人乳头状瘤假病毒中和抗体滴度和ELISA法测定的小鼠血清抗体滴度的相关性研究

The relationship between HPV pseudovirus-neutralizing antibody titers and antibody titer determined by ELISA method

目的探讨两种不同的报告基因纽扣珊瑚绿色荧光蛋白（Zoanthus sp．green fluorescent protein，ZsGreen）和分泌性碱性磷酸酶（secreted alkaline phosphatase，SEAP）测量的人乳头状瘤假病毒中和滴度之间的相关性，以及中和滴度和抗体滴度的相关性。方法将密码子优化的人乳头状瘤病毒（HPV）衣壳蛋白L1、12基因表达质粒和报告基因质粒共转染293FT细胞，48h后收集细胞裂解上清，柱层析纯化假病毒，对假病毒滴度进行测定。采集免疫过候选HPV疫苗和Gardasil疫苗的小鼠血清，测量血清的中和滴度和抗体滴度。结果经统计分析，这两种报告基因系统的假病毒检测的中和滴度结果高度相关（Spearman相关系数r=0．760），而且中和滴度和抗体滴度的相关度很高（Spearman相关系数r=0．577和0．741）。结论两种不同的报告基因ZsGreen和SEAP测量的HPV假病毒中和滴度之间，以及中和滴度和ELISA测定的抗体滴度之间高度相关，揭示了部分HPV疫苗预防病毒入侵的机制，为快速准确鉴定HPV-16和HPV-18候选疫苗的免疫保护效果奠定了基础。

Objective To study the relationship of HPV pseudo-neutralizing titers detected by two different reporter genes： Zoanthus sp. green fluorescent protein （ZsGreen） and secreted alkaline phosphatase （ SEAP）, and the relationship between HPV the pseudovirus-neutralizing antibody titer and the antibody titer determined by ELISA method. Methods The plasmids with expression cassettes of the HPV capsid protein L1 and L2 genes after codon optimization and the plasmid with reporter gene （ZsGreen or SEAP） were co-transfected into 293FT cells. The cell lysate supernatants were collected after 48 h culture, then the pseudovirus was purified through POROS column chromatography from the supernatants. After the titer of pseudovirus bulk were measured, HPV-16 and HPV-18 pseudovirus-neutralization assays were carried out for determining the titer of sera collected from immunized mice with HPV candidate vaccine and Gardasil HPV vaccine. Results In statistical analysis, the two reporter gene systems for the detection of the pseudovirus neutralizing antibody titer are highly relevant to each other （ Spearman coefficient ： r = 0. 760 ）. And their neutralizing antibody titers bear a high degree of correlation with the antibody titer （ Spearman coefficient ： r = 0. 577 and r = 0. 741 ）. Conclusion ZsGreen and SEAP pseudovirus neutralizing antibody titers are highly relevant to each other. The neutralizing antibody and the antibody titer are also relevant. These results reveal some mechanism of HPV vaccines to prevent the virus from invading the host cells, and are absolutely useful in the protection efficiency evaluation of the HPV-16 and HPV-18 candidate vaccines.