利用畢赤酵母系統(tǒng)的丙型肝炎病毒重組E1E2糖蛋白的表達(dá)、純化和性質(zhì)研究
發(fā)布時間：2018-09-03 07:13
【摘要】：丙型肝炎病毒(HCV)是丙型肝炎的致病原因,全球有超過3%的人口被感染。發(fā)展一個有效的HCV疫苗是控制HCV感染的關(guān)鍵。 由于HCV的高變異性,曾經(jīng)有觀點(diǎn)認(rèn)為有效的、可以抑制不同亞型的HCV疫苗是不可能得到的�？墒墙鼇淼难芯拷Y(jié)果指出,HCV包膜糖蛋白上含有保守的HCV細(xì)胞受體結(jié)合位點(diǎn),可以誘導(dǎo)產(chǎn)生廣泛的中和抗體,從而阻止HCV感染。以哺乳動物細(xì)胞表達(dá)產(chǎn)生的HCV包膜糖蛋白作為疫苗成功的在黑猩猩中證實(shí)能夠保護(hù)黑猩猩免受異型的HCV感染。然而,哺乳動物細(xì)胞表達(dá)系統(tǒng)產(chǎn)生的HCV包膜糖蛋白產(chǎn)量很低,難以大規(guī)模應(yīng)用。與細(xì)胞表達(dá)系統(tǒng)相比,大腸桿菌表達(dá)系統(tǒng)中雖然可以產(chǎn)生較高產(chǎn)量的HCV包膜蛋白,但其表達(dá)的蛋白不具備糖基化。這種缺乏糖基化的包膜蛋白在結(jié)構(gòu)、性質(zhì)以及免疫原性上都與天然HCV包膜糖蛋白相差巨大。因此,我們使用畢赤酵母這種經(jīng)濟(jì)的真核表達(dá)系統(tǒng)來表達(dá)HCV包膜糖蛋白。 我們在pPIC9K載體的基礎(chǔ)上構(gòu)建了真核表達(dá)質(zhì)粒pPIC9K-E1E2,其編碼HCV E1 187-346位氨基酸序列和E2 381-699位氨基酸序列,將其電轉(zhuǎn)化進(jìn)入畢赤酵母菌株SMD1168中獲得重組轉(zhuǎn)化子。經(jīng)過甲醇利用型篩選和G418抗性篩選得到的工程菌株能夠在甲醇誘導(dǎo)下高效表達(dá)重組HCV E1E2糖蛋白,產(chǎn)量可達(dá)40mg/L。經(jīng)Q-S-FF柱、P-S-FF柱及G100柱對表達(dá)的包膜糖蛋白進(jìn)行純化,得到了純度較高的重組蛋白。 得到的重組蛋白由于不同的糖基化和不同的聚集度擁有幾種不同的形式：72kD、95kD、145kD和大于200kD的聚集物(Aggregation, Agg), SDS-PAGE顯示其主體為72kD蛋白和Agg。糖苷酶切實(shí)驗(yàn)表明重組蛋白中糖基化的主要形式是甘露糖苷形成的糖基化,但是也存在著復(fù)雜形式的糖基化,而且該復(fù)雜糖基化的糖苷可以很大的影響HCV單克隆抗體A4的識別。此外,我們使用GST—pulldown實(shí)驗(yàn)來驗(yàn)證重組蛋白能否同HCV受體人CD81大胞外環(huán)(LEL)相互作用。我們構(gòu)建了pET-GST-LEL質(zhì)粒并在大腸桿菌BL21中轉(zhuǎn)化表達(dá)GST-LEL融合蛋白,并使用Glutathione Sepharose 4B beads證實(shí)了重組E1E2糖蛋白能同GST-LEL蛋白結(jié)合,表明重組E1E2糖蛋白不僅含有HCV E2 CD81結(jié)合區(qū)的氨基酸序列,而且這結(jié)合區(qū)域形成了正確的構(gòu)象。更進(jìn)一步的是,重組E1E2糖蛋白能夠同核心蛋白core相互作用形成病毒樣粒子。將重組E1E2糖蛋白和大腸桿菌中表達(dá)的core蛋白(氨基酸序列1-137)共同復(fù)性濃縮后,得到了兩種不同直徑的病毒樣粒子。一種粒子的直徑約35nm,推測是core蛋白單獨(dú)形成的粒子；另一種粒子的直徑約55nm,推測該粒子是由重組E1E2蛋白與core共同作用形成的。 將得到的重組E1E2糖蛋白免疫兔子能夠產(chǎn)生高水平的抗體應(yīng)答,并能持續(xù)維持約兩個月,將氫氧化鋁作為佐劑加入后,抗體產(chǎn)生更快,峰值更高,但是持續(xù)維持的時間則較短。在小鼠中我們也得到了類似的結(jié)果。抑制實(shí)驗(yàn)表明,用純化蛋白免疫獲得的兔血清能阻止重組E1E2糖蛋白同CD81的相互作用,且抑制效果呈劑量依賴性,同時對照兔血清則沒有影響。我們應(yīng)用假病毒粒子(PP)系統(tǒng)和HCV細(xì)胞培養(yǎng)系統(tǒng)對血清的中和效果進(jìn)行了研究,結(jié)果表明同對照血清相比,僅用重組E1E2糖蛋白免疫獲得的兔血清對來源于HCV 1a亞型和HCV 1b亞型的HCV假病毒粒子,甚至對HCV 2a亞型的HCV病毒粒子都有中和作用；而用氫氧化鋁作為佐劑免疫獲得的兔血清僅對來源于HCV 1a亞型的HCVpp有強(qiáng)的中和作用,對1b亞型的HCVpp的中和作用較弱,對2a亞型的HCV病毒粒子則沒有中和作用。同時,這些兔血清都對VSVpp不具中和能力,表明該中和能力是HCV特異性的。免疫獲得的小鼠血清中和實(shí)驗(yàn)由于時間關(guān)系尚未完成,仍然在進(jìn)行中。上述結(jié)果初步表明,畢赤酵母系統(tǒng)表達(dá)純化的HCV E1E2糖蛋白能夠誘導(dǎo)產(chǎn)生廣泛的中和抗體,并且憑借其產(chǎn)量高的優(yōu)點(diǎn),有望成為一種可以大規(guī)模生產(chǎn)應(yīng)用的HCV疫苗。
[Abstract]:Hepatitis C virus (HCV) is the cause of hepatitis C, with more than 3% of the world's population infected. The development of an effective HCV vaccine is the key to controlling HCV infection.
