预防性人乳头瘤病毒病毒样颗粒疫苗的研究
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摘要
人乳头瘤病毒(Human Papillomavirus,HPV)有100多型别,根据与致癌性的关系分为高危型和低危型。高危型HPV与人类多种组织恶性肿瘤密切相关,尤其以HPV16、18、31、45、52和58型感染与宫颈癌的发生关系最为密切,其中HPV16占50%以上。
本论文针对HPV开展了以下三方面的研究:(1)利用荧光素酶基因为报告基因与含有HPV6、16、18、31、45、52和58型L1、L2衣壳蛋白基因的真核表达质粒共转染293FT细胞,获得带有荧光素酶基因的七种假病毒,建立了针对七种亚型中和抗体的快速、灵敏、准确的检测方法。(2)利用HPV6、16、18、31、45、52、58等七种真核表达质粒免疫小鼠,考察七种亚型间中和抗体交叉保护情况,结果显示:HPV6、HPV16和HPV18免疫血清对HPV52与HPV58假病毒中和阻断率小于24.1%(p>0.05);HPV58免疫血清对几种假病毒的中和阻断率分别为:HPV6(86.1%,p<0.01)、HPV16(30.9%,p>0.05)、HPV18(60.2%,p<0.005)、HPV45(36.1%,p<0.05)和HPV52(11.7%,p>0.05)。(3)根据中国部分地区和世界范围内相关流行病学研究结果,同时结合中和抗体交叉保护的关系,确定HPV16和58型为中国地区疫苗设计主要型别。分别利用大肠杆菌表达系统和毕赤酵母(Pichia Pastoris)表达系统表达HPV16和58型的L1蛋白,其中原核表达体系表达的L1经过变性和复性过程,可形成L1五聚体;酵母表达体系分泌表达L1蛋白,可形成病毒样颗粒(Virus Like Particles,VLPs),直径约为50nm。免疫动物结果显示:HPV16和58型两种形式疫苗均可产生中和抗体;在免疫血清阻断假病毒感染的能力和疫苗免疫原性方面,病毒样颗粒疫苗优于五聚体疫苗。
本论文研究结果为进一步研制适合中国和东亚地区的HPV预防性疫苗奠定基础。
Since Harold zur Hausen (1981) first linked papillomavirus to cervical cancer, two decades of studies have confirmed that persistent infection with certain human papilloma virus (HPV) genotypes termed‘high risk’is a necessary first carcinogenic step, or‘hit’in the pathogenesis of cervical cancer. HPV is common throughout the world. Although most infections with HPV cause no symptoms and are self-limiting, persistent genital HPV infection can cause cervical cancer in women.
Human papillomaviruses are DNA viruses that infect epithelial (skin or mucosal) cells. There are more than 100 known HPV genotypes. HPV genotypes that infect the genital mucosa are considered“high-risk”or“low-risk”, according to their link with cancer. The high-risk genotypes, genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 66, can lead to cervical cancer, and are also associated with other anogenital, head and neck cancers. Infection with low-risk genotypes very rarely causes cancer, but can cause benign or low-grade changes in cervical cells.
The HPV genome consists of a single molecule of double-stranded, circular DNA containing approximately 7,900 bp associated with histones. All open reading frame (ORF) protein-coding sequences are restricted to one strand. (i) The first is a noncoding upstream regulatory region of 400 to 1,000 bp. This region also contains the highest degree of variation in the viral genome. (ii) The second is an early region, consisting of ORFs E1, E2, E4, E5, E6, and E7, which are involved in viral replication and oncogenesis. (iii) The third is a late region, which encodes the L1 and L2 structural proteins for the viral capsid.
HPV is a relatively small, nonenveloped virus, 55 nm in diameter. It has an icosahedral capsid composed of 72 capsomers, which contain at late two capsid proteins, L1 and L2. Each capsomer is a pentamer of the major capsid protein, L1. Each virion capsid contains several copies (about 12 per virion) of the minor capsid protein, L2.
