摘要
目的 构建HPV 18L1 E6、L1 E7嵌合基因的表达载体 ,并在CHO细胞中表达。方法 克隆HPV 18L1 E6和L1 E7基因 ,插入中介载体 pGEMT Easy中并测序鉴定。采用PCR定点突变法 ,突变L1 E6、L1 E7基因序列中与转化作用相关的位点 ,分别与L1基因连接后插入真核表达载体 pVAX1,构建真核表达质粒 pVAX1 L1E6Mxx、 L1E7Mxx。用磷酸钙沉淀法 ,转染CHO细胞 ,以抗HPV 18L1、抗E6和抗E7特异性单克隆抗体 (mAb)做ELISA和免疫细胞化学法检测。结果 ELISA检测显示 ,转染各种pVAX1 L1E6Mxx、 L1E7Mxx融合蛋白表达质粒的细胞提取物的P N值均 >2 .1;免疫细胞化学检测 ,在胞浆、胞核可见棕黄色颗粒。结论 所构建的 pVAX1 L1E6Mxx E7Mxx融合蛋白表达质粒 ,可在转染细胞内表达相应的L1 E6Mxx和L1 E7Mxx蛋白 ,为今后进行DNA疫苗的研究奠定了基础。
Aim To construct eukaryotic expression vector of HPV18 L1 E6 and L1 E7 chimeric gene. Methods HPV18 L1, E6 and E7 genes were cloned respectively. The genes were separately sequenced and identified by inserting into inter vector pGEMT Easy. The nucleotides within HPV 18 E6 and E7 genes, which are responsible for viral transforming activity, were mutated by PCR site directed mutagenesis method. The correctly mutated E6 and E7 fragments were linked with and cloned into an eukaryotic expression vector pVAX1, HPV 18 L1 gene, generating 12 chimeric recombinants plasmids pVAX1 L1E6Mxx and pVAX1 L1E7Mxx were generated. CHO cells were transiently transfected with the individual construct by calcium phosphate precipitate method. Target protein expression in the lysate of the transfected cells were measured by ELISA and immunocytochemical staining, with HPV 18 L1 E6 and L1 E7 specific monoclonal antibodies. Results ELISA assays showed the P N ratios in the cell lysate transfected with pVAX1 L1E6Mxx L1E7Mxx chimeric recombinant plasmids were larger than 2.1. Immunocytochemical staining revealed brownish precipitant signal in cytoplasm and nuclei of the transfected cells. Conclusion Successful constructions of prophylactic and therapeutic DNA vaccine candidate plasmids lay solid foundation for future animal experiment and clinical trial.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2002年第6期558-561,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助No .3 0 0 70 848
国家高技术研究发展计划 (863 )资助No .2 0 0 1AA2 15 2 2 1
