重组鼠疫耶尔森菌LcrV抗原原液性质研究

目的进行重组鼠疫耶尔森菌LcrV抗原原液二聚体含量及性质研究,确定LcrV原液的相关质控标准。方法在不同缓冲体系(0.85%NaCl(NS)、20 mmol/L PBS),不同蛋白浓度(2.0、1.5、1.0、0.5、0.1 mg/mL)及不同保存温度(4℃、-20℃、-70℃)条件下保存LcrV抗原,采用SDS-PAGE和HPSEC方法定期检测LcrV二聚体含量及纯度。将连续三批检定合格的LcrV抗原原液进行质谱相对分子质量测定、等电点测定、N末端氨基酸序列测定、圆二色(CD)谱、HPLC肽谱及氨基酸组成分析,研究LcrV抗原的相关性质。结果随着保存时间的延长LcrV抗原二聚体含量增加,低温保存时二聚体不易大量形成。在-20℃和-70℃条件下,NS保存的LcrV抗原比PBS体系保存稳定,而在4℃条件下NS保存的LcrV抗原容易降解。LcrV抗原高浓度保存容易发生聚合。LcrV抗原在低质量浓度(0.1 mg/mL)保存时免疫学活性明显下降。质谱检测到LcrV单体和二聚体共同存在,且与理论相对分子质量一致。LcrV原液检测等电点范围为4.6～6.3。N末端测序、CD谱、HPLC肽谱图及氨基酸组成分析与理论结果一致。结论 LcrV抗原原液保存条件确定为:NS体系,蛋白质量浓度1.0～2.0 mg/mL,-20℃以下冻存。制备的LcrV抗原各项检测结果与理论结果一致,抗原性质稳定。

Research on properties of LcrV bulk-antigen of Yersinia pestis

Objective To research on content of dimer of Yersinia pestis LcrV antigen and its properties, and to set up an internal standard of quality control for LcrV antigen. Methods LcrV antigen was preserved in different buffer systems 0.85% NaC1 solution（NS） and 20 mM PBS], with different protein concentrations （2.0, 1.5, 1.0, 0.5,0.1 mg/mL） and different temperatures （4℃, -20℃, -70℃）. The purity and content of dimer of LcrV antigen were detected by u- sing SDS-PAGE and HPSEC at a certain time. Molecular weight by mass spectrum, isoelectric point measurement, N-ter- minal amino sequencing, HPLC peptide mapping and amino acid analysis were carried out in three lots of stock solution of LcrV antigen. Results It was observed that the dimer of LcrV antigen would increase as the preservation time was pro- longed, and the dimer was not easy to form under a low temperature. LcrV antigen was more stable in NS than in PBS un- der -20℃ and -70℃ , whereas it was likely to degregated in NS compared to in PBS under 4℃. LcrV antigen preserved in a higher concentration was easy to polymerize. On the other hand, the immunologic activity of LcrV antigen could de- crease obviously with a low concentration （0. lmg/mL） in storage. Both of monomer and dimer of LcrV antigen were exis- ted in common and the result of mass spectrum determination was equal to thoeretical molecular weight of LcrV antigen. I- soelectric point range was determined in pH 4.6 - 6.3. N-terminal amino sequencing, HPLC peptide mapping and amino acid analysis of LcrV antigen were consistent with theatrical consequences. Conclusion The preservation conditions for stock solution of bulk-LcrV antigen were determined as followings ： in NS solution, with a concentration l. 0 - 2.0 mg/mL,preserved under - 20℃. The properties of purified LcrV antigen were stable, and all of determination results were consist- ent with theoretical requirements in this study.