人乳头瘤病毒6型L1的克隆表达与纯化
发布时间：2018-08-09 10:03
【摘要】： 人乳头瘤病毒6型( human papillomavirus type 6 , HPV6)是尖锐湿疣的主要病因,该病的发病率近年来持续上升,是仅次于淋病的第二位性病,并且其病情顽固,易复发,治疗困难。HPV主要衣壳蛋白L1结构比较保守,是主要的种属特异性抗原,可诱生中和性抗体,用于预防HPV的感染。另外,HPV6是引发性疣最常见的病原体,如尖锐湿疣和扁平疣。尖锐湿疣是世界各地高发的性传播疾病。有报道表明,在尖锐湿疣组织中HPV检出率为100%,其中6和11型占90%以上,HPV6与尖锐湿疣有确切的病因关系,为疫苗的应用提供了可能。基于上述的原因,我们对HPV6 L1基因进行了密码子优化,利用原核表达载体pGEX4T-1构建了HPV6 mL1的原核表达载体,IPTG诱导后可见80kD左右的蛋白,对目的蛋白进行了鉴定,并利用谷胱甘肽琼脂糖凝胶4B介质对目的蛋白进行了纯化。 目的:从质粒pBV105-HPV6扩增获得HPV6 L1片段,将HPV6 L1进行密码子优化。构建HPV6 mL1基因的重组原核表达载体pGEX4T-1/HPV6 mL1,实现HPV6 mL1融合蛋白在大肠杆菌BL21(DE3)原核细胞中的表达,摸索目的蛋白的表达及优化条件,并实现目的蛋白的鉴定和纯化。 方法:用PCR技术对HPV6 L1基因进行扩增和纯化,并将HPV6 L1基因与克隆载体连接,转化入受体菌,实现目的基因的克隆。将L1基因进行测序,并与GenBank中的序列相比较。根据大肠杆菌偏爱密码子表对L1基因的密码子进行优化,合成优化后的序列。用PCR技术对优化后的HPV6 mL1基因进行扩增和纯化,并将HPV6 mL1基因与克隆载体连接,转化入受体菌,实现目的基因克隆。将测序正确的重组克隆质粒pGEM-T/HPV6 mL1和原核表达载体pGEX4T-1分别进行EcoR I和Sal I的双酶切反应,然后将回收得到的目的基因片段与pGEX4T-1载体大片段,进行定向连接,构建成含目的基因HPV6 mL1的重组原核表达载体。将重组质粒pGEX4T-1/HPV6 mL1转化入BL21(DE3)感受态菌,通过IPTG诱导获得表达,表达产物用SDS-PAGE和Western blot等方法鉴定。分别对表达菌株在不同浓度的IPTG(终浓度1、0.5、0.1mmol/L)和不同诱导时间(4、8、12、20小时)及不同温度(37℃、25℃、20℃)下进行表达条件的优化。大量制备目的蛋白,经过洗涤,变性和复性后使用GE Healthcare公司的Glutathione Sepharose 4B介质对目的蛋白进行亲和纯化。 结果:采用PCR方法,从质粒pBV105-HPV6扩增获得HPV6 L1片段。采用T/A克隆法,实现了HPV6 L1的克隆,测序结果表明克隆基因序列与GenBank中HPV6 L1基因序列同源性为99.9%,有2个碱基发生了突变,但编码的氨基酸不改变。将HPV6 L1基因序列进行了密码子优化,优化后序列与优化前序列相比,基因序列的同源性为75%,但编码的氨基酸没有发生变化。密码子优化后,用密码子偏性分析软件CodonW分析,CAI(Codon Adaptation Index)从0.205提高到0.611,CBI(Codon Bias Index)从-0.117提高到0.531,FOP(Frequency of Optimumcodons)从0.356提高到0.730。成功构建了HPV6 mL1克隆基因的重组原核表达载体pGEX4T-1/HPV6 mL1,并用IPTG诱导实现了HPV6 mL1融合蛋白在BL21(DE3)原核细胞中的表达。HPV6 mL1融合蛋白主要以包涵体形式表达,包涵体形式最佳表达条件为浓度为1mmol/L IPTG ,温度37℃,诱导4h。经Glutathione Sepharose 4B柱层析纯化得到HPV6 mL1融合蛋白,为其结构功能研究和疫苗研发提供基础,是否具有生物活性还有待于进一步研究。
[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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R373;Q78
本文编号：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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R373;Q78
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