广东分离株HPV16L1基因克隆及其蛋白的分泌表达

发布时间：2018-07-10 12:45

  本文选题：人乳头瘤病毒16型 + 广东　； 参考：《暨南大学》2008年硕士论文

【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。
[Abstract]:[objective] 1 to clone the L1 gene of human papillomavirus (HPV) type 16 and analyze its structural characteristics in Guangdong, construct the secretory expression vector of Pichia pastoris HPV16 L1 in Guangdong province, and construct the secretory expression vector .2 according to the preference of Pichia pastoris codon. The HPV16 L1 gene was optimized and its expression in Pichia pastoris cells was studied. [methods] 1HPV16 L1 gene was amplified from cervical cancer tissues in Guangdong by PCR. Cloned into Pichia pastoris expression vector pPICZ 伪 C, sequenced and sequenced 2.According to the preference of Pichia pastoris codon usage, we optimized the codon of HPV16L1 gene isolated from Guangdong strain and sequenced the expression vector of Pichia pastoris. 3 PPICZ 伪 C-HPV16L1 and pPICZ 伪 C-HPV16L1m were electrotransformed into Pichia pastoris GS115 and screened for phenotype and Zeocin resistance. The recombinant yeast expression strain integrated with the target gene was identified by PCR. 4. The expression of the target protein was induced by 0.5% (v / v) methanol. SDS-PAGE and Western blot were used to identify the expression of the target protein. [results] 1 The HPV16 L1 gene was amplified from Guangdong province. The sequence of L1 gene of HPV16 in Guangdong was different from that of the German standard strain, the homology was 98.99, the amino acid sequence of HPV16 L1 gene was changed in 8 places, and there were 9 different sequences compared with the Chinese standard strain, the sequence of HPV16 L1 gene was different from that of the German standard strain. The homology was 99.18 and the amino acid sequence of HPV16L1 gene changed in 4 places. 2 the predicted results of protein hydrophobicity and antigenic determinant of HPV16 L1 gene were different from those of German standard strains. 3. The codon optimization of HPV16 L1 gene was carried out in Guangdong province. After sequencing, all 12 base sites were confirmed to be optimized successfully. 4 Pichia pastoris expression vector pPICZ 伪 C-HPV16 L1m.5 was successfully constructed for the expression of HPV16L1 gene by codon. The yeast expression vector pPICZ 伪 C-HPV16L1m.5 the yeast expression of HPV16L1 protein was successfully constructed. At 30 鈩，

本文编号：2113433