significant differences between 3-BrPA,2-DG group,cisplatin combined group and PBS group(P <0.05),and there were significant differences between 3-BrPA,2-DG group,cisplatin combined group and cisplatin group(P < 0.05).Conclusion:(1)in vitro experiments,the inhibitory effect of glycolysis inhibitor on the proliferation and migration of cervical cancer cells was found to be 3-BrPA > 2-DG >clonidamine in vitro.(2)in vivo experiments,it was found that the antitumor effect of 3-BrPA + cisplatin >3-BrPA > 2-DG,3-BrPA and 3-BrPA could decrease the glycolysis rate and the content of lactic acid in serum of mice,and BrPA had the strongest antitumor effect on cervical cancer cells.