The evidence from animal papillomavirus infections, including some of the earliest founding of papillomavirus research, showed that neutralizing antibody was protective. Neutralizing antibodies are directed against the L1 capsid protein and the generation of neutralizing antibody requires the tertiary or native structure of the protein. Data suggested very strongly that generating neutralizing antibody to virus capsid protein would be an effective prophylactic vaccine strategy. The lack of an in vitro propagation system for the production of HPV virions and existence of the E6、E7 oncogenes has hampered the availability of suitable material for prophylactic vaccine development. In 1991,Zhou and many researchers discovered that the major capsid protein L1 can self assembles to virus-like particles (VLPs). The discovery has driven prophylactic HPV vaccine development. The VLPs is similarly to the native virus, These VLPs lack the oncogenic viral DNA, but can elicit high titers of neutralizing antibody that protect against experimental viral infection.
At present, many methods can be applied to detect neutralizing antibody agansist HPV virus in serum. For example, the establishment of the first nude model heterologous tissue transplantation, keratinocytes in vitro, enzyme-linked immunosorbent assay, virus-like particles HI test. However, these methods have many limitations of their own. some are more burdensome and are not suitable for large-scale operation of the testing samples; some have the phenomenon of false-positive; some methods are low sensitivity and can not be used to detect neutralizing antibody in the serum of natural HPV infection; Most of the methods are high cost and time-consuming.
In this article,we use HPV pseudovirus simulating the natural HPV to infect 293FT cell for detecting the antibody in serum. First of all, the reporter gene plasmid and the plasmid containing HPV structure of genes, L1 and L2, co-transfected the 293FT cells. The two proteins, L1 and L2, can self-assembly into virus-like particles , and at the same time can wrapped the virus genome or foreign genes to form pseudovirus that is similar to the structure and immunogenicity of HPV natural viruses. The pseudovirus is infectious, when the pseudovirus infect the 293FT cell, the reporter gene can express in 293FT cell. When pseudovirus and serum containing neutralizing antibody co-incubate before infecting 293FT cell, the pseudovirus will lost its infectious ability, the reporter gene expression level will be decline, so we can detect the neutralizing antibody level by detecting the reporter gene’s expression level.
The high-risk genotypes, HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 66, can lead to cervical cancer, and are also associated with other anogenital, head and neck cancers. So the prophylactic vaccine research must be considered various types of cross-protection provided by vaccine. In order to understand cross-protection between major HPV high-risk genotypes. Seven plasmids, Pshell6, Pshell16, Pshell18, Pshell31, Pshell45, Pshell52, Pshell58, and pcDNA3.1-Luc co-transfect 293FT cells respectively to form seven pseudovirus. The pseudovirus infect 293FT cells, so the Luc gene can express. The model of pesudovirus infect 293FT cells was initially established.The seven pcDNA3.1-HPVL1 plasmids were immuned mice, the serum was got respectively. Neutralizing antibody in the serum can prevent the pseudovirus from infecting 293FT cells. The experiment results show that: when the serum dilution for 1:40, each serum can block the corresponding pesudovirus infecting 293FT cells. Therein,HPV6 pseudovirus can be blocked partially by the other six serums, HPV16 serum (84.4%, p <0.01), HPV18 serum (91.2%, p <0.01), HPV31 serum (90.3%, p <0.01), HPV52 Serum (58.7%, p <0.005), HPV58 serum (86.1%, p <0.01), it was speculated that some of the high-risk HPV infection may inhibit low-risk type of HPV infection or persistent infection; HPV16,HPV31 and HPV52 pseudovirus can not be blocked by the other six serums, it suggests that HPV16 and HPV31 and HPV52 and other six HPVs have low rate cross protection; HPV58 pseudovirus can be blocked partially by HPV45 serum (40%, p <0.05), it suggests that HPV58 and HPV45 have some cross protection; The results show that there are some cross-protection among the seven HPV types, it provide the theoretical basis for the prophylatic vaccine combination of China and Eastasia.
