發(fā)布時(shí)間：2018-07-10 12:45

  本文選題：人乳頭瘤病毒16型 + 廣東��； 參考：《暨南大學(xué)》2008年碩士論文

【摘要】： 【目的】 ①克隆廣東地區(qū)人乳頭瘤病毒(HPV)16型L1基因并分析其結(jié)構(gòu)特點(diǎn);構(gòu)建廣東分離株HPV16 L1畢赤酵母分泌型表達(dá)載體。②按照畢赤酵母密碼子使用偏好,優(yōu)化廣東分離株HPV16 L1基因并對(duì)其在畢赤酵母細(xì)胞中的表達(dá)進(jìn)行研究。 【方法】 ①采用PCR技術(shù)從廣東地區(qū)宮頸癌組織中擴(kuò)增HPV16L1基因,克隆入畢赤酵母表達(dá)載體pPICZαC,測(cè)序并進(jìn)行序列分析。②根據(jù)畢赤酵母密碼子使用偏好對(duì)廣東分離株HPV16L1基因進(jìn)行密碼子優(yōu)化并測(cè)序鑒定。③分別將畢赤酵母表達(dá)載體pPICZαC-HPV16L1及經(jīng)過(guò)密碼子優(yōu)化的畢赤酵母表達(dá)載體pPICZαC-HPV16 L1m電轉(zhuǎn)化入畢赤酵母GS115并進(jìn)行表型篩選和Zeocin抗性篩選;對(duì)整合了目的基因的重組酵母表達(dá)菌株進(jìn)行PCR鑒定。④以0.5%(v/v)甲醇誘導(dǎo)表達(dá)目的蛋白;SDS-PAGE及Western blot鑒定目的蛋白的表達(dá)。 【結(jié)果】 ①成功擴(kuò)增了廣東地區(qū)HPV16L1基因,廣東分離株HPV16 L1基因序列與德國(guó)標(biāo)準(zhǔn)株相比有16處不同,同源性為98.99%,其編碼的氨基酸序列有8處發(fā)生改變;與中國(guó)標(biāo)準(zhǔn)株比較有9處不同,同源性為99.18%,其編碼的氨基酸序列有4處發(fā)生變化。 ②廣東分離株HPV16 L1基因蛋白疏水性及抗原決定簇預(yù)測(cè)結(jié)果與德國(guó)標(biāo)準(zhǔn)株相比較存在差異。 ③對(duì)廣東分離株HPV16 L1基因進(jìn)行了密碼子優(yōu)化,經(jīng)過(guò)測(cè)序鑒定,確認(rèn)12個(gè)堿基位點(diǎn)全部?jī)?yōu)化成功。 ④成功構(gòu)建了經(jīng)密碼子優(yōu)化HPV16L1基因的畢赤酵母表達(dá)載體pPICZαC-HPV16 L1m。 ⑤廣東分離株HPV16 L1蛋白的酵母表達(dá):a.在30℃條件下,以0.5%(v/v)甲醇分別誘導(dǎo)工程菌GS115/pPICZαC-HPV16L1,GS115/pPICZαC-HPV16 L1m。發(fā)酵上清經(jīng)SDS-PAGE電泳檢測(cè)顯示,上清中均有特異蛋白表達(dá);HPV16 L1基因經(jīng)過(guò)密碼子優(yōu)化的工程菌GS115/pPICZαC-HPV16 L1m目的蛋白表達(dá)量要高于HPV16L1基因未經(jīng)過(guò)優(yōu)化的工程菌GS115/pPICZαC-HPV16 L1。b.誘導(dǎo)條件的優(yōu)化:在30℃條件下,甲醇0.5%(v/v)誘導(dǎo)工程菌GS115/pPICZαC-HPV16 L1m72小時(shí),目的蛋白HPV16L1的表達(dá)量可達(dá)到最大值,為107mg/L。 【結(jié)論】 ①?gòu)V東分離株HPV16 L1基因序列與德國(guó)標(biāo)準(zhǔn)株、中國(guó)標(biāo)準(zhǔn)株相比較均存在差異。 ②成功構(gòu)建了經(jīng)密碼子優(yōu)化HPV16L1基因的畢赤酵母表達(dá)載體pPICZαC-HPV16 L1m。 ③目的蛋白HPV16L1在畢赤酵母菌中成功表達(dá)。經(jīng)過(guò)密碼子優(yōu)化的工程菌GS115/pPICZαC-HPV16 L1m目的蛋白表達(dá)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 偽 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 偽 C-HPV16L1 and pPICZ 偽 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 偽 C-HPV16 L1m.5 was successfully constructed for the expression of HPV16L1 gene by codon. The yeast expression vector pPICZ 偽 C-HPV16L1m.5 the yeast expression of HPV16L1 protein was successfully constructed. At 30 鈩，

本文編號(hào)：2113433