本文關(guān)鍵詞： 人類免疫缺陷病毒 膜蛋白 廣譜單克隆中和抗體 序列分析 中和表型　出處：《中國疾病預(yù)防控制中心》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：背景鑒于中和抗體在預(yù)防HIV-1感染中的重要作用,設(shè)計能夠誘導(dǎo)高效廣譜中和抗體應(yīng)答的免疫原是HIV疫苗研究的重要目標(biāo)。膜蛋白是HIV-1中和抗體的靶位,因此對具有廣譜中和活性感染者病毒膜蛋白基因(env)特征的研究有助于HIV疫苗免疫原的設(shè)計。本研究通過探究HIV-1感染者膜蛋白序列的進化特征及假病毒的中和表型,為廣譜中和感染者中毒株進化逃逸研究及設(shè)計誘導(dǎo)廣譜中和抗體的膜蛋白免疫原提供參考信息。目的分析廣譜中和活性感染者HIV-1膜蛋白基因的進化特征;分析基于不同時間點膜蛋白基因的假病毒對自體血漿和代表性廣譜單克隆中和抗體(bmNAbs)的中和敏感性。方法對 HIV-1 廣譜中和活性感染者(CBJC515)20050816、20060418、20070424、20081118和20090519等5個連續(xù)時間點的血漿樣本進行RNA提取、逆轉(zhuǎn)錄、單基因組擴增(SGA)和測序,使用Sequencer、MEGA、BioEdit等軟件和Geno2pheno(coreceptor)、Weblogo等在線工具分析env基因的序列特征。從20050816、20060418和20081118等3個時間點序列中挑選代表性env序列,克隆至pcDNATM3.1 Directional TOPO表達載體并篩選鑒定,將env表達質(zhì)粒與HIV-1骨架質(zhì)粒(pSG3△env)共轉(zhuǎn)染293T/17細胞制備假病毒,并分別與各時間點自體血漿和代表性bmNAbs進行中和實驗,分析假病毒對自體血漿和bmNAbs的中和表型。結(jié)果1.從CBJC515的5個連續(xù)時間點血漿中共擴增獲得env基因SGA序列120條,對20050816、20060418和20081118等3個時間點序列分別構(gòu)建獲得11、4和13個功能性env克隆。所獲得的env基因均與中國RL42參考株聚集,并進化為優(yōu)勢組和劣勢組兩個分支,相同時間點序列多彼此聚集,少數(shù)不同時間點序列呈交叉分布。2.不同年份SGA序列的各可變環(huán)基因距離持續(xù)變化,并隨時間具有增長趨勢。V3環(huán)氨基酸長度和N-糖基化位點數(shù)目高度保守,V1V2可變環(huán)長度和N-糖基化位點數(shù)隨時間具有增長趨勢。X4型輔助受體病毒占比隨病程延長具有增高趨勢,優(yōu)勢毒株組X4型輔助受體的占比一般高于劣勢組。頂端四肽以GPGR為主且其占比隨病程有增高趨勢,優(yōu)勢組頂端四肽均為GPGR,劣勢組中有GLGR和GQGR兩種。3.本研究構(gòu)建的假病毒對8個連續(xù)時間點自體血漿的中和敏感性與使用不同亞型假病毒進行的中和實驗結(jié)果相似,中和敏感性均呈現(xiàn)先升高(第0-15月),后下降(第15-32月),之后再上升(第32-45月)的波動。假病毒對當(dāng)前時間點和該時間點之前的血漿中和敏感性低,對之后時間點血漿中和敏感性增強。4.10E8和VRC01能中和該研究樣本3個時間點構(gòu)建的所有假病毒,PGT135對上述病毒無中和活性;12A21、PGT121、2G12可中和大部分假病毒;X4型假病毒對2G12、12A21較R5型假病毒敏感,對VRC01較R5型假病毒抵抗;優(yōu)勢組病毒對VRC01、10E8較劣勢組抵抗,對2G12、12A21、PGT121較劣勢組敏感。5.假病毒對VRC01的中和敏感性與V1V2長度和gp120上N-糖基化位點數(shù)負相關(guān);對2G12和12A21的中和敏感性與V1V2氨基酸數(shù)正相關(guān);對2G12與PGT121的中和敏感性與N-糖基化位點數(shù)正相關(guān)。結(jié)論1.該感染者體內(nèi)包含優(yōu)勢和劣勢兩組病毒,病毒通過增加V1V2可變環(huán)長度和N-糖基化位點數(shù)、輔助受體轉(zhuǎn)換等方式逃避中和選擇壓力,向不同方向進化和變異。2.病毒對連續(xù)時間點自體血漿的中和敏感性呈現(xiàn)先升高(第0-15月),后下降(第15-32月),之后再上升(第32-45月)的波動,提示感染者體內(nèi)病毒與中和抗體的動態(tài)進化過程。3.在本研究測試的6個bmNAbs中,假病毒對10E8、PGT121和VRC01的敏感性較高,全部對PGT135抵抗;10E8對假病毒的中和能力強于12A21、2G12和VRC01;PGT121對假病毒的中和能力強于12A21和2G12。4.劣勢組毒株對VRC01和10E8的中和敏感性高于優(yōu)勢組病毒;優(yōu)勢組毒株對PGT121、12A21和2G12的敏感性高于劣勢組毒株;假病毒乃至同一時間點假病毒對特定bmNAbs的敏感性差異明顯,提示感染者準種毒株間中和表型的復(fù)雜性。
[Abstract]:In view of the background of neutralizing antibodies in the prevention of an important role in HIV-1 infection, immune design can induce efficient broad-spectrum neutralizing antibody response of the original is an important target of HIV vaccine. The membrane protein is HIV-1 neutralizing antibody targets, so the broad-spectrum neutralizing activity of infected virus membrane protein Bai Jiyin (Env) study on the characteristics of design in HIV vaccine immunogens help. This study through the evolution characteristics of HIV-1 infection of membrane protein sequences and viral neutralization phenotype for broad-spectrum neutralization infected strains escape immune evolutionary membrane protein research and design induce broad-spectrum neutralizing antibodies to provide reference information. Objective to analyze the evolution characteristics of the HIV-1 membrane protein gene of neutralizing activity at different time points of infection; analysis of membrane protein gene pseudotyped virus based on monoclonal autologous plasma and representative broad-spectrum neutralizing antibody (bmNAbs) and the sensitivity method. The infection of HIV-1 broad-spectrum neutralizing activity (CBJC515) and 20050816200604182007042420081118 20090519 plasma samples of 5 consecutive time points for RNA extraction, reverse transcriptase, single genome amplification and sequencing (SGA), Sequencer, MEGA, BioEdit software and Geno2pheno (coreceptor), Weblogo and other online tools to analyze the sequence characteristics of env gene of the selected representative. Env sequence from 2005081620060418 and 20081118 to 3 time points in the sequence, Directional TOPO was cloned into pcDNATM3.1 expression vector and screening and identification, the env expression plasmid and HIV-1 plasmid (pSG3 Env) were transfected into 293T/17 cells for preparing false virus, respectively and each time point of autologous plasma and representative bmNAbs neutralization