During the early 1970s, the methylotrophic yeast Pichia pastoris was developed as a biological tool to convert methanol into high-quality protein that could be used in the livestock industry. Pichia pastoris has been used successfully to express a wide range of heterologous proteins as either intracellular or secreted proteins. Pichia expression system has many advantages of the eukaryotic expression system, such as protein processing, folding, post-translational modification, and so on. Operating with pichia is as simple as E.coli and Saccharomyces cerevisiae. Pichia expression system is more faster, simpler, cheaper, and higher levels of expression than Baculovirus and mammals eukaryotic expression system. As yeast expression systerm, Pichia and Saccharomyces cerevisiae has a similar advantages in operation of molecular and genetic and in foreign protein expression, but Pichia is as 10 times or even 100 times as Saccharomyces cerevisiae in foreign protein expression level.
Secretion requires the presence of a signal sequence on the expressed protein to target it to the secretory pathway. Although several different secretion signal sequences have been used successfully, including the native secretion signal present on some heterologous proteins, success has been variable. The secretion signal sequence from the S. cerevisiae MATαfactor prepro peptide (MF-α1) has been used with the most success. The major advantage of expressing heterologous proteins as secreted proteins is that P. pastoris secretes very low levels of native proteins. That, combined with the very low amount of protein in the minimal Pichia growth medium, means that the secreted heterologous protein comprises the vast majority of the total protein in the medium, and this serves as a first step in purification of the expressed protein.
S. cerevisiae core oligosaccharides have terminalα1-3 glycan linkages, whereas P. pastoris does not. Furthermore, it is believed that theα1-3 glycan linkages of S. cerevisiae are primarily responsible for the hyperantigenic nature of glycosylated proteins from S. cerevisiae that makes them particularly unsuitable for therapeutic use. Although not yet proven, this is predicted to be less of a problem for glycoproteins generated in Pichia, because their glycoprotein structure may resemble that of higher eukaryotes.
In order to obtain high level expression of HPV6,11,16,18,58 L1 protein of recombinant Pichia. First of all, the L1 genes were amplified from the HPV-positive pathological tissue DNA by PCR. The L1 genes were subcloned into pPICZαB expression vector, and then got the pPICZαB-XL1 expression plasmids. Electroporation transformation of X-33 or GS115 or SMD1168H with pPICZαB-XL1 plasmid linearized with SacI restriction enzymes. A quick, direct way to select putative multi-copy recombinants is to plate the transformation mix on increasing concentrations of Zeocin. Select several of Zeocin-resistant Pichia transformants and analyze for the presence of insert using PCR, or protein expression using SDS-PAGE. In this study, we get five types transformants respectively. The clone with the highest protein yield was selected for scale-up protein expression. The high-expression clone that expresses and secretes the 55 kilo Dalton recombinant HPV type 16 and 58L1 proteins in fermentation process and successfully purified the target protein with ion-exchange chromatography in a diethylaminoethanol (DEAE) sepharose column.
HPVL1 protein can also be expressed in E. coli, the expressed HPVL1 usually is in the form of inclusion bodies. In this study, pRSET-B was chosen as expression vector, the pRSET-B vectors is pUC-derived expression vectors designed for high-level protein expression and purification from cloned genes in E. coli. Expression of the gene of interest from pRSET is controlled by the strong phage T7 promoter that drives expression of gene 10 (Φ10). For expression of the gene of interest, it is necessary to deliver T7 RNA polymerase to the cells by inducing expression of the polymerase using the gratuitous inducer isopropylβ-D-thiogalactoside (IPTG). In addition, DNA inserts are positioned downstream and in frame with a sequence that encodes an N-terminal fusion peptide. This sequence includes an ATG translation initiation codon, a polyhistidine tag that functions as a metal binding domain in the translated protein. The metal binding domain of the fusion peptide allows simple purification of recombinant proteins by Immobilized Metal Affinity Chromatography with Invitrogen’s ProBondTM resin. The enterokinase cleavage recognition site in the fusion peptide located between the metal binding domain and the recombinant protein allows for subsequent removal of this N-terminal fusion peptide from the purified recombinant protein.
