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两种HIV非核苷类逆转录酶抑制剂(nonnucleoside reverse transcriptase inhibitors,NNRTIs)JB25和JB26在体外均具有显著的抗HIV活性,为了在临床试验前阐明病毒是否容易对这两种药物产生耐药性及导致耐药性的病毒基因突变,在体外诱导并鉴定了对这两种化合物耐药的HIV-1毒株。以HIV-1NL4.3作为亲代野生型病毒感染MT-2细胞,从两种化合物2倍的IC50开始,以细胞病变(cytopathic effect,CPE)为指标,将病毒在含有浓度逐渐增加的药物的条件下连续传代,每代均在超过75%的MT-2细胞出现CPE后收集病毒培养上清液,然后取1mL接种新的MT-2细胞同时提高药物浓度为原来的2倍,共计传12代。JB25在第6代出现L100I(TTA→ATA)突变,在第12代又变成100M(ATA→ATG),L100M是不常见的突变形式,其对HIV NNRTIs耐药的影响未见报道,在第10代还同时出现了对NNRTIs高度耐药的Y188C(TAT→TGT)突变。JB26在第10代出现了L100I(TTA→ATA)突变。JB25和JB26在体外容易产生耐药性,而对JB26耐药比JB25要困难,导致耐药的主要基因突变发生在病毒逆转录酶基因的188和100位密码子。
Two non-nucleoside reverse transcriptase inhibitors (NNRTIs) JB25 and JB26 have significant anti-HIV activity in vitro, in order to elucidate whether the virus is susceptible to both drugs before clinical trials And mutations in the viral genes that cause drug resistance, in vitro HIV-1 strains that were resistant to both compounds were induced and identified. MT-2 cells were infected with HIV-1NL4.3 as the parental wild-type virus, starting from the 2-fold IC50 of both compounds and using the cytopathic effect (CPE) Were subcultured and subcultured in succession. The virus culture supernatants were harvested from more than 75% of MT-2 cells in each generation after subculturing CPE. The cells were then inoculated with 1 mL of MT-2 cells and the drug concentration was doubled for a total of 12 passages . JB25 had L100I (TTA → ATA) mutation in the 6th generation and became 100M (ATA → ATG) in the 12th generation. L100M was an uncommon mutated form and its effect on HIV NNRTIs resistance was not reported. On the 10th generation, Y188C (TAT → TGT) mutation highly resistant to NNRTIs also appeared. JB26 appeared L100I (TTA → ATA) mutation in the tenth generation. JB25 and JB26 are susceptible to drug resistance in vitro, but resistant to JB26 is more difficult than JB25, and the major genetic mutations leading to drug resistance occur at codons 188 and 100 of the viral reverse transcriptase gene.