人类免疫缺陷病毒1型（HIV-1）假病毒检测体系的建立与应用
发布时间：2019-03-01 18:20
【摘要】： HIV-1具有基因多变性以及ENV结构的复杂性,不同HIV疫苗所诱导的中和抗体的中和特性往往不同,因此,建立一个统一有效的中和抗体检测方法是非常必要的。本课题成功获得31株可以与骨架质粒pSG3△ENV共转染293T细胞形成功能性假病毒的pcDNA3.1-ENV阳性克隆。我们对这31株克隆进行基因分型,其中有19株BC亚型、8株B亚型、4株AE亚型,除了4株假病毒的ENV基因来源于已有质粒外,其他27株中ENV基因均来源于中国HIV-1感染者的阳性血清样本。分析克隆中gp160核苷酸序列可以看出克隆之间存在遗传多样性,31株假病毒的可变区V1、V2、V4和V5中氨基酸数目都有明显差异,但V3区的氨基酸数目没有差异,这可能与假病毒进入细胞的机制有关。不同克隆中N-糖基化位点(PNLG)是有差异的,PNLG的位点数较少,暴露中和位点较多从而表现出较为敏感的中和反应。 假病毒可以感染TZM-bl细胞,诱导细胞内的荧光素酶的表达,根据检测发光值的变化来判定中和活性的强弱。用四种已知的具有中和活性的单克隆抗体(4E10、2F5、2G12和IgG1b12)来分析假病毒的中和活性,结果显示假病毒株中单克隆抗体识别中和表位的氨基酸变化会影响其中和活性。当缺少N-糖基化位点295或392时,假病毒株都表现为对2G12不敏感,因此认为N-糖基化位点295或392是2G12中和活性所必需的识别位点。2F5识别位点包含氨基酸序列ELDKWA,从实验结果可以看出赖氨酸(K)是2F5识别位点所必需的。4E10识别位点紧邻2F5识别位点,包含氨基酸序列NWFDIT,序列中N被S取代,D被S/N取代,T被S取代均不影响假病毒对4E10的敏感性。 用假病毒检测体系检测43份血清样本,结果显示19株BC亚型的假病毒与BC亚型血清样本有明显的、广泛的中和活性,而与B、AE亚型血清样本反应时仅个别反应表现出中和活性,但抑制率都偏低。同样8株B亚型假病毒与同亚型血清样本反应表现出较高的中和活性,4株AE亚型假病毒与同亚型血清样本反应表现出相对较高的中和活性,各亚型间没有明显的交叉活性。从检测血清样本的结果可以看出,假病毒检测体系应用于血清样本的检测是可行的,而且假病毒检测体系容易标准化,其结果重复性较好。 假病毒检测体系成功的应用于药物AZT、3TC、d4T和ddI检测,检测药物的实验结果与已报道的结果相一致,可以看出假病毒检测体系可应用于抗HIV药物检测。假病毒检测体系既可以定性得到半数有效浓度EC50又可以定量得到半数有效剂量ED50,并且相对于活病毒法具有重复性好、灵敏、精确、易操作的优点。因此,假病毒法在检测抗HIV-1药物的研究中具有很好的发展前景。
[Abstract]:HIV-1 is characterized by genetic variability and complexity of ENV structure. The neutralization characteristics of neutralizing antibodies induced by different HIV vaccines are often different. Therefore, it is necessary to establish a uniform and effective detection method for neutralizing antibodies. In this study, 31 pcDNA3.1-ENV positive clones which could co-transfect 293T cells with cytoskeleton plasmid pSG3 ENV were obtained. We genotyped these 31 clones, including 19 strains of BC subtype, 8 strains of B subtype and 4 strains of AE subtype. Except for 4 strains of pseudovirus, the ENV gene of these clones was derived from the existing plasmids. The other 27 strains of ENV gene were derived from positive serum samples of Chinese HIV-1-infected individuals. Analysis of the nucleotide sequence of gp160 showed that there was genetic diversity among clones. The amino acid numbers in V1, V2, V4 and V5 regions of 31 pseudovirus strains were significantly different, but there was no difference in amino acid number of V3 region. This may be related to the mechanism of pseudovirus entering cells. The (PNLG) of N-glycosylation sites in different clones was different. The number of sites in PNLG was less and the number of exposed neutralization sites was more. Therefore, the N-glycosylation sites showed a more sensitive neutralization reaction. Pseudovirus can infect TZM-bl cells, induce the expression of luciferase in the cells, and determine the neutralization activity according to the change of luminescence value. Four known neutralizing monoclonal antibodies (4E10, 2F5, 2G12 and IgG1b12) were used to analyze the neutralization activity of pseudoviruses. The results showed that the change of amino acids in the neutralizing epitopes of monoclonal antibodies in pseudoviruses affected neutralization activity. In the absence of N-glycosylation site 295 or 392, pseudovirus strains were insensitive to 2G12, so it was considered that N-glycosylation site 295 or 392 was necessary for neutralizing 2G12. 2F5 recognition site contained amino acid sequence ELDKWA,. From the experimental results, we can see that lysine (K) is necessary for 2F5 recognition site. 4E10 recognition site is adjacent to 2F5 recognition site, including amino acid sequence NWFDIT, sequence N is replaced by S, D is replaced by S, and D is substituted by S and D, and the amino acid sequence is substituted by S and D, respectively. The substitution of T with S did not affect the sensitivity of pseudovirus to 4E10. The results showed that 19 strains of BC subtype and BC subtype had obvious and extensive neutralization activity, but with that of B, B and B, respectively. Only a few of the serum samples of AE subtype showed neutralization activity, but the inhibition rate was low. The same 8 strains of pseudoviruses of subtype B showed higher neutralization activity with serum samples of the same subtype, 4 strains of AE subtype of pseudovirus showed relatively high neutralization activity with serum samples of the same subtype, and there was no cross activity among the subtypes. From the results of serum samples detection, it can be seen that the pseudo-virus detection system is feasible, and the pseudo-virus detection system is easy to standardize, and the results are reproducible. The pseudo-virus detection system has been successfully applied to the detection of drug AZT,3TC,d4T and ddI. The experimental results are consistent with the reported results. It can be seen that the pseudo-virus detection system can be applied to the detection of anti-HIV drugs. The pseudo-virus detection system can obtain both qualitative and quantitative half-effective concentration of EC50 and quantitative half-effective dose of ED50, which has the advantages of good repeatability, sensitivity, accuracy and ease of operation compared with the live virus method. Therefore, pseudo-virus method in the detection of anti-HIV-1 drugs has a good prospect of development.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392.11