In Michael.P’s research about quantitative disassembly and Reassembly of HPV11L1, homogeneous capsomeres (pentamers) averaging 11.3S±1.5S (n=5) in average sedimentation coefficient and 9.7±1.2nm(n=15)in diameter, with occasionally a few larger aggregated structures apparent. The pentamers migrated on 5 to 20% linear sucrose gradients. The purified VLP, migrated to10 to 65% linear sucrose gradients, was distributed across the gradient, with many particles migrating to the position expected for intact VLPs (150S to 160S) but with the majority of the protein further down the gradient and in the pellet .It is clear from Michael.P’s studies that at physiological ionic strength, disulfide bonds are necessary to hold HPV-11 L1 VLPs together. In this experiment, there is a little DTT in refolding solution, DTT can prevent the HPVL1 from forming the VLPs by blocking the disulfide bonds, which are essential to maintaining long-term HPVL1 VLP structure at physiological ionic strength, the HPVL1 can form pentamers without dislfide bonds. The purified HPV16L1 and HPV58L (0.5MNaCl,PBS solution) were layered to 40% sucrose cushion, and then we analysed the supernatant and pellets with western-blot. HPV16L1 and HPV58L1 exist in supernatant. With electron microscope , the HPV16L1 and HPV58L1 in supernatant are in the form of pentamers (10nm), barely VLP(50nm). The study results showed that the HPVL1 protein expressed in E.coil,usually form monomer or pentamer,can form a virus-like particles, but the yield is very low. It is not suitable for industrial production.
In immunogenicity research, six kinds of vaccines (HPV16L1 pentamer vaccine, HPV58L1 pentamer vaccine, HPV16L1 and HPV58L1 pentamers bivalent vaccine, HPV16L1 VLPs vaccine, HPV58L1 VLPs vaccine, HPV16L1 and HPV58L1 VLPs bivalent vaccine) are employed. The six kinds of sera was got respectively. Neutralizing antibody in the six kinds of sera can prevent the corresponding pseudovirus from infecting 293FT cells. When the blocking rate is 50%, the four mono-vaccines corresponding neutralizing antibody titres are 1:25000 (HPV16L1 VLPs vaccine), 1:7800 ( HPV58L1 VLPs vaccine), 1:3800 (HPV16L1 pentamer vaccine), 1:3300 (HPV58L1 pentamer vaccine). The two bivalent vaccines corresponding neutralizing antibody titres are high too, the neutralizing antibody titers of VLPs vaccine are significantly higher than pentamer vaccine’s, the six kinds of vaccine show a good immunogenicity. When the two bivalent vaccines’sera dilution for 1:40, two kinds of sera can block the corresponding pesudovirus infecting 293FT cells, the VLPs bivalent vaccine’s blocking rates for corresponding types are 97.5% (HPV16) and 98.7% (HPV58), the pentamers bivalent vaccine’s blocking rates for corresponding types are 98% (HPV16) and 98.4% (HPV58), there was no significant difference between them. The VLPs bivalent vaccine’s blocking rates for other types are 89% (HPV6) , 49.8% (HPV18), 20.9% (HPV45) and 35.8%(HPV52), the pentamers bivalent vaccine’s blocking rates for other types are 87.4% (HPV6) , 37.8% (HPV18), 14.3% (HPV45) and 42.6% (HPV52). Both of two kinds of sera can not prevent the HPV31 pesudovirus from infecting 293FT cells. The results show that HPVL1 vaccine can prevent corresponding HPV infection, and also provide partical cross-protection for other type of HPV.
