二价HPV病毒样颗粒在血清中和抗体检测与卵黄IgY抗体制备中的应用

doi:10.19586/j.2095-2341.2025.0052

生物技术进展 ›› 2025, Vol. 15 ›› Issue (5): 913-920.DOI: 10.19586/j.2095-2341.2025.0052

二价HPV病毒样颗粒在血清中和抗体检测与卵黄IgY抗体制备中的应用

刘改娟¹(), 刘薇², 颜廷宝², 周立敬¹, 强亚欣¹, 邢龙龙¹, 陈鑫健², 夏振民¹()

^1.河北健海医学检验实验室有限公司，石家庄 050000
^2.河北健海生物芯片技术有限责任公司，石家庄 050000

收稿日期:2025-04-18 接受日期:2025-05-30 出版日期:2025-09-25 发布日期:2025-11-11
通讯作者: 夏振民
作者简介:刘改娟 E-mail: 279752363@qq.com；
基金资助:
石家庄市科技计划项目(231200034A)

Applications of Bivalent HPV Virus-like Particles in Neutralizing Antibody Detection and Yolk IgY Antibody Production

Gaijuan LIU¹(), Wei LIU², Tingbao YAN², Lijing ZHOU¹, Yaxin QIANG¹, Longlong XING¹, Xianjian CHEN², Zhenmin XIA¹()

^1.Hebei Jianhai Medical Laboratory Co. ，Ltd. ，Shijiazhuang 050000，China
^2.Hebei Jianhai Biochip Technology Co. ，Ltd. ，Shijiazhuang 050000，China

Received:2025-04-18 Accepted:2025-05-30 Online:2025-09-25 Published:2025-11-11
Contact: Zhenmin XIA

摘要/Abstract

摘要：

人乳头状瘤病毒（human papillomavirus, HPV）感染与多种疾病相关，制备了二价HPV假病毒并建立了中和抗体检测体系，并探究了卵黄IgY抗体的制备方法及中和效力。采用同源重组构建HPV16/18-L1/L2表达质粒，制备假病毒通过透射电镜观察形态；制备卵黄IgY抗体，建立双色荧光检测体系。结果发现，制备的HPV病毒样（virus-like particles，VLPs）颗粒滴度为4.0×10?~5.7×10? IU·mL^-1，电镜观察呈二十面体对称，直径55±5 nm，与天然HPV一致。中和抗体检测特异性好，HPV16和HPV18抗体标准品ID₅₀分别为177和463，阴性血清无反应。双色与单色荧光检测体系一致性好。假病毒免疫鸡群后，制备的卵黄IgY抗体纯度约90%，中和效价在免疫后第6周达峰值，分别为1∶5 424（HPV16）和1∶3 304（HPV18）。HPV假病毒和中和抗体检测体系的建立及卵黄IgY抗体的制备，为疫苗效果评价和新型抗体药物的开发提供了重要支撑。

关键词: 二价HPV病毒样颗粒, 中和抗体, 卵黄IgY抗体, 疫苗评价

Abstract:

Human papillomavirus （HPV） infection is associated with various diseases. This study aimed to prepare bivalent HPV pseudovirus， establish a neutralizing antibody detection system， and investigate the preparation method and neutralizing efficacy of yolk IgY antibodies. Eukaryotic expression plasmids of HPV16/18-L1/L2 were constructed by homologous recombination. Pseudovirus particles were prepared and observed under transmission electron microscopy. Yolk IgY antibodies were extracted， and a dual-color fluorescence detection system was established. The prepared HPV virus-like particles （VLPs） had a titer of 4.0×10? to 5.7×10? IU·mL^-1. Electron microscopy showed icosahedral symmetry with a diameter of 55 ± 5 nm， consistent with natural HPV. The neutralizing antibody detection system exhibited high specificity， with ID₅₀ values of 177 and 463 for HPV16 and HPV18 antibody standards， respectively， and no reaction was observed with negative serum. The dual-color and single-color fluorescence systems showed good agreement. After immunizing chickens with the pseudovirus， yolk IgY antibodies with approximately 90% purity were obtained. The neutralizing titers peaked at week 6 post-immunization， reaching 1∶5 424 for HPV16 and 1∶3 304 for HPV18. The successful preparation of HPV pseudovirus， establishment of the neutralizing antibody detection system， and production of yolk IgY antibodies provide important support for vaccine evaluation and development of novel antibody-based drugs.

