【作者】 杨坤宇；

【导师】 夏宁邵；

【作者基本信息】 厦门大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 高致病性禽流感病毒H5N1的爆发不仅给全世界养禽业造成了毁灭性打击,也严重危害到人类的生命安全,开发一种安全有效且广谱的疫苗是控制禽流感病毒传播的最有效的手段。毕赤酵母具有分子遗传操作简单、外源蛋白表达水平高、具有近似于哺乳动物细胞翻译后修饰与加工的功能,并且能进行高密度发酵。本研究利用毕赤酵母表达系统高效分泌表达了重组HA1蛋白,通过多种实验证明rHA1具有开发成H5N1禽流感广谱疫苗的可能性,在分子对接表位预测的基础上,鉴定了HA1上几个重要的中和表位的关键氨基酸,培养出了13D4-rHA1复合物晶体,为阐明H5N1病毒的保守中和抗体表位结构奠定了良好的基础。首先,将构建好的表达载体pPIC9K-HA1电转化进入毕赤酵母菌株GS115中,构建工程菌株,进行分泌表达。用G418筛选不同拷贝数的重组菌株,考察基因剂量对重组HA1蛋白表达的影响,实验证明适量提高拷贝数有利于提高rHA1的分泌表达量。优化了重组菌株的摇瓶培养条件,得到最佳诱导条件:初始pH值为5.4,甲醇浓度为1.0%,培养温度为25℃。在摇瓶培养的基础上,用10L发酵罐进行高密度发酵的工艺研究,发酵最终菌体浓度OD₆₀₀约为320,rHA1的表达量为120mg/L比摇瓶培养提高了10倍左右,表明我们建立起了高效稳定的高密度发酵。缺失HA1N-端前48个氨基酸残基后,rHA1的表达量提高了～5倍（～550mg/L）,且保留了与代表性中和抗体的反应性,以及与HA1相当的免疫原性。发酵培养液上清通过切向流浓缩、更换缓冲液后,利用离子交换层析柱纯化,获得了较高纯度的重组HA1蛋白,蛋白回收率为24%左右。其次,利用本实验室已经筛选到的93株H5N1禽流感病毒单抗盘进行活性检测,结果显示重组蛋白HA1具有良好的抗原性。用重组HA1和HA1-48aa蛋白免疫小鼠5周后,anti-rHA1抗体滴度达到1.2×10⁴和1.3×10⁴,并具有较好的血凝抑制（HI）活性。体外阻断实验表明小鼠血清能较好地阻断13D4、8H5、8G9、10F7等广谱中和单抗与YU22病毒的结合,说明rHA1具备开发成H5N1禽流感的广谱疫苗的潜力。在本实验已有的广谱中和表位分子对接预测的工作基础上进一步应用突变分析方法进行实验验证。结果显示,Glu¹¹²,Lys¹¹³,Ile¹¹⁴对HA1上的构象表位的维持较为重要;Pro¹¹⁸和Tyr¹³⁷是1A6和13H8构象表位的关键氨基酸;Asn¹⁶⁵的突变可使广谱中和单抗13D4的结合活性下降40倍左右,但对其他广谱中和单抗的活性无显著的影响;aa123～aa132 loop区对HA1的构象活性很重要。最后,获得rHA1与广谱性中和单抗13D4Fab复合物的晶体培养条件,培养出可用于X-射线晶体衍射实验的体积约为0.2mm×0.2mm×0.05mm的方块状晶体,为HA1-13D4复合物空间结构的解析奠定了基础。更多还原

【Abstract】 The recent outbreaks of highly pathogenic H5N1 influenza A virus not only cause a large amount of economic loss to farms but also endanger people’s life,A broad spectrum vaccine will be the most effective way to limit the spread of highly pathogenic H5N1 influenza virues.P.pastoris as a cellular host for expression of recombinant proteins is easier to genetically manipulate and culture than mammalian cells and can be grown to high cell densities.Equally important,P.pastoris is also a eukar yote,and thereby provides the potential for producing soluble,correctly folded recom binant proteins that have undergone all the post-translational modifications required for functionality.Here we report the high-level secretory expression of active recombinant HA1 protein in p.pastoris and the recombinant HA1 proteins was proved to be a good candidate vaccine.A series of important epitopes were predicted and verified with the help of bioinformatics.The crystal of 13D4 Fab-rHA1 co mplex was obtained,this provided a probability to elucidate the conservative epitope of H5N1 virus.The expression plasmid pPIC9K-HA1 was transformed into P.pastoris host cell GS115,and the recombinant engineering strains for secretory expression were constructed and screened.The positive strains containing multiple gene dosages were screened out by G418-YPD plates,and the results showed that the higher gene dosages had the effect of increasing the amount of rHA1 expression.Then the influence of different factors on biomass and recombinant HA1 protein production during induction phase in shake flask were studied.The optimized cultivation conditions were original pH at 5.4,concentration of methanol at 1.0%and cultivation temperature at 25℃.Based on the research in shake flask,the recombinant HA1 protein was produced by high-cell density fermentation,after optimizing the fermentation process,the rHA1 yield in 10L-fermentor（about 120mg/L） was 10 times higher than that in shake flask（about 11mg/L）,with the final OD₆₀₀ at about 320.The expression level of rHA1 could improve 5 times by deleting N-terminal 48aa.The supernatant of the fermentation culture was concentrated and buffer-exchanged by crossflow filtration,and then purified by ion-exchange chromatography.The purified protein recovery was about 24%.After detected by a panel of 93 anti-H5 HA monoclonal antibodies（mAbs）,the recombinant HA1 was proved to have good antigenicity.Mice was immuned with recombinant HA1 protein,after 5 weeks,the titers of anti-rHA1 serum reached 1.2×10⁴ and the anti-rHA1 serum had high Hemagglutination inhibition（HI） titers to YU22 and 2439 avian virus but relatively low Hemagglutination inhibition titers to Yu324,213 avian virus.Anti-rHA1 serum was tested against 4 neutralization monoclonal antibodies（13D4、8H5、8G9、10F7） in blocking assay,anti-rHA1 serum could block the reaction between neutralization monoclonal antibodies and YU22 virus.The deletion of N-terminal 48aa won’t lower the biologic activity of rHA1.This suggested that the recombinant HA1 protein produced by P.pastoris was a good candidate vaccine.In the former study,our research group had predicted many important epitopes of HA1 by bioinformatics.In this study,we did site-directed mutagenesis of these specific amino acids,and compared the biological activity bewteen normal rHA1 and mutated rHA1,finally we found that Glu¹¹²,Lys¹¹³,Ile¹¹⁴ was important for the correct folding of HA1,Pro¹¹⁸ and Tyr¹³⁷ were the key amino acids of epitopes of 1A6 and 13H8 respectively,Asn¹⁶⁵ was an important amino acid for HA1 to react with 13D4, an a mutaion of a loop from aa123 to aa132 would greatly lower the biological activity of rHA1.After deleting 48aa from N-terminal,the expression level of rHA1 mutant was improved and was up to 520mg/L,and the rHA1 mutant remained the original biologic activity of HA1.At last,we studied the crystallization of 13D4 Fab-rHA1 complex,and got some cuboid crystals of 13D4 Fab-rHA1 complex with the size 0.2mm×0.2mm×0.05mm. This lay a foundation for the analysis of 13D4 Fab-rHA1 structure by X-ray.更多还原