In conclusion, we establish initially the evaluation method for HPV neutralizing antibody and investigate the cross-protection among the HPV6 and other six types. In this study, we design the prophylatic vaccine combination for China and Eastasia areas accroding to the cross-protection results and epidemiological findings about HPV. All vaccine combination show a good immunogenicity, but cross-protection is limited.
本论文针对HPV开展了以下三方面的研究:(1)利用荧光素酶基因为报告基因与含有HPV6、16、18、31、45、52和58型L1、L2衣壳蛋白基因的真核表达质粒共转染293FT细胞,获得带有荧光素酶基因的七种假病毒,建立了针对七种亚型中和抗体的快速、灵敏、准确的检测方法。(2)利用HPV6、16、18、31、45、52、58等七种真核表达质粒免疫小鼠,考察七种亚型间中和抗体交叉保护情况,结果显示:HPV6、HPV16和HPV18免疫血清对HPV52与HPV58假病毒中和阻断率小于24.1%(p>0.05);HPV58免疫血清对几种假病毒的中和阻断率分别为:HPV6(86.1%,p<0.01)、HPV16(30.9%,p>0.05)、HPV18(60.2%,p<0.005)、HPV45(36.1%,p<0.05)和HPV52(11.7%,p>0.05)。(3)根据中国部分地区和世界范围内相关流行病学研究结果,同时结合中和抗体交叉保护的关系,确定HPV16和58型为中国地区疫苗设计主要型别。分别利用大肠杆菌表达系统和毕赤酵母(Pichia Pastoris)表达系统表达HPV16和58型的L1蛋白,其中原核表达体系表达的L1经过变性和复性过程,可形成L1五聚体;酵母表达体系分泌表达L1蛋白,可形成病毒样颗粒(Virus Like Particles,VLPs),直径约为50nm。免疫动物结果显示:HPV16和58型两种形式疫苗均可产生中和抗体;在免疫血清阻断假病毒感染的能力和疫苗免疫原性方面,病毒样颗粒疫苗优于五聚体疫苗。
本论文研究结果为进一步研制适合中国和东亚地区的HPV预防性疫苗奠定基础。
Since Harold zur Hausen (1981) first linked papillomavirus to cervical cancer, two decades of studies have confirmed that persistent infection with certain human papilloma virus (HPV) genotypes termed‘high risk’is a necessary first carcinogenic step, or‘hit’in the pathogenesis of cervical cancer. HPV is common throughout the world. Although most infections with HPV cause no symptoms and are self-limiting, persistent genital HPV infection can cause cervical cancer in women.
Human papillomaviruses are DNA viruses that infect epithelial (skin or mucosal) cells. There are more than 100 known HPV genotypes. HPV genotypes that infect the genital mucosa are considered“high-risk”or“low-risk”, according to their link with cancer. The high-risk genotypes, genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 66, can lead to cervical cancer, and are also associated with other anogenital, head and neck cancers. Infection with low-risk genotypes very rarely causes cancer, but can cause benign or low-grade changes in cervical cells.
The HPV genome consists of a single molecule of double-stranded, circular DNA containing approximately 7,900 bp associated with histones. All open reading frame (ORF) protein-coding sequences are restricted to one strand. (i) The first is a noncoding upstream regulatory region of 400 to 1,000 bp. This region also contains the highest degree of variation in the viral genome. (ii) The second is an early region, consisting of ORFs E1, E2, E4, E5, E6, and E7, which are involved in viral replication and oncogenesis. (iii) The third is a late region, which encodes the L1 and L2 structural proteins for the viral capsid.
HPV is a relatively small, nonenveloped virus, 55 nm in diameter. It has an icosahedral capsid composed of 72 capsomers, which contain at late two capsid proteins, L1 and L2. Each capsomer is a pentamer of the major capsid protein, L1. Each virion capsid contains several copies (about 12 per virion) of the minor capsid protein, L2.
