人類免疫缺陷病毒1型（HIV-1）假病毒檢測體系的建立與應(yīng)用
發(fā)布時間：2019-03-01 18:20
【摘要】： HIV-1具有基因多變性以及ENV結(jié)構(gòu)的復(fù)雜性,不同HIV疫苗所誘導(dǎo)的中和抗體的中和特性往往不同,因此,建立一個統(tǒng)一有效的中和抗體檢測方法是非常必要的。本課題成功獲得31株可以與骨架質(zhì)粒pSG3△ENV共轉(zhuǎn)染293T細(xì)胞形成功能性假病毒的pcDNA3.1-ENV陽性克隆。我們對這31株克隆進(jìn)行基因分型,其中有19株BC亞型、8株B亞型、4株AE亞型,除了4株假病毒的ENV基因來源于已有質(zhì)粒外,其他27株中ENV基因均來源于中國HIV-1感染者的陽性血清樣本。分析克隆中g(shù)p160核苷酸序列可以看出克隆之間存在遺傳多樣性,31株假病毒的可變區(qū)V1、V2、V4和V5中氨基酸數(shù)目都有明顯差異,但V3區(qū)的氨基酸數(shù)目沒有差異,這可能與假病毒進(jìn)入細(xì)胞的機(jī)制有關(guān)。不同克隆中N-糖基化位點(diǎn)(PNLG)是有差異的,PNLG的位點(diǎn)數(shù)較少,暴露中和位點(diǎn)較多從而表現(xiàn)出較為敏感的中和反應(yīng)。 假病毒可以感染TZM-bl細(xì)胞,誘導(dǎo)細(xì)胞內(nèi)的熒光素酶的表達(dá),根據(jù)檢測發(fā)光值的變化來判定中和活性的強(qiáng)弱。用四種已知的具有中和活性的單克隆抗體(4E10、2F5、2G12和IgG1b12)來分析假病毒的中和活性,結(jié)果顯示假病毒株中單克隆抗體識別中和表位的氨基酸變化會影響其中和活性。當(dāng)缺少N-糖基化位點(diǎn)295或392時,假病毒株都表現(xiàn)為對2G12不敏感,因此認(rèn)為N-糖基化位點(diǎn)295或392是2G12中和活性所必需的識別位點(diǎn)。2F5識別位點(diǎn)包含氨基酸序列ELDKWA,從實(shí)驗(yàn)結(jié)果可以看出賴氨酸(K)是2F5識別位點(diǎn)所必需的。4E10識別位點(diǎn)緊鄰2F5識別位點(diǎn),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取代均不影響假病毒對4E10的敏感性。 用假病毒檢測體系檢測43份血清樣本,結(jié)果顯示19株BC亞型的假病毒與BC亞型血清樣本有明顯的、廣泛的中和活性,而與B、AE亞型血清樣本反應(yīng)時僅個別反應(yīng)表現(xiàn)出中和活性,但抑制率都偏低。同樣8株B亞型假病毒與同亞型血清樣本反應(yīng)表現(xiàn)出較高的中和活性,4株AE亞型假病毒與同亞型血清樣本反應(yīng)表現(xiàn)出相對較高的中和活性,各亞型間沒有明顯的交叉活性。從檢測血清樣本的結(jié)果可以看出,假病毒檢測體系應(yīng)用于血清樣本的檢測是可行的,而且假病毒檢測體系容易標(biāo)準(zhǔn)化,其結(jié)果重復(fù)性較好。 假病毒檢測體系成功的應(yīng)用于藥物AZT、3TC、d4T和ddI檢測,檢測藥物的實(shí)驗(yàn)結(jié)果與已報道的結(jié)果相一致,可以看出假病毒檢測體系可應(yīng)用于抗HIV藥物檢測。假病毒檢測體系既可以定性得到半數(shù)有效濃度EC50又可以定量得到半數(shù)有效劑量ED50,并且相對于活病毒法具有重復(fù)性好、靈敏、精確、易操作的優(yōu)點(diǎn)。因此,假病毒法在檢測抗HIV-1藥物的研究中具有很好的發(fā)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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R392.11
本文編號：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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R392.11
【引證文獻(xiàn)】
相關(guān)博士學(xué)位論文 前1條
1 張明順;中國HIV-1包膜蛋白中和免疫原性研究[D];南京醫(yī)科大學(xué);2011年
，本文編號：2432682
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