The objective of this study is to map epitopes on HPMAPV16 L1 protein and provide information to the design of HPV16 prophylactic peptide vaccine. The epitopes on L1 protein were screenedby polyclonal and two monocl...The objective of this study is to map epitopes on HPMAPV16 L1 protein and provide information to the design of HPV16 prophylactic peptide vaccine. The epitopes on L1 protein were screenedby polyclonal and two monoclonal antibodies (BS and F4G3) against RPV16 L1 protin from a 6-merfd phage display epitope library with the method or immuuo-afrinity screening (Biopauuing). Aferthree rounds or Bio-Panning, the Positive phages were detected by L1 antibodies again with ELISA.The positive phages reacted strongly with L1 antibodies were then identified by DNA sequencing.Three mimotopes have been screened by polycloual and two monoclonal antibodies. The mimotope(LSLFSC) reacted with mouoclonal antibody B8 showed 50% pomology with the sequence 270275a. a (DSLFFY) of prototype HPV16 L1. Another mimotope (LTSSYS) reacted with polyclonalantibodies had 66% pomology with the L1 sequence 516~521a. A(TTSSTS), also a mimotope (DRWDRF) was found had the bomologic RF with the known L1 sequence 441 ~446a. a. The mimotopesLSLFSC and DRWDRF were adjacent to the epitopes at 267~269a. a and 422~441 a. a reported byother researchers Previously. Our results suggest that there might be a batch of epitopes on HPV16L1 ppotein, and the predominant epitopes of HPV16 L1 protein are located in the above two domains. These results will be helpful for design or HPV16 prophylactic peatide vaccines and HPVpolyvalent vaccines.展开更多
目的:优化人乳头瘤病毒16型主要衣壳蛋白L1(human papillomavirus type 16 major capsid protein L1,HPV16L1)在毕赤酵母中的表达,并考察可能的影响因素。方法:四个不同序列特征的HPV16L1基因M16、Y16、P16、W16(其中,M16和Y16按酵母密...目的:优化人乳头瘤病毒16型主要衣壳蛋白L1(human papillomavirus type 16 major capsid protein L1,HPV16L1)在毕赤酵母中的表达,并考察可能的影响因素。方法:四个不同序列特征的HPV16L1基因M16、Y16、P16、W16(其中,M16和Y16按酵母密码子优化,P16为哺乳动物细胞密码子优化,而W16为野生型序列)分别克隆于毕赤酵母表达质粒p PinkTM-HC(高基因拷贝菌落筛选)和p PinkTM-LC(低基因拷贝菌落筛选),并转化不同蛋白酶缺陷的宿主菌。甲醇诱导24小时后,取菌体样品经Western blot分析L1蛋白的表达。结果:M16显示了最高的表达水平,其次是Y16与P16,而W16几乎无表达。基因序列密码子应用特征分析显示,4个基因的密码子适应指数从高到低依次为Y16、M16、W16和P16。通过自由能和GC含量分析4个序列的mRNA二级结构,Y16为-409.40 kcal/mol和43.85%;M16为-451.50 kcal/mol和47.83%;P 16为-606.50kcal/mol and 64.10%;W16为-384.70 kcal/mol and 38.01%。蛋白酶缺陷菌株L1表达高于野生型菌株,质粒p PinkTM-HC与p PinkTM-LC介导的表达无明显区别。结论:密码子优化操作显著改善了HPV16L1在毕赤酵母中的表达,但表达水平与密码子利用优劣并不完全对应,提示密码子优化仅是部分原因,而mRNA结构与稳定性变化值得探讨。蛋白酶缺陷菌株提高了HPV16L1蛋白的稳定性,显著影响了表达水平。研究证明基因剂量对HPV16L1的表达未产生明显影响。展开更多
文摘The objective of this study is to map epitopes on HPMAPV16 L1 protein and provide information to the design of HPV16 prophylactic peptide vaccine. The epitopes on L1 protein were screenedby polyclonal and two monoclonal antibodies (BS and F4G3) against RPV16 L1 protin from a 6-merfd phage display epitope library with the method or immuuo-afrinity screening (Biopauuing). Aferthree rounds or Bio-Panning, the Positive phages were detected by L1 antibodies again with ELISA.The positive phages reacted strongly with L1 antibodies were then identified by DNA sequencing.Three mimotopes have been screened by polycloual and two monoclonal antibodies. The mimotope(LSLFSC) reacted with mouoclonal antibody B8 showed 50% pomology with the sequence 270275a. a (DSLFFY) of prototype HPV16 L1. Another mimotope (LTSSYS) reacted with polyclonalantibodies had 66% pomology with the L1 sequence 516~521a. A(TTSSTS), also a mimotope (DRWDRF) was found had the bomologic RF with the known L1 sequence 441 ~446a. a. The mimotopesLSLFSC and DRWDRF were adjacent to the epitopes at 267~269a. a and 422~441 a. a reported byother researchers Previously. Our results suggest that there might be a batch of epitopes on HPV16L1 ppotein, and the predominant epitopes of HPV16 L1 protein are located in the above two domains. These results will be helpful for design or HPV16 prophylactic peatide vaccines and HPVpolyvalent vaccines.
文摘目的:优化人乳头瘤病毒16型主要衣壳蛋白L1(human papillomavirus type 16 major capsid protein L1,HPV16L1)在毕赤酵母中的表达,并考察可能的影响因素。方法:四个不同序列特征的HPV16L1基因M16、Y16、P16、W16(其中,M16和Y16按酵母密码子优化,P16为哺乳动物细胞密码子优化,而W16为野生型序列)分别克隆于毕赤酵母表达质粒p PinkTM-HC(高基因拷贝菌落筛选)和p PinkTM-LC(低基因拷贝菌落筛选),并转化不同蛋白酶缺陷的宿主菌。甲醇诱导24小时后,取菌体样品经Western blot分析L1蛋白的表达。结果:M16显示了最高的表达水平,其次是Y16与P16,而W16几乎无表达。基因序列密码子应用特征分析显示,4个基因的密码子适应指数从高到低依次为Y16、M16、W16和P16。通过自由能和GC含量分析4个序列的mRNA二级结构,Y16为-409.40 kcal/mol和43.85%;M16为-451.50 kcal/mol和47.83%;P 16为-606.50kcal/mol and 64.10%;W16为-384.70 kcal/mol and 38.01%。蛋白酶缺陷菌株L1表达高于野生型菌株,质粒p PinkTM-HC与p PinkTM-LC介导的表达无明显区别。结论:密码子优化操作显著改善了HPV16L1在毕赤酵母中的表达,但表达水平与密码子利用优劣并不完全对应,提示密码子优化仅是部分原因,而mRNA结构与稳定性变化值得探讨。蛋白酶缺陷菌株提高了HPV16L1蛋白的稳定性,显著影响了表达水平。研究证明基因剂量对HPV16L1的表达未产生明显影响。