Because of the high variability of HCV, it was once thought that effective vaccines against different subtypes of HCV were impossible to obtain. However, recent studies have shown that HCV envelope glycoproteins contain conserved receptor binding sites for HCV cells, which can induce a wide range of neutralizing antibodies to prevent HCV infection. The production of HCV envelope glycoprotein as a vaccine has been successfully demonstrated in chimpanzees to protect chimpanzees from heterotypic HCV infection. However, the production of HCV envelope glycoprotein produced by mammalian cell expression system is very low and is difficult to be used on a large scale. This glycosylation-deficient envelope protein differs greatly from natural HCV envelope glycoproteins in structure, properties and immunogenicity. Therefore, we use Pichia pastoris as an economical eukaryotic expression system to express HCV envelope glycoproteins.
The eukaryotic expression plasmid pPIC9K-E1E2 was constructed on the basis of pPIC9K vector. It encoded the 187-346 amino acid sequence of HCV E1 and the E2 381-699 amino acid sequence of HCV E1, and was electrotransformed into Pichia pastoris strain SMD1168 to obtain the recombinant transformants. The recombinant HCV E1E2 glycoprotein was highly expressed and the yield was up to 40mg/L. The recombinant protein was purified by Q-S-FF column, P-S-FF column and G100 column.
Because of different glycosylation and different aggregation degree, the recombinant protein has several different forms: 72 kD, 95 kD, 145 kD and 200 kD aggregates (Aggregation, Agg). SDS-PAGE showed that 72 kD protein and Agg. In addition, we used GST-pulldown assay to verify whether the recombinant protein could interact with human CD81 extracellular loop (LEL) of HCV receptor. We constructed pET-GST-LEL plasmid and expressed it in E. coli BL21. Glutathione Sepharose 4B beads confirmed that recombinant E1E2 glycoprotein could bind to GST-LEL protein, indicating that the recombinant E1E2 glycoprotein contained not only the amino acid sequence of the binding region of HCV E2 CD81, but also the correct conformation of the binding region. The recombinant E1E2 glycoprotein and the core protein (amino acid sequence 1-137) expressed in E. coli were renatured and concentrated together, and two kinds of virus-like particles with different diameters were obtained. It is speculated that the particle is formed by the interaction of recombinant E1E2 protein and core.
