对奈韦拉平耐药的HIV-1毒株诱导研究

Cultivation of NVP-resistant HIV-1 Stain

目的探讨对NVP耐药的I型艾滋病病毒（HIV-1）毒株的诱导方法，获得能够产生连续稳定传代的对NVP高度耐药的HIV-1毒株。方法MT4细胞中先后加入终浓度为100TCID50的HIV-1实验室毒株89．6与终浓度为10倍Ic，。的NVP药物溶液，在药物浓度不变的条件下传代，培育HIV-1病毒。提取每代培养液上清病毒RNA，逆转录后使用巢式PCR扩增HIV-1pol区基因，收集目标片段并进行测序分析。结果诱导培养所得耐药株由第5代开始与野生株相比，出现NVPIC。变化，倍数约为3倍，培养11代后，变化倍数高达178倍。此外，发生Y181C和Y188Y位点突变。结论本实验中在体外经诱导所得耐药株对NVP具有较高的耐药性，并可获得稳定传代。

Objective To investigate the method and process of the introduction of NVP-resistant HIV-1 strain, meanwhile, obtain the stable strain in vitro. Methods MT4 cells were added into the final concentration of 100 times the median tissue culture infective dose （TCIDs0） of the HIV-1 laboratory strain 89.6, and half tissue culture infective dose at concentration of 10 times the median inhibitory concentration （ ICso ） of the NVP drug solution, at the same concentration under the condition of subculture, cultivation of HIV-1 virus. Extract the virus RNA of the cutture supernatant from each generation, after reverse transcrip- tion used nested PCR to amplification of HIV-1 gene pol region, collecting the target fragment and sequencing analysis. Results The induction of the cultivation of resistant strains from the fifth generation, compared with wild strains, NVP-ICs0 began to raise about 3 times, after 11 generation of culture, change ratio as high as 178 times. In addition, Y181C and Y188Y mutation occured. Conclusions In this study, we obtain the strains highly resistant to NVP, and it can go down to the future generation stably.