【作者】 刘红；

【导师】 宇丽；

【作者基本信息】 暨南大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 【目的】①克隆广东地区人乳头瘤病毒(HPV)16型L1基因并分析其结构特点;构建广东分离株HPV16 L1毕赤酵母分泌型表达载体。②按照毕赤酵母密码子使用偏好,优化广东分离株HPV16 L1基因并对其在毕赤酵母细胞中的表达进行研究。【方法】①采用PCR技术从广东地区宫颈癌组织中扩增HPV16L1基因,克隆入毕赤酵母表达载体pPICZαC,测序并进行序列分析。②根据毕赤酵母密码子使用偏好对广东分离株HPV16L1基因进行密码子优化并测序鉴定。③分别将毕赤酵母表达载体pPICZαC-HPV16L1及经过密码子优化的毕赤酵母表达载体pPICZαC-HPV16 L1m电转化入毕赤酵母GS115并进行表型筛选和Zeocin抗性筛选;对整合了目的基因的重组酵母表达菌株进行PCR鉴定。④以0.5%(v/v)甲醇诱导表达目的蛋白;SDS-PAGE及Western blot鉴定目的蛋白的表达。【结果】①成功扩增了广东地区HPV16L1基因,广东分离株HPV16 L1基因序列与德国标准株相比有16处不同,同源性为98.99%,其编码的氨基酸序列有8处发生改变;与中国标准株比较有9处不同,同源性为99.18%,其编码的氨基酸序列有4处发生变化。②广东分离株HPV16 L1基因蛋白疏水性及抗原决定簇预测结果与德国标准株相比较存在差异。③对广东分离株HPV16 L1基因进行了密码子优化,经过测序鉴定,确认12个碱基位点全部优化成功。④成功构建了经密码子优化HPV16L1基因的毕赤酵母表达载体pPICZαC-HPV16 L1m。⑤广东分离株HPV16 L1蛋白的酵母表达:a.在30℃条件下,以0.5%(v/v)甲醇分别诱导工程菌GS115/pPICZαC-HPV16L1,GS115/pPICZαC-HPV16 L1m。发酵上清经SDS-PAGE电泳检测显示,上清中均有特异蛋白表达;HPV16 L1基因经过密码子优化的工程菌GS115/pPICZαC-HPV16 L1m目的蛋白表达量要高于HPV16L1基因未经过优化的工程菌GS115/pPICZαC-HPV16 L1。b.诱导条件的优化:在30℃条件下,甲醇0.5%(v/v)诱导工程菌GS115/pPICZαC-HPV16 L1m72小时,目的蛋白HPV16L1的表达量可达到最大值,为107mg/L。【结论】①广东分离株HPV16 L1基因序列与德国标准株、中国标准株相比较均存在差异。②成功构建了经密码子优化HPV16L1基因的毕赤酵母表达载体pPICZαC-HPV16 L1m。③目的蛋白HPV16L1在毕赤酵母菌中成功表达。经过密码子优化的工程菌GS115/pPICZαC-HPV16 L1m目的蛋白表达量要高于工程菌GS115/pPICZαC-HPV16L1。更多还原

【Abstract】 [Objective]①To clone the human papillomavirus(HPV) type 16 L1 gene in Guangdong area and to investigate its structure specificity;to construct its Pichia pastories secretion type expression vector.②To optimize the HPV16 L1 gene,which was amplified from human cervical carcinoma tissues in Guangdong area,accordine to the codon bias of Pichia pastories and to evaluate the expression of the optimized gene in Pichia pastories.[Methods]①To amplify the HPV16 L1 gene from human cervical carcinoma tissues in Guangdong area by PCR,and then cloned the HPV16L1 gene into Pichia pastories expression vectors.The HPV16 L1 gene were sequenced and analyzed.②To optimize the HPV16L1 gene,accordine to the codon bias of Pichia pastories,then sequencing to evaluate.②The recombinant vectors pPICZαC-HPV16L1 and the gene modified recombinant vectors pPICZαC-HPV16L1m were transformed into Pichia GS115 by electroporation.Positive recombinant were screened through phenotype and Zeocin resistance.Recombinant yeast expression strains were analysised by PCR.④Add 100%methanol to a final concentration of 0.5%(v/v) methanol to maintain induction.Detected the expression of HPV16L1 protein in Pichia pastories by SDS-PAGE and Western blot.[Results]①Successfully amplified the HPV16L1 gene from human cervical carcinoma tissues in Guangdong area.There were 16 nucleotide differences between Guangdong strain and Germanic standard strain,the sequences were 98.99%homology,subsequently changed 8 amino acids it coded.There were 9 nucleotide differences between Guangdong strain and Chinese standard strain,the sequences were 99.18%homology,subsequently changed 4 amino acids it coded.②The hydrophobicity prediction and predicted antigen determinants results of Guangdong strain are different from that of Germanic standard strain.③Successfully optimized the HPV16 L1 gene.After sequenced,all of the 12 sites which need to be mutated were optimized.④The codon optimized recombinant plasmid pPICZαC-HPV16 L1m were constructed.⑤The expression of HPV16 L1 protein:a.Under the condition of 30℃,adding 0.5%(v/v) methanol to induce the GS115/pPICZαC-HPV16 L1 and GS115/pPICZαC-HPV16 L1m to express the HPV16 L1 protein.The result of SDS-PAGE indicated that the aimed protein band can be detected and the expression level of codon optimized GS115/pPICZαC-HPV16L1m was higher than that of GS115/pPICZαC-HPV16L1.b.The optimal induction condition were found to be:30℃for induction temperature,0.5%(v/v)methanol, and 72 hours for induction time.Expression of HPV16L1 was found to be satisfactory (107mg/L)in Pichia pastories,and the product counted for about 45%of the total proteins in the expression system.[Conclusion]①There are nucleotide differences of HPV16 L1 gene among Germanic standard strain, Chinese standard strain and Guangdong strain.②The codon optimized recombinant plasmid pPICZαC-HPV16 L1m were constructed.③The HPV16 L1 protein was expressed in Pichia pastories.And the expression level of codon optimized GS115/pPICZαC-HPV16L1m was higher than that of GS115/pPICZαC-HPV16 L1.更多还原