experiment analysis of autologous plasma and bmNAbs pseudotyped virus neutralization phenotype. The results of 5 consecutive time points of the 1. plasma from CBJC515 env gene was amplified SGA sequence 12 0, 2005081620060418 and 20081118 of the 3 time sequences were constructed to obtain 11,4 and 13 functional env clones. Env gene were obtained by clustering and China RL42 reference strains, and evolutionary advantage and disadvantage group group of two branches, at the same time sequence with each other together, a few different time sequence is the variable cross distribution of.2. in different years SGA loop sequence gene distance continues to change with time, and with the growth trend of.V3 loop amino acid length and N- glycosylation sites are highly conserved and variable length V1V2 ring and N- glycosylation sites over time with the growth trend of.X4 auxiliary receptor virus accounted for with the extension of the course of the increasing tendency, the dominant strains were type X4 coreceptor proportion is generally higher than the inferior group. The top four peptides based on GPGR and the proportion with the course of disease has increased, the dominant group of top four peptides were GPGR, GLGR inferior group Two GQGR and.3. constructed in this study pseudovirion of 8 consecutive time points of autologous plasma neutralization sensitivity with different subtypes of the virus neutralization test false similar results, and sensitivity were increased (0-15 months), then decreased (15-32 months), and then on the rise (32-45 month) fluctuations. Plasma pseudovirus of prior to a current time point and the point of time and the sensitivity is low, for all time points after the virus in plasma and increased sensitivity to.4.10E8 and VRC01 and the study sample of 3 time points of PGT135, no neutralizing activity of the virus; 12A21, PGT121,2G12 and most of the false virus; X4 pseudotyped virus pseudotyped virus with 2G12,12A21 R5 on VRC01 sensitive than R5 pseudotyped virus resistance; virus of VRC01,10E8 was the dominant group of group 2G12,12A21, inferior resistance, PGT121 is sensitive to.5. pseudovirion disadvantage group VRC01 and sensitive And the length of V1V2 and gp120 on N- glycosylation sites are negatively correlated; positive correlation of neutralization sensitivity and V1V2 amino acid number 2G12 and 12A21; on 2G12 and PGT121 and N- sensitivity and glycosylation sites are related to the infection in vivo. Conclusion 1. contains the advantages and disadvantages of the two group of viruses, the virus through the increase of V1V2 variable length and ring N- glycosylation sites, coreceptor conversion of neutralization escape selection pressure to evolve in different directions and the variation of the.2. virus on the continuous time autologous plasma neutralization sensitivity increased (0-15 months), then decreased (15-32 months), then increased (32-45 1) fluctuations, suggesting that virus infection in vivo and the dynamic evolution process of.3. neutralizing antibody in 6 bmNAbs test in this study, the false virus on 10E8, PGT121 had higher sensitivity and VRC01, all of PGT135 resistance; 10E8 on false virus neutralizing ability is stronger than that of 12A2 1,2G12 and VRC01 PGT121; the false virus neutralizing ability is stronger than that of 12A21 and 2G12.4. strains were sensitive to VRC01 and inferior and 10E8 higher than that of the dominant group virus; sensitivity of PGT121,12A21 and 2G12 were the dominant group was higher than that of inferior group strains; sensitivity and false virus at the same time point of pseudotyped virus specific bmNAbs, suggesting that infection those kind of quasi complexity strains and phenotype.

【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R512.91

【參考文獻】

相關(guān)期刊論文 前4條

1 王敏連;梁冰玉;葉力;潘沛江;陳榮鳳;趙芳凝;蔣俊俊;黃頡剛;王洪;劉潔;周波;梁浩;;廣西HIV-1流行株env基因C2V3區(qū)序列特征和亞型研究[J];中華疾病控制雜志;2015年12期

2 洪坤學(xué);鄒森;邵一鳴;;艾滋病生物醫(yī)學(xué)預(yù)防策略現(xiàn)狀及挑戰(zhàn)[J];中國熱帶醫(yī)學(xué);2016年11期

3 胡園園;鄒森;齊中偉;侯佳麗;張岱;任莉;邵一鳴;郝彥玲;;HIV-1 B′亞型毒株感染者廣譜中和活性樣本不同時間點env基因變異研究[J];中國艾滋病性病;2017年01期

4 ;2016年12月全國艾滋病性病疫情[J];中國艾滋病性病;2017年02期

，

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