利用毕赤酵母系统的丙型肝炎病毒重组E1E2糖蛋白的表达、纯化和性质研究
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摘要
丙型肝炎病毒(HCV)是丙型肝炎的致病原因,全球有超过3%的人口被感染。发展一个有效的HCV疫苗是控制HCV感染的关键。
由于HCV的高变异性,曾经有观点认为有效的、可以抑制不同亚型的HCV疫苗是不可能得到的。可是近来的研究结果指出,HCV包膜糖蛋白上含有保守的HCV细胞受体结合位点,可以诱导产生广泛的中和抗体,从而阻止HCV感染。以哺乳动物细胞表达产生的HCV包膜糖蛋白作为疫苗成功的在黑猩猩中证实能够保护黑猩猩免受异型的HCV感染。然而,哺乳动物细胞表达系统产生的HCV包膜糖蛋白产量很低,难以大规模应用。与细胞表达系统相比,大肠杆菌表达系统中虽然可以产生较高产量的HCV包膜蛋白,但其表达的蛋白不具备糖基化。这种缺乏糖基化的包膜蛋白在结构、性质以及免疫原性上都与天然HCV包膜糖蛋白相差巨大。因此,我们使用毕赤酵母这种经济的真核表达系统来表达HCV包膜糖蛋白。
我们在pPIC9K载体的基础上构建了真核表达质粒pPIC9K-E1E2,其编码HCV E1 187-346位氨基酸序列和E2 381-699位氨基酸序列,将其电转化进入毕赤酵母菌株SMD1168中获得重组转化子。经过甲醇利用型筛选和G418抗性筛选得到的工程菌株能够在甲醇诱导下高效表达重组HCV E1E2糖蛋白,产量可达40mg/L。经Q-S-FF柱、P-S-FF柱及G100柱对表达的包膜糖蛋白进行纯化,得到了纯度较高的重组蛋白。
得到的重组蛋白由于不同的糖基化和不同的聚集度拥有几种不同的形式：72kD、95kD、145kD和大于200kD的聚集物(Aggregation, Agg), SDS-PAGE显示其主体为72kD蛋白和Agg。糖苷酶切实验表明重组蛋白中糖基化的主要形式是甘露糖苷形成的糖基化,但是也存在着复杂形式的糖基化,而且该复杂糖基化的糖苷可以很大的影响HCV单克隆抗体A4的识别。此外,我们使用GST—pulldown实验来验证重组蛋白能否同HCV受体人CD81大胞外环(LEL)相互作用。我们构建了pET-GST-LEL质粒并在大肠杆菌BL21中转化表达GST-LEL融合蛋白,并使用Glutathione Sepharose 4B beads证实了重组E1E2糖蛋白能同GST-LEL蛋白结合,表明重组E1E2糖蛋白不仅含有HCV E2 CD81结合区的氨基酸序列,而且这结合区域形成了正确的构象。更进一步的是,重组E1E2糖蛋白能够同核心蛋白core相互作用形成病毒样粒子。将重组E1E2糖蛋白和大肠杆菌中表达的core蛋白(氨基酸序列1-137)共同复性浓缩后,得到了两种不同直径的病毒样粒子。一种粒子的直径约35nm,推测是core蛋白单独形成的粒子；另一种粒子的直径约55nm,推测该粒子是由重组E1E2蛋白与core共同作用形成的。
将得到的重组E1E2糖蛋白免疫兔子能够产生高水平的抗体应答,并能持续维持约两个月,将氢氧化铝作为佐剂加入后,抗体产生更快,峰值更高,但是持续维持的时间则较短。在小鼠中我们也得到了类似的结果。抑制实验表明,用纯化蛋白免疫获得的兔血清能阻止重组E1E2糖蛋白同CD81的相互作用,且抑制效果呈剂量依赖性,同时对照兔血清则没有影响。我们应用假病毒粒子(PP)系统和HCV细胞培养系统对血清的中和效果进行了研究,结果表明同对照血清相比,仅用重组E1E2糖蛋白免疫获得的兔血清对来源于HCV 1a亚型和HCV 1b亚型的HCV假病毒粒子,甚至对HCV 2a亚型的HCV病毒粒子都有中和作用；而用氢氧化铝作为佐剂免疫获得的兔血清仅对来源于HCV 1a亚型的HCVpp有强的中和作用,对1b亚型的HCVpp的中和作用较弱,对2a亚型的HCV病毒粒子则没有中和作用。同时,这些兔血清都对VSVpp不具中和能力,表明该中和能力是HCV特异性的。免疫获得的小鼠血清中和实验由于时间关系尚未完成,仍然在进行中。上述结果初步表明,毕赤酵母系统表达纯化的HCV E1E2糖蛋白能够诱导产生广泛的中和抗体,并且凭借其产量高的优点,有望成为一种可以大规模生产应用的HCV疫苗。
Hepatitis C virus (HCV) is the pathogen of hepatitis C. It infects over 3% of the world population. Development of an effective vaccine may be the key in the control of HCV infection.