Key words: bivalent HPV virus-like particles, neutralizing antibodies, yolk IgY antibodies, vaccine evaluation

中图分类号:

Q939.4

刘改娟, 刘薇, 颜廷宝, 周立敬, 强亚欣, 邢龙龙, 陈鑫健, 夏振民. 二价HPV病毒样颗粒在血清中和抗体检测与卵黄IgY抗体制备中的应用[J]. 生物技术进展, 2025, 15(5): 913-920.

Gaijuan LIU, Wei LIU, Tingbao YAN, Lijing ZHOU, Yaxin QIANG, Longlong XING, Xianjian CHEN, Zhenmin XIA. Applications of Bivalent HPV Virus-like Particles in Neutralizing Antibody Detection and Yolk IgY Antibody Production[J]. Current Biotechnology, 2025, 15(5): 913-920.

图/表 9

图1 HPV16/HPV18重组质粒转染效率

Fig. 1 Transfection efficiency of HPV16/HPV18 recombinant plasmids

表1 不同批次假病毒滴度测定结果

Table 1 Titration results of different batches of pseudoviruses

假病毒型别	假病毒滴度（TCID₅₀/·mL^-1）			CV/%
假病毒型别	第1批	第2批	第3批	CV/%
HPV16	5.62×10⁷	4.33×10⁷	5.62×10⁷	11.74
HPV18	4.33×10⁷	4.64×10⁷	5.62×10⁷	11.33

图2 HPV16和HPV18假病毒颗粒透射电镜观察

Fig. 2 Transmission electron microscopy observation of HPV16 and HPV18 pseudovirus particles

图3 HPV假病毒中和试验不同稀释梯度荧光采集A：HPV16；B:HPV18。细胞对照为未加假病毒颗粒的HEK293FT细胞空白对照，病毒对照为单加假病毒颗粒的HEK293FT细胞病毒对照。

Fig. 3 Fluorescence acquisition of HPV pseudovirus neutralization test at different dilution gradients

图4 混合HPV16/18中和抗体检测

Fig. 4 Detection of mixed HPV16/18 neutralizing antibodies

图5 Bland-Altman分析单检和混合检平均值A:单色荧光与双色混合荧光HPV16假病毒检测体系一致性评价；B:单色荧光与双色混合荧光HPV18假病毒检测体系一致性评价

Fig. 5 Bland-Altman analysis of the average values of single test and mixed test

图6 卵黄IgY纯化检测

Fig. 6 Purification and detection of yolk IgY

图7 卵黄IgY抗体中和效价评价

Fig. 7 Evaluation of neutralizing titer of egg yolk IgY antibodies

表2 Bland-Altman描述统计

Table 2 Bland-Altman descriptive statistics

项	HPV16值	HPV18值
有效样本量	47	47
均值（第1种方法-单检）	2 046.766	2 046.766
均值（第2种方法-混合检）	2 031.787	2 021.149
均值（差值）	14.979	25.617
标准差（差值）	109.876	122.691
95% CI（差值均值）	-17.282~47.239	-10.406~61.641
95% CI（差值）	-200.374~230.331	-214.854~266.088
t值（H0：差值的平均值=0)	0.935	1.431
P值（H0：差值的平均值=0)	0.355	0.159

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二价HPV病毒样颗粒在血清中和抗体检测与卵黄IgY抗体制备中的应用

Applications of Bivalent HPV Virus-like Particles in Neutralizing Antibody Detection and Yolk IgY Antibody Production

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