CD137L基因大肠杆菌表达、纯化及功能研究

发布时间：2018-04-10 23:26

本文选题：CD137L + 大肠杆菌　；参考：《浙江大学》2017年硕士论文

【摘要】：目的:研究CD137L基因在大肠杆菌中的表达,通过构建表达CD137L重组蛋白的原核表达系统,优化CD137L原核表达菌的表达条件,通过纯化、透析复性并对蛋白的功能进行初步研究,为CD137L蛋白的开发提供工作基础。方法:通过密码子优化的CD137L基因插入PUC18质粒中,先将CD137L基因进行PCR扩增,构建亚克隆载体pGEIM/CD137LT质粒,通过NdeI和XhoI双酶切得到目的基因,然后插入pET30a空载体,对所得重组载体进行PCR和酶切鉴定,并转入BL21(DE3)进行表达。通过改变表达系统生长诱导过程中的单个条件因素,来寻找最优化的表达条件:(1)培养基pH条件;(2)诱导时机;(3)KANA浓度;(4)接种量;(5)诱导剂浓度;(6)诱导温度。在确定培养条件后,通过色谱柱纯化蛋白,并对蛋白的功能进行初步研究。结果:通过6方面条件的实验,最终初步得出工程菌株的最适培养、诱导表达条件为:(1)培养基pH条件:7.09;(2)诱导时机:OD600≈1.1;(3)KANA浓度:50mg/L;(4)接种量:5%;(5)诱导剂浓度:0.25mM;(6)诱导温度:370℃。将CD137L与人乳头瘤病毒16型(HPV16)重组蛋白疫苗共同免疫肿瘤模型小鼠,观察CD137L对HPV16重组蛋白疫苗抗肿瘤免疫效果的影响。结果表明:阴性对照组、CD137L11μg、CD137L5μg、CD137L50μg四个组中的小鼠肿瘤模型抑制率10%,HPV疫苗组的小鼠肿瘤模型抑制率为40%,HPV疫苗加CD137L1μg和HPV疫苗加CD137L5μg两个组的小鼠肿瘤模型抑制率为90%,HPV疫苗加CD137L50μg的小鼠肿瘤模型抑制率为50%。结论:通过实验研究,构建并筛选得到了能有效表达CD137L蛋白的重组工程菌株。通过6个方面条件的实验,最终得出工程菌株的最适培养、诱导表达条件。且以该培养条件为基础表达的CD137L重组蛋白,通过纯化、透析复性,可以提高人乳头瘤病毒16型(HPV16)重组蛋白疫苗的免疫效果。
[Abstract]:Objective: to study the expression of CD137L gene in Escherichia coli and to optimize the expression conditions of CD137L prokaryotic expression strain by constructing a prokaryotic expression system expressing CD137L recombinant protein.To provide the basis for the development of CD137L protein.Methods: the CD137L gene optimized by codon was inserted into the PUC18 plasmid. The CD137L gene was amplified by PCR and subcloned vector pGEIM/CD137LT plasmid was constructed. The target gene was digested by NdeI and XhoI, then inserted into the empty pET30a vector.The recombinant vector was identified by PCR and restriction endonuclease digestion.By changing a single conditional factor in the process of growth induction of the expression system, the optimal expression condition was found to be the optimal expression condition: 1) the medium pH condition was 2) the time for inducing Kana was 4) the inoculation quantity was 5)) the concentration of inducer was 0. 6) induction temperature.After the culture conditions were determined, the protein was purified by chromatographic column, and the function of the protein was preliminarily studied.CD137L and human papillomavirus type 16 (HPV16) recombinant protein vaccine were used to immunize tumor model mice to observe the effect of CD137L on the anti-tumor immunity of HPV16 recombinant protein vaccine.The inhibition rate of tumor model was 50%.Conclusion: the recombinant engineering strain expressing CD137L protein was constructed and screened.Through the experiments of 6 aspects, the optimal culture and expression conditions of the engineering strain were obtained.The recombinant CD137L protein expressed on the basis of this culture condition can improve the immune effect of recombinant protein vaccine of human papillomavirus type 16 (HPV16) by purification and dialysis renaturation.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78

【相似文献】