H5亚型禽流感病毒广谱治疗性单抗13D4中试工艺的建立，及其晶体培养与表位预测
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摘要
高致病性禽流感(Avian influenza,AI)是由A型流感病毒H5N1引起的烈性传染病。近年来,该疾病随着候鸟迁徙不断向各国传播,在造成巨大的经济损失的同时,也引起了多例人感染禽流感的病例,因此,对禽流感防治手段的研究引起了广泛重视。中和单抗是防治病毒性疾病的一种很有效手段。但是由于禽流感病毒基因突变频率高,尤其以血凝素(HA)基因为最,因此针对禽流感病毒的中和单抗中和谱较窄,往往只能中和少数遗传变异亚系的病毒,这就极大限制了这些中和单抗的应用。
本实验室所制备的单抗13D4具有广谱中和活性,能有效中和41株不同遗传变异亚系的H5N1病毒代表株,在禽流感的防治中具有广阔的应用前景。本研究通过实验摸索,建立了一套低成本,简便高效的13D4全抗体和F(ab)'_2的纯化制备方法,在中试规模生产中能够制备出符合国家检定标准的产品,纯度达到99%左右,得率为60%,为该抗体药物的成功奠定了基础。
为了进一步揭示13D4广谱中和活性的机制,本研究还进行了13D4 Fab片段晶体的培养。通过对13D4 Fab片段培养条件的筛选和优化,制备出了能用于X射线晶体衍射的单晶。
另外,本研究还通过分子模拟的方法,得到了抗体13D4 Fab片段以及A/Ck/HK/Yu22/02(H5N1)HA1的三维结构,并通过Fab分子依次与已有晶体结构的3个H5亚型HA以及模拟的HA1分子进行对接,预测单抗13D4可能结合表位为:由13个Glu~(112),Lys~(113),Ile~(114),Pro~(118),Ser~(120),Ser~(121),Ser~(123),Lys~(161)t,Arg~(162),Ser~(163),Tyr~(164),Asn~(168),Lys~(255)不连续氨基酸残基组成的构象表位。还利用分子对接结果分析H5亚型禽流感的广谱中和表位和,综合单个Fab分子与4个HA分子的对接结果用于寻找该单抗识别的HA抗原表位,用于寻找与抗原良好契合的多肽结构。13D4 Fab片段上可能用于治疗禽流感的多肽片段为Gln~(27)-Thr~(28)-Ser~(31)-Gly~(31)-Leu~(52)-Gly~(54)-Ser~(55)-Asn~(57)-Thr~(100)-Thr~(101)-Ala~(102)-Val~(103)。
Highly pathogenic avian influenza(AI),one of syndrome disease caused by influenza A virus H5N1,has recently spread to many countries by the migratory birds. It severely led heavy loss of national economy in these countries,and even caused fatal disease on human.Study on prevention and treatment of avian influenza has aroused extensive attention.Neutralization antibody becomes one of effective means to contral virus disease.Because avian influenza virus mutants frequently,especially in the hemagglutinin gene,most H5N1 mab only neutralize narrow spectrum of virus.
Our research group has indentified a strain of broad spectum neutralization antibody,named 13D4,which binds to 41 representative strains of H5N1 virus from various clades.In this study,a easilyscaled -up and high-effective process with low cost was established to purify 13D4 and F(ab)'_2 fragments.The purity of final product was about 99%and the recoverity rate is about 60%.All the test ruesults fulfiled the requirements of PHARMACOPOEIA of PRC.
In this study,the vapor diffusion technique was used to crystallize 13D4 Fab fragment,and sigle crystals suitable for X-ray diffraction were obtained in some conditions after screening and Optimization.
The structures of 13D4 Fab fragment and HA1 from A/Ck/HK/Yu22/02 (H5N1) were modeled by homology modeling.Then 13D4 Fab was subject to dock with three HA structures found in PDB(PDB ID:ljsm,2ibx and 2fk0) and HA1 from A/Ck/HK/Yu22/02(H5N1).The binding patterns between the antibody and the HA were analyzed according to the docking results.The binding pattern of 13D4 Fab with the 4 HAs was used to search the broad neutral epitopes which the antibody binding to and the interacting polypeptideslocated in the antibody which may be made use of the reference of the anti-virus drug.The polypeptide located in 13D4 Fab fragment is Gln~(27)-Thr~(28)-Ser~(30)-Gly(31)-Leu~(52)-Gly~(54)-Ser~(55)-Asn~(57)-Thr~(100)-Thr~(101)-Ala~(102)-Val~(103).
