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目的分析哈尔滨市新确诊人类免疫缺陷病毒1型(HIV-1)CRF01_AE亚型nef基因及nef蛋白重要功能区氨基酸的变异性。方法从56例2014-2015年新确诊感染者外周血单核细胞(peripheral blood mononuclear cell,PBMC)中提取基因组DNA,扩增全长nef基因,并测序。构建nef基因系统发育树,分析基因特点;将nef氨基酸序列与中国和国际CRF01_AE共享序列进行比对,分析nef蛋白重要功能区氨基酸的变异性。结果 56株nef基因序列与不同CRF01_AE亚簇聚集,其中26株与CRF01-4亚簇聚集,24株与CRF01-5亚簇聚集,6株与CRF01-1亚簇聚集。亚簇内基因距离分析发现,CRF01-5亚簇最小,CRF01-4亚簇次之,CRF01-1亚簇最大。nef蛋白多个功能区氨基酸序列高度保守,但长度可变区、凋亡结构域、酸性区和C端PAK结合区变异率较高,其中凋亡区M50I突变和PAK结合区K192R突变多见于CD4数较低的样本(P<0.05)。结论哈尔滨市新确诊CRF01_AE亚型nef基因的组成较为复杂,可能来源于多个地区和多种感染途径;凋亡结构域和PAK结合区氨基酸变异与疾病进展相关。
Objective To analyze the variability of nef gene and important functional domain of nef protein of newly diagnosed human immunodeficiency virus type 1 (HIV-1) in Harbin. Methods Genomic DNA was extracted from 56 newly diagnosed peripheral blood mononuclear cells (PBMCs) from 2014 to 2015, and the full-length nef gene was amplified and sequenced. The phylogenetic tree of nef gene was constructed and the characteristics of genes were analyzed. The amino acid sequence of nef was compared with the shared sequence of CRF01_AE in China and in the world to analyze the variability of amino acids in important functional domains of nef protein. Results The sequences of 56 nef genes were clustered with different subtypes of CRF01_AE. Among them, 26 were clustered with CRF01-4, 24 with CRF01-5 and 6 with CRF01-1. Sub-cluster gene distance analysis found that CRF01-5 sub-cluster smallest, CRF01-4 sub-cluster, CRF01-1 sub-cluster largest. The amino acid sequences of multiple functional domains of nef protein were highly conserved. However, the mutation rates of variable length region, apoptotic domain, acidic region and C-terminal PAK binding region were higher. The M50I mutation in apoptosis region and K192R mutation in PAK binding region were more common in CD4 A lower number of samples (P <0.05). Conclusion The nef gene of newly diagnosed CRF01_AE subtypes in Harbin is more complicated, which may originated from multiple regions and multiple infection routes. The amino acid variation of apoptosis domain and PAK binding region is related to disease progression.