人乳头瘤病毒假病毒制备方法的优化

Optimization of Preparation Method for Human Papillomavirus Pseudovirion

人乳头瘤病毒假病毒可广泛应用于中和抗体鉴定和疫苗免疫性评估等研究.本研究对多质粒共转染293FT细胞生产HPV假病毒的方法进行了深入优化,结果显示不同的HPV结构蛋白表达质粒转染比例对于假病毒生产效率有显著的影响,HPV主要结构蛋白L1的表达水平是影响生产效率的主要因素,L2表达质粒的用量过高或过低均不利于假病毒的生产,而报告质粒的用量对假病毒生产效率的影响相对较小.综合比较显示,在该体系中L1和L2表达质粒和报告质粒以合适比例(如1∶1/10∶1/2)进行共转染可获得更为理想的生产效率.本研究同时也在293FT细胞中对磷酸钙转染方法进行了优化,通过磷酸钙与质粒用量的交叉配比试验获得了较优的转染条件.通过优化,建立了更为高效的HPV假病毒大量制备方法,在HPV16、HPV18、HPV6、HPV11假病毒生产中的应用结果显示较原方法可显著提高生产效率.本研究为HPV假病毒中和实验的规模化应用提供了有利条件.

The inability to produce HPV efficiently in animal models or cell culture systems led to the development of alternative approaches for the evaluation of virus-neutralizing antibodies and the study of HPV. In previous research, we have reported that infectious HPV pseudovirion could be economically produced by calcium phosphate eo-transfection of codon optimized HPV16 capsid genes L1 and L2 and reporter plasmid in 293FT cells. Extensive applications of HPV pseudovirion rise the requirement of producing large quantity of pseudovirion. In this report, the pseudovirion producing method was further optimized by testing different quantity ratios of the three plasmids and concentration of DNA in calcium phosphate reagents. Results showed that the producing efficiency of HPV pseudovirion was markedly influenced by different quantity ratios of L1 and L2 expression plasmids,but slightly by different quantity ratio of reporter plasmid. Excessive transfection of either reporter plasmid or L2 expression plasmid led to a decline of L1 expression,along with a lower producing efficiency of pseudovirion, but L2 protein was necessary to generate infectious pseudovirion efficiently. The 1 ： 1/10 ： 1/2 would be the ideal quantity ratio of L1 and L2 expression plasmids and reporter plasmid in the producing system. The producing efficiency of HPV pseudovirion could be further improved by optimizing the volume of calcium phosphate reagents and the concentration of total plasmids. Results of the cross experiment showed that 37.5 μg/mL and 800 μL would be the ideal concentration of total plasmids and volume of calcium phosphate reagents for 9. 0 × 10^5 293FT cells cultured in 6-well plate. Compared with previous method, the optimized method could achieve higher producing efficiency of HPV pseudovirion, including HPV16, HPV18, HPV11, HPV6 pseudovirion. The optimized method developed in this study facilitate the application of pseudovirion-based human papillomavirus neutralization assay in large scale.