HIV-1假病毒包装及感染条件优化

Optimization of package and infection of the HIV-1 pseudotyped virus

目的对人类免疫缺陷病毒1型(HIV-1)假病毒包装及感染条件进行优化,获得高重复性、高稳定性和高滴度的假病毒包装方法和较高的感染效率。方法采用pSG3△env质粒与pcDNA3.1-SF162rev/env质粒共转染293T细胞包装病毒,对HIV假病毒骨架质粒与env真核表达质粒转染比例、转染试剂与质粒比例、假病毒收获时间对HIV假病毒包装滴度的影响进行实验,同时对感染过程中假病毒接种剂量与二乙氨乙基葡聚糖(DEAE-Dextran)使用浓度进行了优化。结果在包装过程中当HIV假病毒骨架质粒与env真核表达质粒转染比例为4∶1、转染试剂与质粒比例为3∶1,转染后培养48hHIV-1假病毒的滴度最高;在假病毒感染过程中,15μg/mL的DEAE-Dextran可有效增加假病毒的感染效率且不会对细胞造成较大毒性。结论通过对包装和感染过程中关键条件的优化,可以有效提高HIV-1假病毒的包装和感染效率,增加实验技术稳定性,为新型抗艾滋病药物筛选和艾滋病疫苗评价等研究奠定了基础。

Objective To optimize the package and infection conditions of human immunodeficiency virus type I（HIV-1） pseud- ovirus,in order to obtain a high-repeatability, high-stability and high-infection titer packaging method of HIV-1 pseudovirus and in- crease the efficiency of infection. Methods In view of the transfection efficiency factors,through different transfection ways,pSG3 /kenv plasmid and pcDNA3.1-SF162 rev/env plasmid were transfected into 293T cells packaging virus,and the HIV-1 pseudotyped virus were collected after 24 h,48 h and 72 h, respectively,then reporter gene after infecting TZM-bl cells for 48 h were detected. Results The HIV-1 pseudovirus was packaged at highest efficiency and titer under optimized conditions, in which the ratio of transfection backbone plasmid and envelope eukaryotic expression plasmid was at 4 ： 1, plasmid mixture and transfection reagent was at 3 ： 1 ,and the time of harvest of HIV pseudovirus was at 48 h after transfection. During the infection of HIV-1 pseudovirus, diethylaminoethyl dextran（DEAE-Dextran） at the concentration of 15 μg/mL could increase the efficiency of infection,causing relatively lower toxicity to 293T cell line. Conclusion The package and infection efficiency of HIV-1 pseudovirus are increased and the stability of experiment is improved with optimized key conditions. The results have established foundation for the stability of various applications,such as HIV-1 pseudovirus-based new types of anti-AIDS drug screening and AIDS vaccine evaluation.