HCV is hypervariable, so it used to be suspectable to get an efficient vaccine which is able to neutralize different HCV subgenotypes. However, recent studies have shown that HCV envelope proteins contain conserved receptor binding domain, which can induce broadly neutralizing antibodies against HCV. One report showed that E1E2 glycoproteins expressed by mammalian cells can protect chimpanzees from heterogeneous HCV infection as a HCV vaccine. But the yield of HCV envelope glycoprotein expressed by mammalian cells is too low to be applied to mass production. More HCV envelope protein can be produced by E.coli expression system, but the protein hasn't glycan. Hence, this HCV envelope protein is very different from native HCV envelope glycoprotein in structures, functions and antigenicity. Herein, we used Pichia pastoris yeast, an efficient and cheap eukaryotic expression system, to express HCV E1E2 recombinant glycoproteins (rE1E2s).
We constructed eukaryotic expression plasmid pPIC9K-E1E2, which encodes HCV E1 (aa187-aa346) and E2 (aa381-aa699). Then It was electroporated into Pichia pastoris strain SMD1168. A single colony of multiple inserted His+ Mut+ SMD1168 recombinants was selected and confirmed. It can express recombinant HCV E1E2 glycoproteins (rElE2s), the yield of rElE2s was 40mg/L. We got purified rElE2s by chromatography with Q-S-FF column, P-S-FF column and G100 column.
There are four forms of rE1E2s:72kD,95kD,145kD and over 200kD. SDS-PAGE shows the major rElE2s are the 72kD protein and over 200kD aggregation(Agg). It was shown that the major glycans of rE1E2s are high mannose oligosaccharides and the minor glycans are the complex forms. The complex glycans can affect the recognition of monoclonal antibody A4. To assess whether rElE2s are able to bind CD81 large extracelluar loop(LEL), we employed GST-pulldown assay. We constructed pET-GST-LEL plasmid and transform into E.coli BL21 strain. GST-LEL protein was expressed by BL21 and purified by Glutathione Sepharose 4B beads. GST-pulldown assay shows rE1E2s can bind GST-LEL. It suggest that rE1E2s does not only contain the sequence of HCV E2 CD81 binding domain, but also has the domain with correct conformation. Moreover, rElE2s can assemble into virus-like particles with HCV core protein. We mixed rE1E2s and core protein (aal-137), then renatured and concentrated the proteins. Two kinds of particles were produced, the diameter of smaller particles is about 35nm, and the diameter of bigger particles is about 55nm. So we suggest that the smaller particles are only assembled by core protein and the bigger particles are assembled by core protein and rE1E2s.
High humoral response was induced by vaccination with rE1E2s, and could hold two months. Comparing with vaccination with only rElE2s, higher humoral response was induced more rapidly, but more transient by vaccination with Al(OH)3 as adjuvant. Similar results were obtained in immunized mice. Blocking assay showed the rabbit serum induced by rE1E2s can block the interaction between rE1E2s with CD81 as a dose-dependent manner. Furthermore, we employed HCV pseudotype particles(HCVpp) system and HCV cell culture(HCVcc) system to confirm whether the serum can neutralize HCV. The results indicated the rabbit serum which induced by only rE1E2s can neutralize two kinds of HCVpp derived from HCV genotype la or lb,even HCV virions derived from HCV genotype 2a. And the rabbit serum which induced by rElE2s addition with Al(OH)3 as adjuvant can neutralize HCVpp derived from genotype la, can neutralize HCVpp derived from genotype lb with lower efficiency. The ability of neutralizing virus is HCV-specific because of all the serum disable to neutralize VSVpp. The inhibit assay of mouse serum is not complete. As a conclusion, rE1E2s expressed by Pichia pastoris yeast can induce broad neutralizing antibodies against HCV, and is hopeful for mass production as a HCV vaccine.
