人乳頭瘤病毒6型L1的克隆表達與純化
發(fā)布時間：2018-08-09 10:03
【摘要】： 人乳頭瘤病毒6型( human papillomavirus type 6 , HPV6)是尖銳濕疣的主要病因,該病的發(fā)病率近年來持續(xù)上升,是僅次于淋病的第二位性病,并且其病情頑固,易復發(fā),治療困難。HPV主要衣殼蛋白L1結(jié)構(gòu)比較保守,是主要的種屬特異性抗原,可誘生中和性抗體,用于預防HPV的感染。另外,HPV6是引發(fā)性疣最常見的病原體,如尖銳濕疣和扁平疣。尖銳濕疣是世界各地高發(fā)的性傳播疾病。有報道表明,在尖銳濕疣組織中HPV檢出率為100%,其中6和11型占90%以上,HPV6與尖銳濕疣有確切的病因關(guān)系,為疫苗的應(yīng)用提供了可能。基于上述的原因,我們對HPV6 L1基因進行了密碼子優(yōu)化,利用原核表達載體pGEX4T-1構(gòu)建了HPV6 mL1的原核表達載體,IPTG誘導后可見80kD左右的蛋白,對目的蛋白進行了鑒定,并利用谷胱甘肽瓊脂糖凝膠4B介質(zhì)對目的蛋白進行了純化。 目的:從質(zhì)粒pBV105-HPV6擴增獲得HPV6 L1片段,將HPV6 L1進行密碼子優(yōu)化。構(gòu)建HPV6 mL1基因的重組原核表達載體pGEX4T-1/HPV6 mL1,實現(xiàn)HPV6 mL1融合蛋白在大腸桿菌BL21(DE3)原核細胞中的表達,摸索目的蛋白的表達及優(yōu)化條件,并實現(xiàn)目的蛋白的鑒定和純化。 方法:用PCR技術(shù)對HPV6 L1基因進行擴增和純化,并將HPV6 L1基因與克隆載體連接,轉(zhuǎn)化入受體菌,實現(xiàn)目的基因的克隆。將L1基因進行測序,并與GenBank中的序列相比較。根據(jù)大腸桿菌偏愛密碼子表對L1基因的密碼子進行優(yōu)化,合成優(yōu)化后的序列。用PCR技術(shù)對優(yōu)化后的HPV6 mL1基因進行擴增和純化,并將HPV6 mL1基因與克隆載體連接,轉(zhuǎn)化入受體菌,實現(xiàn)目的基因克隆。將測序正確的重組克隆質(zhì)粒pGEM-T/HPV6 mL1和原核表達載體pGEX4T-1分別進行EcoR I和Sal I的雙酶切反應(yīng),然后將回收得到的目的基因片段與pGEX4T-1載體大片段,進行定向連接,構(gòu)建成含目的基因HPV6 mL1的重組原核表達載體。將重組質(zhì)粒pGEX4T-1/HPV6 mL1轉(zhuǎn)化入BL21(DE3)感受態(tài)菌,通過IPTG誘導獲得表達,表達產(chǎn)物用SDS-PAGE和Western blot等方法鑒定。分別對表達菌株在不同濃度的IPTG(終濃度1、0.5、0.1mmol/L)和不同誘導時間(4、8、12、20小時)及不同溫度(37℃、25℃、20℃)下進行表達條件的優(yōu)化。大量制備目的蛋白,經(jīng)過洗滌,變性和復性后使用GE Healthcare公司的Glutathione Sepharose 4B介質(zhì)對目的蛋白進行親和純化。 結(jié)果:采用PCR方法,從質(zhì)粒pBV105-HPV6擴增獲得HPV6 L1片段。采用T/A克隆法,實現(xiàn)了HPV6 L1的克隆,測序結(jié)果表明克隆基因序列與GenBank中HPV6 L1基因序列同源性為99.9%,有2個堿基發(fā)生了突變,但編碼的氨基酸不改變。將HPV6 L1基因序列進行了密碼子優(yōu)化,優(yōu)化后序列與優(yōu)化前序列相比,基因序列的同源性為75%,但編碼的氨基酸沒有發(fā)生變化。密碼子優(yōu)化后,用密碼子偏性分析軟件CodonW分析,CAI(Codon Adaptation Index)從0.205提高到0.611,CBI(Codon Bias Index)從-0.117提高到0.531,FOP(Frequency of Optimumcodons)從0.356提高到0.730。成功構(gòu)建了HPV6 mL1克隆基因的重組原核表達載體pGEX4T-1/HPV6 mL1,并用IPTG誘導實現(xiàn)了HPV6 mL1融合蛋白在BL21(DE3)原核細胞中的表達。HPV6 mL1融合蛋白主要以包涵體形式表達,包涵體形式最佳表達條件為濃度為1mmol/L IPTG ,溫度37℃,誘導4h。經(jīng)Glutathione Sepharose 4B柱層析純化得到HPV6 mL1融合蛋白,為其結(jié)構(gòu)功能研究和疫苗研發(fā)提供基礎(chǔ),是否具有生物活性還有待于進一步研究。
[Abstract]:The human papillomavirus 6 (human papillomavirus type 6, HPV6) is the main cause of condyloma acuminata. The incidence of this disease has continued to rise in recent years. It is the second STD only second only to gonorrhea, and its disease is stubborn and easy to relapse. The.HPV main capsid protein of.HPV is relatively conservative and is the main species specific antigen and can be induced to be induced. Neutralizing antibodies are used to prevent HPV infection. In addition, HPV6 is the most common pathogen of verruca provoking, such as condyloma acuminata and verruca plana. Condyloma acuminata is a high incidence of sexually transmitted diseases all over the world. It is reported that the detection rate of HPV in condyloma acuminata is 100%, of which 6 and 11 are more than 90%, HPV6 and condyloma acuminata have definite etiological factors. On the basis of the above reasons, we have optimized the HPV6 L1 gene and constructed the prokaryotic expression vector of HPV6 mL1 using the prokaryotic expression vector pGEX4T-1. IPTG induced the protein of 80kD around 80kD, the target protein was determined, and the glutathione agarose gel 4B medium was used. The protein was purified.