The rabbits immunized with the recombinant E1E2 glycoprotein produced a high level of antibody response and maintained it for about two months. Adding aluminium hydroxide as an adjuvant, the antibody production was faster and the peak value was higher, but the duration was shorter. Similar results were obtained in mice. Inhibition experiments showed that the purified protein was used as an adjuvant. The immunized rabbit serum could prevent the interaction between recombinant E1E2 glycoprotein and CD81 in a dose-dependent manner, while the control rabbit serum had no effect. The serum neutralization effect was studied by using pseudovirus particle (PP) system and HCV cell culture system. The results showed that the recombinant E1E2 glycoprotein only used as control serum. Rabbit serum immunized with glycoprotein can neutralize HCV pseudovirus particles derived from HCV 1a and HCV 1b subtypes, even HCV 2A subtypes, while rabbit serum immunized with aluminium hydroxide as an adjuvant can only neutralize HCV PP derived from HCV 1A subtype and can neutralize HCV PP derived from HCV 1b subtype. At the same time, these rabbit sera did not neutralize VSVpp, suggesting that the neutralization ability was HCV-specific. The neutralization experiment of the immunized mice sera was still in progress because of the unfinished time relationship. V E1E2 glycoprotein can induce a wide range of neutralizing antibodies, and with its high yield, it is expected to become a large-scale production of HCV vaccine.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R346
[Abstract]:Hepatitis C virus (HCV) is the cause of hepatitis C, with more than 3% of the world's population infected. The development of an effective HCV vaccine is the key to controlling HCV infection.
Because of the high variability of HCV, it was once thought that effective vaccines against different subtypes of HCV were impossible to obtain. However, recent studies have shown that HCV envelope glycoproteins contain conserved receptor binding sites for HCV cells, which can induce a wide range of neutralizing antibodies to prevent HCV infection. The production of HCV envelope glycoprotein as a vaccine has been successfully demonstrated in chimpanzees to protect chimpanzees from heterotypic HCV infection. However, the production of HCV envelope glycoprotein produced by mammalian cell expression system is very low and is difficult to be used on a large scale. This glycosylation-deficient envelope protein differs greatly from natural HCV envelope glycoproteins in structure, properties and immunogenicity. Therefore, we use Pichia pastoris as an economical eukaryotic expression system to express HCV envelope glycoproteins.
The eukaryotic expression plasmid pPIC9K-E1E2 was constructed on the basis of pPIC9K vector. It encoded the 187-346 amino acid sequence of HCV E1 and the E2 381-699 amino acid sequence of HCV E1, and was electrotransformed into Pichia pastoris strain SMD1168 to obtain the recombinant transformants. The recombinant HCV E1E2 glycoprotein was highly expressed and the yield was up to 40mg/L. The recombinant protein was purified by Q-S-FF column, P-S-FF column and G100 column.
Because of different glycosylation and different aggregation degree, the recombinant protein has several different forms: 72 kD, 95 kD, 145 kD and 200 kD aggregates (Aggregation, Agg). SDS-PAGE showed that 72 kD protein and Agg. In addition, we used GST-pulldown assay to verify whether the recombinant protein could interact with human CD81 extracellular loop (LEL) of HCV receptor. We constructed pET-GST-LEL plasmid and expressed it in E. coli BL21. Glutathione Sepharose 4B beads confirmed that recombinant E1E2 glycoprotein could bind to GST-LEL protein, indicating that the recombinant E1E2 glycoprotein contained not only the amino acid sequence of the binding region of HCV E2 CD81, but also the correct conformation of the binding region. The recombinant E1E2 glycoprotein and the core protein (amino acid sequence 1-137) expressed in E. coli were renatured and concentrated together, and two kinds of virus-like particles with different diameters were obtained. It is speculated that the particle is formed by the interaction of recombinant E1E2 protein and core.
The rabbits immunized with the recombinant E1E2 glycoprotein produced a high level of antibody response and maintained it for about two months. Adding aluminium hydroxide as an adjuvant, the antibody production was faster and the peak value was higher, but the duration was shorter. Similar results were obtained in mice. Inhibition experiments showed that the purified protein was used as an adjuvant. The immunized rabbit serum could prevent the interaction between recombinant E1E2 glycoprotein and CD81 in a dose-dependent manner, while the control rabbit serum had no effect. The serum neutralization effect was studied by using pseudovirus particle (PP) system and HCV cell culture system. The results showed that the recombinant E1E2 glycoprotein only used as control serum. Rabbit serum immunized with glycoprotein can neutralize HCV pseudovirus particles derived from HCV 1a and HCV 1b subtypes, even HCV 2A subtypes, while rabbit serum immunized with aluminium hydroxide as an adjuvant can only neutralize HCV PP derived from HCV 1A subtype and can neutralize HCV PP derived from HCV 1b subtype. At the same time, these rabbit sera did not neutralize VSVpp, suggesting that the neutralization ability was HCV-specific. The neutralization experiment of the immunized mice sera was still in progress because of the unfinished time relationship. V E1E2 glycoprotein can induce a wide range of neutralizing antibodies, and with its high yield, it is expected to become a large-scale production of HCV vaccine.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R346
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