由于HCV的高变异性,曾经有观点认为有效的、可以抑制不同亚型的HCV疫苗是不可能得到的。可是近来的研究结果指出,HCV包膜糖蛋白上含有保守的HCV细胞受体结合位点,可以诱导产生广泛的中和抗体,从而阻止HCV感染。以哺乳动物细胞表达产生的HCV包膜糖蛋白作为疫苗成功的在黑猩猩中证实能够保护黑猩猩免受异型的HCV感染。然而,哺乳动物细胞表达系统产生的HCV包膜糖蛋白产量很低,难以大规模应用。与细胞表达系统相比,大肠杆菌表达系统中虽然可以产生较高产量的HCV包膜蛋白,但其表达的蛋白不具备糖基化。这种缺乏糖基化的包膜蛋白在结构、性质以及免疫原性上都与天然HCV包膜糖蛋白相差巨大。因此,我们使用毕赤酵母这种经济的真核表达系统来表达HCV包膜糖蛋白。
我们在pPIC9K载体的基础上构建了真核表达质粒pPIC9K-E1E2,其编码HCV E1 187-346位氨基酸序列和E2 381-699位氨基酸序列,将其电转化进入毕赤酵母菌株SMD1168中获得重组转化子。经过甲醇利用型筛选和G418抗性筛选得到的工程菌株能够在甲醇诱导下高效表达重组HCV E1E2糖蛋白,产量可达40mg/L。经Q-S-FF柱、P-S-FF柱及G100柱对表达的包膜糖蛋白进行纯化,得到了纯度较高的重组蛋白。
得到的重组蛋白由于不同的糖基化和不同的聚集度拥有几种不同的形式：72kD、95kD、145kD和大于200kD的聚集物(Aggregation, Agg), SDS-PAGE显示其主体为72kD蛋白和Agg。糖苷酶切实验表明重组蛋白中糖基化的主要形式是甘露糖苷形成的糖基化,但是也存在着复杂形式的糖基化,而且该复杂糖基化的糖苷可以很大的影响HCV单克隆抗体A4的识别。此外,我们使用GST—pulldown实验来验证重组蛋白能否同HCV受体人CD81大胞外环(LEL)相互作用。我们构建了pET-GST-LEL质粒并在大肠杆菌BL21中转化表达GST-LEL融合蛋白,并使用Glutathione Sepharose 4B beads证实了重组E1E2糖蛋白能同GST-LEL蛋白结合,表明重组E1E2糖蛋白不仅含有HCV E2 CD81结合区的氨基酸序列,而且这结合区域形成了正确的构象。更进一步的是,重组E1E2糖蛋白能够同核心蛋白core相互作用形成病毒样粒子。将重组E1E2糖蛋白和大肠杆菌中表达的core蛋白(氨基酸序列1-137)共同复性浓缩后,得到了两种不同直径的病毒样粒子。一种粒子的直径约35nm,推测是core蛋白单独形成的粒子；另一种粒子的直径约55nm,推测该粒子是由重组E1E2蛋白与core共同作用形成的。
将得到的重组E1E2糖蛋白免疫兔子能够产生高水平的抗体应答,并能持续维持约两个月,将氢氧化铝作为佐剂加入后,抗体产生更快,峰值更高,但是持续维持的时间则较短。在小鼠中我们也得到了类似的结果。抑制实验表明,用纯化蛋白免疫获得的兔血清能阻止重组E1E2糖蛋白同CD81的相互作用,且抑制效果呈剂量依赖性,同时对照兔血清则没有影响。我们应用假病毒粒子(PP)系统和HCV细胞培养系统对血清的中和效果进行了研究,结果表明同对照血清相比,仅用重组E1E2糖蛋白免疫获得的兔血清对来源于HCV 1a亚型和HCV 1b亚型的HCV假病毒粒子,甚至对HCV 2a亚型的HCV病毒粒子都有中和作用；而用氢氧化铝作为佐剂免疫获得的兔血清仅对来源于HCV 1a亚型的HCVpp有强的中和作用,对1b亚型的HCVpp的中和作用较弱,对2a亚型的HCV病毒粒子则没有中和作用。同时,这些兔血清都对VSVpp不具中和能力,表明该中和能力是HCV特异性的。免疫获得的小鼠血清中和实验由于时间关系尚未完成,仍然在进行中。上述结果初步表明,毕赤酵母系统表达纯化的HCV E1E2糖蛋白能够诱导产生广泛的中和抗体,并且凭借其产量高的优点,有望成为一种可以大规模生产应用的HCV疫苗。
Hepatitis C virus (HCV) is the pathogen of hepatitis C. It infects over 3% of the world population. Development of an effective vaccine may be the key in the control of HCV infection.