Objective: to obtain the HPV6 L1 fragment from plasmid pBV105-HPV6, optimize the codon of HPV6 L1, construct the Recombinant Prokaryotic expression vector pGEX4T-1/HPV6 mL1 of the HPV6 mL1 gene, realize the expression of HPV6 mL1 fusion protein in the Escherichia coli prokaryotic cell, explore the expression of the target protein and optimize the conditions, and realize the identification of the target protein. And purify.
Methods: the HPV6 L1 gene was amplified and purified by PCR technology, and the HPV6 L1 gene was connected with the clone vector and transformed into the receptor bacteria to realize the clone of the target gene. The L1 gene was sequenced and compared with the sequence in the GenBank. The codon of the L1 gene was optimized and the optimized sequence was synthesized according to the Escherichia coli preference cipher table. The optimized HPV6 mL1 gene was amplified and purified by PCR technology, and the HPV6 mL1 gene was connected with the clone vector and transformed into the receptor bacteria to achieve the target gene cloning. The double enzyme digestion reaction of the correct recombinant cloned plasmid pGEM-T/HPV6 mL1 and the prokaryotic expression vector pGEX4T-1 for EcoR I and Sal I, respectively, would be recovered and then recovered. The target gene fragment was linked with a large fragment of the pGEX4T-1 vector to construct a Recombinant Prokaryotic expression vector containing the target gene HPV6 mL1. The recombinant plasmid pGEX4T-1/HPV6 mL1 was transformed into BL21 (DE3) receptive bacteria and expressed by IPTG, and the expression products were identified by SDS-PAGE and Western blot. The expression conditions of different concentrations of IPTG (final concentration 1,0.5,0.1mmol/L) and different induction time (4,8,12,20 hours) and different temperatures (37, 25, 20) were optimized. A large number of target proteins were prepared. After washing, denaturing and refolding, the target protein was purified by GE Healthcare Glutathione Sepharose 4B medium.
Results: the HPV6 L1 fragment was obtained from plasmid pBV105-HPV6 by PCR method. The cloning of HPV6 L1 was realized by T/A cloning. The sequence results showed that the homology of the cloned gene sequence and the HPV6 L1 gene sequence in GenBank was 99.9%, and the 2 bases were mutated, but the encoded amino acids did not change. The HPV6 L1 gene sequence was cipher. Compared with the pre optimized sequence, the homology of the gene sequence is 75%, but the encoded amino acids are not changed. After the codon optimization, the CAI (Codon Adaptation Index) is improved from 0.205 to 0.611, and CBI (Codon Bias Index) is raised from -0.117 to 0.531 from -0.117 (Codon Bias Index). Mumcodons) from 0.356 to 0.730., the Recombinant Prokaryotic expression vector pGEX4T-1/HPV6 mL1 of HPV6 mL1 cloned gene was successfully constructed, and the expression of HPV6 mL1 fusion protein in BL21 (DE3) prokaryotic cells was induced by IPTG, and the fusion protein was expressed mainly in the form of inclusion body. The optimum expression of inclusion body form was concentration as concentration. IPTG, at 37 degrees centigrade, induced 4h. mL1 fusion protein purified by Glutathione Sepharose 4B column chromatography, which provides a basis for its structural function research and vaccine research and development, and whether it has biological activity remains to be further studied.
