噬菌体随机肽库分析HIV-1 p24抗原表位

Analysis of the antigenic epitopes of HIV-1 p24 using phage display peptide library

目的 利用噬菌体随机肽库分析抗HIV 1核心区抗原p2 4单抗在抗原上的识别位点。方法 用抗HIV 1p2 4单抗 2C7和 3H10作为筛选分子 ,对噬菌体肽库进行生物淘洗 (biopanning) ,并通过DNA测序、ELISA效价测定等对所获得的噬菌体克隆进行鉴定 ,最后对合成的 7肽位点通过间接ELISA及竞争抑制试验进行血清学分析。结果 序列分析结果表明 ,单抗 2C7和 3H10在HIV 1p2 4上的抗原识别表位的保守序列分别为DHPXPXX和XXXXKAF。分别合成这 2个 7肽氨基酸序列P C1(DHPSPWG)和P H3(SPWLKAFGGGS) ,并分析其免疫学结合特性 ,结果表明与P H3相比 ,单抗 2C7的抗原识别表位P C1的固相结合特性较好 ,固相P C1检测血样 ,13份抗HIV阳性样本中 ,12份为阳性(检出率为 92 .3% ) ,19份抗HIV阴性样本中 ,仅 1份为假阳性结果 (特异性为 94.7% )。与P C1相比 ,单抗 3H10的抗原表位P H3的固相结合能力极差 ,但液相结合活性较好 ,血样与P H3的抑制试验表明 ,13份抗HIV阳性样本中 12份样本对P H3的抑制率大于 6 0 % (12 13) ,而 9份抗HIV阴性样本中仅 1份对P H3的抑制率大于 5 0 %。结论 用抗HIV 1p2 4单抗筛选噬菌体随机肽库 ,获得单抗在p2 4抗原上的识别表位的氨基酸序列 ,血清学结果表明这 2个抗原表位存在于p2 4自然抗原上 。

Objective To obtain the antigenic epitopes of the HIV-1 core antigen p24 recognized by the monoclonal antibodies (McAbs) of 2C7 and 3H10 using the phage display peptide library. Methods With the 2C7 and 3H10 as the target proteins, the phage peptide library was biopanned and the positive clones were identificated by the ELISA and DNA sequence. Finally the 7-aa peptides were sythesised and analyzed with the samples by indirect and competition ELISA. Results The consensus sequences of the epitopes on the HIV-1 p24 recognized by 2C7 and 3H10 were DHPXPXX and XXXXKAF separately obtained by DNA sequencing and the two peptide of P-C1 (DHPSPWG) and P-H3 (SPWLKAFGGGS) were synthesized. Compared with P-H3, the P-C1 was coated on the solid phase well. Using coated P-C1 to screen the samples, 12 of 13 anti-HIV positive were detected (the positive ratio was 92.3%) and among 19 anti-HIV negative samples there was only one to be false-positive (specificity was 94.7%). In comparison with P-C1, the P-H3 was reacted with 3H10 well in the liquid phase and among 13 anti-HIV positive samples there were 12 to inhibit P-H3 binding to 3H10 (inhibition ratio was more than 60%) and 1 of 9 anti-HIV negative could inhibit the P-H3 (inhibition ratio was more than 50%). Conclusion Screened the phage peptide library with anti-HIV p24 McAbs of 2C7 and 3H10, there were obtained the consensus amino acid sequences of the antigenic epitopes on p24 and these two epitopes were on the natural p24 in the HIV infected samples. They could have potential role in the detection of the infection of HIV. [