一种重组戊型肝炎疫苗抗原的一级结构分析

目的: 对一种重组戊型肝炎疫苗抗原的一级结构进行分析。方法: 采用液质联用的方法测定其相对分子质量;采用变性剂使重组戊肝疫苗抗原的空间结构充分打开,用胰蛋白酶对其进行酶解,液质联用测定酶解后样品的液质肽图;结合质谱相对分子质量的测定及液质肽图的解析对其一级结构进行分析。结果: 该抗原的氨基酸序列与理论一致,部分蛋白的N-末端缬氨酸存在乙酰化修饰,修饰率约为40%;链内唯一1个半胱氨酸处于还原状态,未参与链间二硫键的形成。结论: 采用合适的酶解方法结合液质联用技术,对该疫苗抗原的氨基酸序列进行了确证,并对翻译后修饰进行了鉴定,所用方法可为其他重组疫苗抗原的一级结构分析提供参考。

Objective: To analyze the primary structure of a recombinant antigen of HEV vaccine. Methods: The relative molecular mass of the HEV antigen was measured by LC-MS.The tertiary structure of the antigen was fully opened by denaturant and the antigen was enzymolyzed by trypsin,the peptide mass mapping of the HEV antigen was also detected using LC-MS.Primary structure of the antigen was analyzed from results of above approaches. Results: The amino acid sequence was consistent with the theoretical sequence,40% of proteins were acetylated at the N-terminal Val,and the sole cysteine in protein remained in the reducing state which did not take part in the formation of inter-chain disulfide bonds. Conclusion: The amino acid sequence of the antigen was verified and the post translational modification was identified by proper enzymolysis combined with LC-MS analysis; the established methods could be referred for the primary structure characterization of other recombinant antigens.

[1] Dellagostin OA,Grassmann AA,Hartwig DD,et al.Recombinant vaccines against leptospirosis[J].Hum Vaccin,2011,7(11):1215
[2] Sharma A,Krause A,Worgall S.Recent developments for Pseudomonas vaccines[J].Hum Vaccin,2011,7(10):999
[3] Whitehead SS.Next-generation dengue vaccines: novel strategies currently under development[J].Viruses,2011,3(10):1800
[4] Kang SM,Song JM,Compans RW.Novel vaccines against influenza viruses[J]. Virus Res,2011,162(1-2):31
[5] Sharma A,Krause A,Worgall S.Recent developments for Pseudomonas vaccines[J].Hum Vaccin,2011,7(10):999
[6] Li T,Lin H,Zhang Y,et al.Improved characteristics and protective efficacy in an animal model of E.coli-derived recombinant double-layered rotavirus virus-like particles[J].Vaccine,2014,32(17):1921
[7] Lua LH,Connors NK,Sainsbury F,et al.Bioengineering virus-like particles as vaccines[J].Biotechnol Bioeng,2014,111(3):425
[8] RAO Chun-ming(饶春明),TAO Lei(陶磊),SHI Xin-chang(史新昌),et al.Peptide mass mapping analysis of rhIFN-α1b and localization of disulfide bonds(重组人干扰素α1b质量肽图分析及二硫键定位)[J].Chin J Pharm Anal(药物分析杂志),2007,27(10):1505
[9] TAO Lei(陶磊),RAO Chun-ming(饶春明),GAO Kai(高凯), et al.Structure verification of a recombinant chimeric anti-CD20 IgG1 monoclonal antibody(重组嵌合抗CD20 IgG1型单克隆抗体的结构验证)[J].Acta Pharm Sin(药学学报),2010,45(6): 752
[10] Rogeberg M,Furlund CB,Moe MK,et al.Identification of peptide products from enzymatic degradation of amyloid beta[J].Biochimie,2014.105:106
[11] Yin J,Shao C,Jia L,et al.Comparison at the peptide level with post-translational modification consideration reveals more differences between two unenriched samples[J].Rapid Commun Mass Spectrom,2014,28(12):1364
[12] Raftery MJ.Determination of oxidative protein modifications using mass spectrometry[J].Redox Rep,2014,19(4):140
[13] Soppa J.Protein acetylation in archaea,bacteria,and eukaryotes[J].Archaea,2010 Sep 16;2010.pii: 820681.doi: 10.1155/2010/820681
[14] Tang X,Wen S,Zheng D, et al.Acetylation of drosha on the N-terminus inhibits its degradation by ubiquitination[J]. PLos One,2013,8(8):e72503
[15] Dikiy I,Eliezer D.N-terminal acetylation stabilizes N-terminal helicity in lipid- and micelle-bound α-synuclein and increases its affinity for physiological membranes[J].J Biol Chem,2014,289(6):3652