【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R373;Q78
本文編號：2173731
[Abstract]:The human papillomavirus 6 (human papillomavirus type 6, HPV6) is the main cause of condyloma acuminata. The incidence of this disease has continued to rise in recent years. It is the second STD only second only to gonorrhea, and its disease is stubborn and easy to relapse. The.HPV main capsid protein of.HPV is relatively conservative and is the main species specific antigen and can be induced to be induced. Neutralizing antibodies are used to prevent HPV infection. In addition, HPV6 is the most common pathogen of verruca provoking, such as condyloma acuminata and verruca plana. Condyloma acuminata is a high incidence of sexually transmitted diseases all over the world. It is reported that the detection rate of HPV in condyloma acuminata is 100%, of which 6 and 11 are more than 90%, HPV6 and condyloma acuminata have definite etiological factors. On the basis of the above reasons, we have optimized the HPV6 L1 gene and constructed the prokaryotic expression vector of HPV6 mL1 using the prokaryotic expression vector pGEX4T-1. IPTG induced the protein of 80kD around 80kD, the target protein was determined, and the glutathione agarose gel 4B medium was used. The protein was purified.
Objective: to obtain the HPV6 L1 fragment from plasmid pBV105-HPV6, optimize the codon of HPV6 L1, construct the Recombinant Prokaryotic expression vector pGEX4T-1/HPV6 mL1 of the HPV6 mL1 gene, realize the expression of HPV6 mL1 fusion protein in the Escherichia coli prokaryotic cell, explore the expression of the target protein and optimize the conditions, and realize the identification of the target protein. And purify.
Methods: the HPV6 L1 gene was amplified and purified by PCR technology, and the HPV6 L1 gene was connected with the clone vector and transformed into the receptor bacteria to realize the clone of the target gene. The L1 gene was sequenced and compared with the sequence in the GenBank. The codon of the L1 gene was optimized and the optimized sequence was synthesized according to the Escherichia coli preference cipher table. The optimized HPV6 mL1 gene was amplified and purified by PCR technology, and the HPV6 mL1 gene was connected with the clone vector and transformed into the receptor bacteria to achieve the target gene cloning. The double enzyme digestion reaction of the correct recombinant cloned plasmid pGEM-T/HPV6 mL1 and the prokaryotic expression vector pGEX4T-1 for EcoR I and Sal I, respectively, would be recovered and then recovered. The target gene fragment was linked with a large fragment of the pGEX4T-1 vector to construct a Recombinant Prokaryotic expression vector containing the target gene HPV6 mL1. The recombinant plasmid pGEX4T-1/HPV6 mL1 was transformed into BL21 (DE3) receptive bacteria and expressed by IPTG, and the expression products were identified by SDS-PAGE and Western blot. The expression conditions of different concentrations of IPTG (final concentration 1,0.5,0.1mmol/L) and different induction time (4,8,12,20 hours) and different temperatures (37, 25, 20) were optimized. A large number of target proteins were prepared. After washing, denaturing and refolding, the target protein was purified by GE Healthcare Glutathione Sepharose 4B medium.
Results: the HPV6 L1 fragment was obtained from plasmid pBV105-HPV6 by PCR method. The cloning of HPV6 L1 was realized by T/A cloning. The sequence results showed that the homology of the cloned gene sequence and the HPV6 L1 gene sequence in GenBank was 99.9%, and the 2 bases were mutated, but the encoded amino acids did not change. The HPV6 L1 gene sequence was cipher. Compared with the pre optimized sequence, the homology of the gene sequence is 75%, but the encoded amino acids are not changed. After the codon optimization, the CAI (Codon Adaptation Index) is improved from 0.205 to 0.611, and CBI (Codon Bias Index) is raised from -0.117 to 0.531 from -0.117 (Codon Bias Index). Mumcodons) from 0.356 to 0.730., the Recombinant Prokaryotic expression vector pGEX4T-1/HPV6 mL1 of HPV6 mL1 cloned gene was successfully constructed, and the expression of HPV6 mL1 fusion protein in BL21 (DE3) prokaryotic cells was induced by IPTG, and the fusion protein was expressed mainly in the form of inclusion body. The optimum expression of inclusion body form was concentration as concentration. IPTG, at 37 degrees centigrade, induced 4h. mL1 fusion protein purified by Glutathione Sepharose 4B column chromatography, which provides a basis for its structural function research and vaccine research and development, and whether it has biological activity remains to be further studied.
【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R373;Q78
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