The evidence from animal papillomavirus infections, including some of the earliest founding of papillomavirus research, showed that neutralizing antibody was protective. Neutralizing antibodies are directed against the L1 capsid protein and the generation of neutralizing antibody requires the tertiary or native structure of the protein. Data suggested very strongly that generating neutralizing antibody to virus capsid protein would be an effective prophylactic vaccine strategy. The lack of an in vitro propagation system for the production of HPV virions and existence of the E6、E7 oncogenes has hampered the availability of suitable material for prophylactic vaccine development. In 1991,Zhou and many researchers discovered that the major capsid protein L1 can self assembles to virus-like particles (VLPs). The discovery has driven prophylactic HPV vaccine development. The VLPs is similarly to the native virus, These VLPs lack the oncogenic viral DNA, but can elicit high titers of neutralizing antibody that protect against experimental viral infection.
At present, many methods can be applied to detect neutralizing antibody agansist HPV virus in serum. For example, the establishment of the first nude model heterologous tissue transplantation, keratinocytes in vitro, enzyme-linked immunosorbent assay, virus-like particles HI test. However, these methods have many limitations of their own. some are more burdensome and are not suitable for large-scale operation of the testing samples; some have the phenomenon of false-positive; some methods are low sensitivity and can not be used to detect neutralizing antibody in the serum of natural HPV infection; Most of the methods are high cost and time-consuming.
In this article,we use HPV pseudovirus simulating the natural HPV to infect 293FT cell for detecting the antibody in serum. First of all, the reporter gene plasmid and the plasmid containing HPV structure of genes, L1 and L2, co-transfected the 293FT cells. The two proteins, L1 and L2, can self-assembly into virus-like particles , and at the same time can wrapped the virus genome or foreign genes to form pseudovirus that is similar to the structure and immunogenicity of HPV natural viruses. The pseudovirus is infectious, when the pseudovirus infect the 293FT cell, the reporter gene can express in 293FT cell. When pseudovirus and serum containing neutralizing antibody co-incubate before infecting 293FT cell, the pseudovirus will lost its infectious ability, the reporter gene expression level will be decline, so we can detect the neutralizing antibody level by detecting the reporter gene’s expression level.
The high-risk genotypes, HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 66, can lead to cervical cancer, and are also associated with other anogenital, head and neck cancers. So the prophylactic vaccine research must be considered various types of cross-protection provided by vaccine. In order to understand cross-protection between major HPV high-risk genotypes. Seven plasmids, Pshell6, Pshell16, Pshell18, Pshell31, Pshell45, Pshell52, Pshell58, and pcDNA3.1-Luc co-transfect 293FT cells respectively to form seven pseudovirus. The pseudovirus infect 293FT cells, so the Luc gene can express. The model of pesudovirus infect 293FT cells was initially established.The seven pcDNA3.1-HPVL1 plasmids were immuned mice, the serum was got respectively. Neutralizing antibody in the serum can prevent the pseudovirus from infecting 293FT cells. The experiment results show that: when the serum dilution for 1:40, each serum can block the corresponding pesudovirus infecting 293FT cells. Therein,HPV6 pseudovirus can be blocked partially by the other six serums, HPV16 serum (84.4%, p <0.01), HPV18 serum (91.2%, p <0.01), HPV31 serum (90.3%, p <0.01), HPV52 Serum (58.7%, p <0.005), HPV58 serum (86.1%, p <0.01), it was speculated that some of the high-risk HPV infection may inhibit low-risk type of HPV infection or persistent infection; HPV16,HPV31 and HPV52 pseudovirus can not be blocked by the other six serums, it suggests that HPV16 and HPV31 and HPV52 and other six HPVs have low rate cross protection; HPV58 pseudovirus can be blocked partially by HPV45 serum (40%, p <0.05), it suggests that HPV58 and HPV45 have some cross protection; The results show that there are some cross-protection among the seven HPV types, it provide the theoretical basis for the prophylatic vaccine combination of China and Eastasia.