本文编号：2432682
[Abstract]:HIV-1 is characterized by genetic variability and complexity of ENV structure. The neutralization characteristics of neutralizing antibodies induced by different HIV vaccines are often different. Therefore, it is necessary to establish a uniform and effective detection method for neutralizing antibodies. In this study, 31 pcDNA3.1-ENV positive clones which could co-transfect 293T cells with cytoskeleton plasmid pSG3 ENV were obtained. We genotyped these 31 clones, including 19 strains of BC subtype, 8 strains of B subtype and 4 strains of AE subtype. Except for 4 strains of pseudovirus, the ENV gene of these clones was derived from the existing plasmids. The other 27 strains of ENV gene were derived from positive serum samples of Chinese HIV-1-infected individuals. Analysis of the nucleotide sequence of gp160 showed that there was genetic diversity among clones. The amino acid numbers in V1, V2, V4 and V5 regions of 31 pseudovirus strains were significantly different, but there was no difference in amino acid number of V3 region. This may be related to the mechanism of pseudovirus entering cells. The (PNLG) of N-glycosylation sites in different clones was different. The number of sites in PNLG was less and the number of exposed neutralization sites was more. Therefore, the N-glycosylation sites showed a more sensitive neutralization reaction. Pseudovirus can infect TZM-bl cells, induce the expression of luciferase in the cells, and determine the neutralization activity according to the change of luminescence value. Four known neutralizing monoclonal antibodies (4E10, 2F5, 2G12 and IgG1b12) were used to analyze the neutralization activity of pseudoviruses. The results showed that the change of amino acids in the neutralizing epitopes of monoclonal antibodies in pseudoviruses affected neutralization activity. In the absence of N-glycosylation site 295 or 392, pseudovirus strains were insensitive to 2G12, so it was considered that N-glycosylation site 295 or 392 was necessary for neutralizing 2G12. 2F5 recognition site contained amino acid sequence ELDKWA,. From the experimental results, we can see that lysine (K) is necessary for 2F5 recognition site. 4E10 recognition site is adjacent to 2F5 recognition site, including amino acid sequence NWFDIT, sequence N is replaced by S, D is replaced by S, and D is substituted by S and D, and the amino acid sequence is substituted by S and D, respectively. The substitution of T with S did not affect the sensitivity of pseudovirus to 4E10. The results showed that 19 strains of BC subtype and BC subtype had obvious and extensive neutralization activity, but with that of B, B and B, respectively. Only a few of the serum samples of AE subtype showed neutralization activity, but the inhibition rate was low. The same 8 strains of pseudoviruses of subtype B showed higher neutralization activity with serum samples of the same subtype, 4 strains of AE subtype of pseudovirus showed relatively high neutralization activity with serum samples of the same subtype, and there was no cross activity among the subtypes. From the results of serum samples detection, it can be seen that the pseudo-virus detection system is feasible, and the pseudo-virus detection system is easy to standardize, and the results are reproducible. The pseudo-virus detection system has been successfully applied to the detection of drug AZT,3TC,d4T and ddI. The experimental results are consistent with the reported results. It can be seen that the pseudo-virus detection system can be applied to the detection of anti-HIV drugs. The pseudo-virus detection system can obtain both qualitative and quantitative half-effective concentration of EC50 and quantitative half-effective dose of ED50, which has the advantages of good repeatability, sensitivity, accuracy and ease of operation compared with the live virus method. Therefore, pseudo-virus method in the detection of anti-HIV-1 drugs has a good prospect of development.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392.11
【引证文献】
相关博士学位论文 前1条
1 张明顺;中国HIV-1包膜蛋白中和免疫原性研究[D];南京医科大学;2011年
，本文编号：2432682
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