有限稀释病人PBMCs共培养法分离培养HIV-1 CRF07_BC生物性克隆病毒

Isolation and culture of HIV-1 CRF-07 bio-clone strains by limiting dilution of patient's peripheral blood mononuclear cells

目的通过有限稀释艾滋病病毒(HIV)感染者外周血单个核细胞(PBMCs)与正常供体PBMCs共培养,分离培养HIV-1生物性克隆毒株。方法选择6位新疆HIV-1CRF07_BC感染者,分离病人PBMCs,以不同浓度的病人PBMCs细胞数梯度(推荐浓度分别为5×103、2×104、4×104/孔)与健康供体的1×105PBMCs共培养,每个浓度设置32个复孔,每周检测HIV p24抗原。当某一浓度的复孔中p24阳性率低于33%时,可认为在这些阳性孔中的病毒是起源于同一个感染细胞。同时进行env区V1-V5基因扩增、测序和分析。结果经过有限稀释法共培养后,样本XJZK007在1×104细胞/孔的浓度下,各复孔HIV-1p24阳性率为31.3%;样本CBJB539在4×104细胞/孔的浓度下,得到9.4%分离阳性率;样本CBJB540在2×104细胞/孔的浓度下,得到25%分离阳性率;样本CBJB541在2×104细胞/孔的浓度下,得到12.5%分离阳性率;样本CBJB543在1×104细胞/孔的浓度下,得到21.9%分离阳性率;样本CBJB544在2×104细胞/孔的浓度下,得到25%分离阳性率。同一样本分离的不同生物克隆表现出不同的生物表型。对样本XJZK007分离得到的12株生物克隆的序列分析显示,各序列相互之间存在氨基酸的变异、插入及缺失,从而验证了生物性克隆方法的正确性与可行性。结论有限稀释病人PBMCs共培养方法,以常规分离方法十分之一的细胞数即可分离得到个体内多样性的病毒,即生物克隆病毒。

Objective To isolate and propagate HIV1 biological cloning strains by co-cultivating limiting diluition of patient＇s peripheral blood mononuclear cells （PBMCs） with PBMCs from health donors. Methods HIV-1 infected individuals were selected from XinJiang. PBMCs were isolated from patients＇whole blood. PBMCs were diluted into a series of concentration（5 × 10^3 , 1 × 10^4 , 2 × 10^4 and 4 × 10^4 per well recommended）, and co-cultivated with 1× 10^5 PHA-stimulated PBMCs from donors blood. Each concentration set 32 duplicate wells. HIV p24 antigen in the culture suspension was monitored weekly. When the P24 positive rates of the duplicate wells in one of the four concentrions were below 33% , it was considered that the virus in each well were originated from the same PB- MC of the infected patients. Simultaneously, the V1 / V5 gene amplification, sequencing and analysis were conducted. Results Co-cultivating in a limiting dilution system, the HIV-1 p24 positive rate of XJZK007 and CBJB543 at 1 × 10^4 cell/well concentration were 31.3% and 21.9 % respectively, while CBJB40 and CBJB544 at 2 × 10^4 cell/well concentration were both 25%. And at 4× 10^4 cell/well concentration, the HIV-1 p24 positive rate of CBJB41 and CBJB539 were 12.5% and 9.4% respectively. Different biological clonal vairants separated from the same patient showed different phenotypes. Sequence analysis of the 12 variants separated from XJZK007 showed that there were variation, insert and loss of amino acids among sequences, which testified the correctness and feasibility of biological cloning method. Conclusion Only with 1/10 PBMCs of conventional separation methods, the co-cultivation of limitig dilution PBMCs from patients could isolate and obtain various biological clone viruses from individual patients.