During the early 1970s, the methylotrophic yeast Pichia pastoris was developed as a biological tool to convert methanol into high-quality protein that could be used in the livestock industry. Pichia pastoris has been used successfully to express a wide range of heterologous proteins as either intracellular or secreted proteins. Pichia expression system has many advantages of the eukaryotic expression system, such as protein processing, folding, post-translational modification, and so on. Operating with pichia is as simple as E.coli and Saccharomyces cerevisiae. Pichia expression system is more faster, simpler, cheaper, and higher levels of expression than Baculovirus and mammals eukaryotic expression system. As yeast expression systerm, Pichia and Saccharomyces cerevisiae has a similar advantages in operation of molecular and genetic and in foreign protein expression, but Pichia is as 10 times or even 100 times as Saccharomyces cerevisiae in foreign protein expression level.
Secretion requires the presence of a signal sequence on the expressed protein to target it to the secretory pathway. Although several different secretion signal sequences have been used successfully, including the native secretion signal present on some heterologous proteins, success has been variable. The secretion signal sequence from the S. cerevisiae MATαfactor prepro peptide (MF-α1) has been used with the most success. The major advantage of expressing heterologous proteins as secreted proteins is that P. pastoris secretes very low levels of native proteins. That, combined with the very low amount of protein in the minimal Pichia growth medium, means that the secreted heterologous protein comprises the vast majority of the total protein in the medium, and this serves as a first step in purification of the expressed protein.
S. cerevisiae core oligosaccharides have terminalα1-3 glycan linkages, whereas P. pastoris does not. Furthermore, it is believed that theα1-3 glycan linkages of S. cerevisiae are primarily responsible for the hyperantigenic nature of glycosylated proteins from S. cerevisiae that makes them particularly unsuitable for therapeutic use. Although not yet proven, this is predicted to be less of a problem for glycoproteins generated in Pichia, because their glycoprotein structure may resemble that of higher eukaryotes.
In order to obtain high level expression of HPV6,11,16,18,58 L1 protein of recombinant Pichia. First of all, the L1 genes were amplified from the HPV-positive pathological tissue DNA by PCR. The L1 genes were subcloned into pPICZαB expression vector, and then got the pPICZαB-XL1 expression plasmids. Electroporation transformation of X-33 or GS115 or SMD1168H with pPICZαB-XL1 plasmid linearized with SacI restriction enzymes. A quick, direct way to select putative multi-copy recombinants is to plate the transformation mix on increasing concentrations of Zeocin. Select several of Zeocin-resistant Pichia transformants and analyze for the presence of insert using PCR, or protein expression using SDS-PAGE. In this study, we get five types transformants respectively. The clone with the highest protein yield was selected for scale-up protein expression. The high-expression clone that expresses and secretes the 55 kilo Dalton recombinant HPV type 16 and 58L1 proteins in fermentation process and successfully purified the target protein with ion-exchange chromatography in a diethylaminoethanol (DEAE) sepharose column.
HPVL1 protein can also be expressed in E. coli, the expressed HPVL1 usually is in the form of inclusion bodies. In this study, pRSET-B was chosen as expression vector, the pRSET-B vectors is pUC-derived expression vectors designed for high-level protein expression and purification from cloned genes in E. coli. Expression of the gene of interest from pRSET is controlled by the strong phage T7 promoter that drives expression of gene 10 (Φ10). For expression of the gene of interest, it is necessary to deliver T7 RNA polymerase to the cells by inducing expression of the polymerase using the gratuitous inducer isopropylβ-D-thiogalactoside (IPTG). In addition, DNA inserts are positioned downstream and in frame with a sequence that encodes an N-terminal fusion peptide. This sequence includes an ATG translation initiation codon, a polyhistidine tag that functions as a metal binding domain in the translated protein. The metal binding domain of the fusion peptide allows simple purification of recombinant proteins by Immobilized Metal Affinity Chromatography with Invitrogen’s ProBondTM resin. The enterokinase cleavage recognition site in the fusion peptide located between the metal binding domain and the recombinant protein allows for subsequent removal of this N-terminal fusion peptide from the purified recombinant protein.