本实验室所制备的单抗13D4具有广谱中和活性,能有效中和41株不同遗传变异亚系的H5N1病毒代表株,在禽流感的防治中具有广阔的应用前景。本研究通过实验摸索,建立了一套低成本,简便高效的13D4全抗体和F(ab)'_2的纯化制备方法,在中试规模生产中能够制备出符合国家检定标准的产品,纯度达到99%左右,得率为60%,为该抗体药物的成功奠定了基础。
为了进一步揭示13D4广谱中和活性的机制,本研究还进行了13D4 Fab片段晶体的培养。通过对13D4 Fab片段培养条件的筛选和优化,制备出了能用于X射线晶体衍射的单晶。
另外,本研究还通过分子模拟的方法,得到了抗体13D4 Fab片段以及A/Ck/HK/Yu22/02(H5N1)HA1的三维结构,并通过Fab分子依次与已有晶体结构的3个H5亚型HA以及模拟的HA1分子进行对接,预测单抗13D4可能结合表位为:由13个Glu~(112),Lys~(113),Ile~(114),Pro~(118),Ser~(120),Ser~(121),Ser~(123),Lys~(161)t,Arg~(162),Ser~(163),Tyr~(164),Asn~(168),Lys~(255)不连续氨基酸残基组成的构象表位。还利用分子对接结果分析H5亚型禽流感的广谱中和表位和,综合单个Fab分子与4个HA分子的对接结果用于寻找该单抗识别的HA抗原表位,用于寻找与抗原良好契合的多肽结构。13D4 Fab片段上可能用于治疗禽流感的多肽片段为Gln~(27)-Thr~(28)-Ser~(31)-Gly~(31)-Leu~(52)-Gly~(54)-Ser~(55)-Asn~(57)-Thr~(100)-Thr~(101)-Ala~(102)-Val~(103)。
Highly pathogenic avian influenza(AI),one of syndrome disease caused by influenza A virus H5N1,has recently spread to many countries by the migratory birds. It severely led heavy loss of national economy in these countries,and even caused fatal disease on human.Study on prevention and treatment of avian influenza has aroused extensive attention.Neutralization antibody becomes one of effective means to contral virus disease.Because avian influenza virus mutants frequently,especially in the hemagglutinin gene,most H5N1 mab only neutralize narrow spectrum of virus.
Our research group has indentified a strain of broad spectum neutralization antibody,named 13D4,which binds to 41 representative strains of H5N1 virus from various clades.In this study,a easilyscaled -up and high-effective process with low cost was established to purify 13D4 and F(ab)'_2 fragments.The purity of final product was about 99%and the recoverity rate is about 60%.All the test ruesults fulfiled the requirements of PHARMACOPOEIA of PRC.
In this study,the vapor diffusion technique was used to crystallize 13D4 Fab fragment,and sigle crystals suitable for X-ray diffraction were obtained in some conditions after screening and Optimization.
The structures of 13D4 Fab fragment and HA1 from A/Ck/HK/Yu22/02 (H5N1) were modeled by homology modeling.Then 13D4 Fab was subject to dock with three HA structures found in PDB(PDB ID:ljsm,2ibx and 2fk0) and HA1 from A/Ck/HK/Yu22/02(H5N1).The binding patterns between the antibody and the HA were analyzed according to the docking results.The binding pattern of 13D4 Fab with the 4 HAs was used to search the broad neutral epitopes which the antibody binding to and the interacting polypeptideslocated in the antibody which may be made use of the reference of the anti-virus drug.The polypeptide located in 13D4 Fab fragment is Gln~(27)-Thr~(28)-Ser~(30)-Gly(31)-Leu~(52)-Gly~(54)-Ser~(55)-Asn~(57)-Thr~(100)-Thr~(101)-Ala~(102)-Val~(103).
引文
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