HCV is hypervariable, so it used to be suspectable to get an efficient vaccine which is able to neutralize different HCV subgenotypes. However, recent studies have shown that HCV envelope proteins contain conserved receptor binding domain, which can induce broadly neutralizing antibodies against HCV. One report showed that E1E2 glycoproteins expressed by mammalian cells can protect chimpanzees from heterogeneous HCV infection as a HCV vaccine. But the yield of HCV envelope glycoprotein expressed by mammalian cells is too low to be applied to mass production. More HCV envelope protein can be produced by E.coli expression system, but the protein hasn't glycan. Hence, this HCV envelope protein is very different from native HCV envelope glycoprotein in structures, functions and antigenicity. Herein, we used Pichia pastoris yeast, an efficient and cheap eukaryotic expression system, to express HCV E1E2 recombinant glycoproteins (rE1E2s).
We constructed eukaryotic expression plasmid pPIC9K-E1E2, which encodes HCV E1 (aa187-aa346) and E2 (aa381-aa699). Then It was electroporated into Pichia pastoris strain SMD1168. A single colony of multiple inserted His+ Mut+ SMD1168 recombinants was selected and confirmed. It can express recombinant HCV E1E2 glycoproteins (rElE2s), the yield of rElE2s was 40mg/L. We got purified rElE2s by chromatography with Q-S-FF column, P-S-FF column and G100 column.
There are four forms of rE1E2s:72kD,95kD,145kD and over 200kD. SDS-PAGE shows the major rElE2s are the 72kD protein and over 200kD aggregation(Agg). It was shown that the major glycans of rE1E2s are high mannose oligosaccharides and the minor glycans are the complex forms. The complex glycans can affect the recognition of monoclonal antibody A4. To assess whether rElE2s are able to bind CD81 large extracelluar loop(LEL), we employed GST-pulldown assay. We constructed pET-GST-LEL plasmid and transform into E.coli BL21 strain. GST-LEL protein was expressed by BL21 and purified by Glutathione Sepharose 4B beads. GST-pulldown assay shows rE1E2s can bind GST-LEL. It suggest that rE1E2s does not only contain the sequence of HCV E2 CD81 binding domain, but also has the domain with correct conformation. Moreover, rElE2s can assemble into virus-like particles with HCV core protein. We mixed rE1E2s and core protein (aal-137), then renatured and concentrated the proteins. Two kinds of particles were produced, the diameter of smaller particles is about 35nm, and the diameter of bigger particles is about 55nm. So we suggest that the smaller particles are only assembled by core protein and the bigger particles are assembled by core protein and rE1E2s.
High humoral response was induced by vaccination with rE1E2s, and could hold two months. Comparing with vaccination with only rElE2s, higher humoral response was induced more rapidly, but more transient by vaccination with Al(OH)3 as adjuvant. Similar results were obtained in immunized mice. Blocking assay showed the rabbit serum induced by rE1E2s can block the interaction between rE1E2s with CD81 as a dose-dependent manner. Furthermore, we employed HCV pseudotype particles(HCVpp) system and HCV cell culture(HCVcc) system to confirm whether the serum can neutralize HCV. The results indicated the rabbit serum which induced by only rE1E2s can neutralize two kinds of HCVpp derived from HCV genotype la or lb,even HCV virions derived from HCV genotype 2a. And the rabbit serum which induced by rElE2s addition with Al(OH)3 as adjuvant can neutralize HCVpp derived from genotype la, can neutralize HCVpp derived from genotype lb with lower efficiency. The ability of neutralizing virus is HCV-specific because of all the serum disable to neutralize VSVpp. The inhibit assay of mouse serum is not complete. As a conclusion, rE1E2s expressed by Pichia pastoris yeast can induce broad neutralizing antibodies against HCV, and is hopeful for mass production as a HCV vaccine.
引文
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