In Michael.P’s research about quantitative disassembly and Reassembly of HPV11L1, homogeneous capsomeres (pentamers) averaging 11.3S±1.5S (n=5) in average sedimentation coefficient and 9.7±1.2nm(n=15)in diameter, with occasionally a few larger aggregated structures apparent. The pentamers migrated on 5 to 20% linear sucrose gradients. The purified VLP, migrated to10 to 65% linear sucrose gradients, was distributed across the gradient, with many particles migrating to the position expected for intact VLPs (150S to 160S) but with the majority of the protein further down the gradient and in the pellet .It is clear from Michael.P’s studies that at physiological ionic strength, disulfide bonds are necessary to hold HPV-11 L1 VLPs together. In this experiment, there is a little DTT in refolding solution, DTT can prevent the HPVL1 from forming the VLPs by blocking the disulfide bonds, which are essential to maintaining long-term HPVL1 VLP structure at physiological ionic strength, the HPVL1 can form pentamers without dislfide bonds. The purified HPV16L1 and HPV58L (0.5MNaCl,PBS solution) were layered to 40% sucrose cushion, and then we analysed the supernatant and pellets with western-blot. HPV16L1 and HPV58L1 exist in supernatant. With electron microscope , the HPV16L1 and HPV58L1 in supernatant are in the form of pentamers (10nm), barely VLP(50nm). The study results showed that the HPVL1 protein expressed in E.coil,usually form monomer or pentamer,can form a virus-like particles, but the yield is very low. It is not suitable for industrial production.
In immunogenicity research, six kinds of vaccines (HPV16L1 pentamer vaccine, HPV58L1 pentamer vaccine, HPV16L1 and HPV58L1 pentamers bivalent vaccine, HPV16L1 VLPs vaccine, HPV58L1 VLPs vaccine, HPV16L1 and HPV58L1 VLPs bivalent vaccine) are employed. The six kinds of sera was got respectively. Neutralizing antibody in the six kinds of sera can prevent the corresponding pseudovirus from infecting 293FT cells. When the blocking rate is 50%, the four mono-vaccines corresponding neutralizing antibody titres are 1:25000 (HPV16L1 VLPs vaccine), 1:7800 ( HPV58L1 VLPs vaccine), 1:3800 (HPV16L1 pentamer vaccine), 1:3300 (HPV58L1 pentamer vaccine). The two bivalent vaccines corresponding neutralizing antibody titres are high too, the neutralizing antibody titers of VLPs vaccine are significantly higher than pentamer vaccine’s, the six kinds of vaccine show a good immunogenicity. When the two bivalent vaccines’sera dilution for 1:40, two kinds of sera can block the corresponding pesudovirus infecting 293FT cells, the VLPs bivalent vaccine’s blocking rates for corresponding types are 97.5% (HPV16) and 98.7% (HPV58), the pentamers bivalent vaccine’s blocking rates for corresponding types are 98% (HPV16) and 98.4% (HPV58), there was no significant difference between them. The VLPs bivalent vaccine’s blocking rates for other types are 89% (HPV6) , 49.8% (HPV18), 20.9% (HPV45) and 35.8%(HPV52), the pentamers bivalent vaccine’s blocking rates for other types are 87.4% (HPV6) , 37.8% (HPV18), 14.3% (HPV45) and 42.6% (HPV52). Both of two kinds of sera can not prevent the HPV31 pesudovirus from infecting 293FT cells. The results show that HPVL1 vaccine can prevent corresponding HPV infection, and also provide partical cross-protection for other type of HPV.
In conclusion, we establish initially the evaluation method for HPV neutralizing antibody and investigate the cross-protection among the HPV6 and other six types. In this study, we design the prophylatic vaccine combination for China and Eastasia areas accroding to the cross-protection results and epidemiological findings about HPV. All vaccine combination show a good immunogenicity, but cross-protection is limited.
引文
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