HPV16假病毒制作方法优化

Optimize the construction method of HPV16 pseudovirus

目的为了优化HPV16假病毒制作方法。方法本实验用L1/L2结构蛋白质粒和p CMV-Gluc质粒,从融合试剂,结构质粒与报告质粒比例,细胞接种密度和收获时间等4个方面优化了HPV16假病毒的制作过程,ELISA验证不同抗体水平下假病毒检出效果。结果最优条件分别为E498b,结构质粒与报告质粒2:1,293FT细胞接种密度50%,收获时间48h或72h能够达到最佳包装效率。优化后的假病毒TCID50能够达到105.55,血清ELISA滴度分别为1:2000;1:8000;1:16000,中和实验ID50分别为90,270和810,具有很好的区分度。结论证明该优化方法现实可行,为HPV假病毒生产和中和抗体检测提供了一定的参考意义,为后续病毒试验提供了依据。

Objective： To optimize the construction method of HPV16 pseudovirus, Methods： In this experiment, we used L1 / L2 structural protein plasmids and pCMV-Gluc plasmids to optimize the construction method of HPV16 pseudovirus from four aspects as fusion reagents, the ratio of structural plasmids and reporter plasmids, density of cell seeding and harvest time. We used ELISA to verify the detection effect of pseudovirus under different antibody levels. Results： Optimal conditions were E498b, structural plasmid and reporter plasmid 2： 1, density of 293FT cell seeding was 50%, the harvest time was 48h or 72h, which could achieve the best efficiency. TCID50 of optimized pseudovirus reached 105.55. The serum ELISA titer were 1： 2000; 1：8000 and 1：16 000 respectively, and neutralization experiment ID50 were 90, 270 and 810 respectively, with good discrimination. Conclusion： This optimization method proves feasible, and it provides some referenced value for the production of HPV16 pseudovirus and the detection of neutralizing antibody, and provides basis for the virus test.