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目的利用B细胞培养和RT-PCR技术从我国人类免疫缺陷病毒1型(HIV-1)感染者中筛选出具有广谱中和活性的人单克隆抗体。方法本研究采集HIV-1感染者抗凝全血,分离出外周血单个核细胞,用磁珠分选纯化记忆性B细胞,在体外选择3T3-hCD40L作为饲养细胞,并使用细胞因子白细胞介素(IL)-2和IL-21诱导记忆性B细胞分泌IgG,用HIV假病毒微量中和的方法进行筛选。从有中和活性的孔中分别扩增出B细胞IgG的轻链和重链基因,并将其克隆到含IgG恒定区基因的载体上。然后将携带重链和轻链基因的质粒进行大量表达,得到人单克隆抗体,并进行抗体结合活性、中和活性及与抗原Gp120亲和力的鉴定。结果从我国3例HIV-1感染者记忆性B细胞中筛选出3株对HIV-1Env抗原有结合活性的单克隆抗体。其中8E7和55B3主要与Gp120结合,19GF3与Gp120和FLSC均可结合且结合活性无明显差异;基因变异大的8E7抗体对A、B、C三种亚型的假病毒均有一定的中和活性,55B3对SF162有中和活性,19GF3未检测到中和活性;这三种单抗的亲和力都很强,8E7、19GF3、55B3与抗原Gp120的KD值分别为6.45×10~(-10) mol/L、1.12×10~(-10) mol/L、2.76×10~(-10) mol/L。结论成功建立了在96孔板中高效刺激人B细胞在体外分泌IgG的方法,并利用HIV假病毒微量中和实验和RT-PCR技术筛选得到对HIV-1具有中和活性的人单克隆抗体。
OBJECTIVE: To screen human monoclonal antibodies with broad-spectrum neutralization activity from human immunodeficiency virus type 1 (HIV-1) infected patients in China by B cell culture and RT-PCR. Methods In this study, anticoagulant whole blood was collected from HIV-1 infected individuals. Peripheral blood mononuclear cells (PBMCs) were isolated and purified by magnetic beads. 3T3-hCD40L cells were selected as feeder cells in vitro and cultured with cytokines interleukin (IL) -2 and IL-21 induce the secretion of IgG by memory B cells, screening by micro-neutralization of HIV pseudovirus. The light and heavy chain genes of B cell IgG were separately amplified from the wells with neutralizing activity and cloned into the vector containing the constant region IgG gene. Then, the heavy chain and light chain gene plasmids were overexpressed to obtain human monoclonal antibodies, and the antibody binding activity, neutralizing activity and affinity with the antigen Gp120 were identified. Results Three monoclonal antibodies against HIV-1 Env antigen were screened from memory B cells of 3 HIV-1 infected patients in our country. Among them, 8E7 and 55B3 mainly bind to Gp120, 19GF3 bind to Gp120 and FLSC, and there is no significant difference in binding activity. 8E7 antibody with large gene mutation has certain neutralizing activity against the three subtypes of A, B and C , 55B3 had neutralizing activity on SF162 and no neutralizing activity on 19GF3. The affinity of these three McAbs was very strong. The KD values of 8E7, 19GF3 and 55B3 and antigen Gp120 were 6.45 × 10 ~ (-10) mol /L,1.12 × 10 ~ (10) mol / L, 2.76 × 10 ~ (-10) mol / L. Conclusion The method of efficiently stimulating human B cells to secrete IgG in 96-well plates was successfully established. Human monoclonal antibody with neutralizing activity to HIV-1 was screened by micro-neutralization test of HIV pseudovirus and RT-PCR technology .