福建省HIV-1CRF01_AE流行株的表型分析、基因序列特征、膜蛋白表達及感染性克隆構(gòu)建的研究
發(fā)布時間：2018-09-10 11:33
【摘要】： 人類免疫缺陷病毒(Human Immunodeficiency virus,HIV),是逆轉(zhuǎn)錄病毒科慢病毒屬靈長類慢病毒亞屬中的一類病毒。據(jù)全國兩次分子流行病學調(diào)查結(jié)果表明HIV CRF01_AE重組型株正在從南部及沿海省市向周圍省市擴散,CRF01_AE重組型在全國所占比例也有所增加,在福建省HIV CRF01_AE重組型為主要的流行株。本研究首次在國內(nèi)開展了HIV CRF01_AE流行株的表型分析、全長膜基因的特征和表達、全長基因的特征和感染性克隆構(gòu)建的研究,為本型病毒的病原學、變異特征、疫苗、診斷方法和致病機理的研究提供了堅實的基礎(chǔ),本研究分為五個部分。 一.福建省HIV-1 CRF01_AE感染者的病毒分離及其生物學表型初步研究 1.成功分離4份HIV-1 CRF01_AE感染者的毒株,Fj200604在MT-2細胞誘導了典型的合胞體(SI),另外3個毒株沒有形成明顯的合胞體(NSI);分離株Fj200601-Fj200603利用CCR5輔助受體,Fj200604利用CXCR4輔助受體。 2.分離株的生物學表型與V3環(huán)序列變異密切相關(guān),表明通過對V3環(huán)關(guān)鍵氨基酸的分析可以預測HIV-1輔助受體的使用情況。 二.福建省HIV-1 CRF01_AE亞型毒株全長gp120基因序列特征分析 1.基因系統(tǒng)樹分析顯示本研究所有的樣本屬于HIV-1 CRF01_AE重組型,遺傳變異較大,組內(nèi)基因距離為9.5±2.5 %。 2. V3環(huán)頂端四肽存在四種類型:GPGQ(72.24 %),GPGR(19.04 %),GPGH(4.76 %),GQGQ(4.76 %)。16份(76.19 %)樣本可能使用CCR5作為輔助受體,1份(4.76 %)樣本可能使用CCR5/CXCR4受體,4份樣本(19.04 %)不能做出預測。 3. C1-C5區(qū)比較保守,而V1-V5環(huán)變化較大,其中V3相對保守。 4. N糖基化位點分析發(fā)現(xiàn)大多數(shù)位點較為保守,少數(shù)糖基化位點發(fā)生丟失和增加。 三.HIV膜蛋白gp120基因在果蠅S2細胞中的表達 1.構(gòu)建了全長gp120基因和缺失V1/V2環(huán)的表達重組載體,在果蠅S2細胞中瞬時表達成功。 2.建立了穩(wěn)定表達的篩選方法,篩選了穩(wěn)定分泌表達全長gp120基因和缺失V1/V2環(huán)的重組果蠅S2細胞。 四.福建省13條HIV-1 CRF01_AE亞型毒株全長基因克隆及其序列特征分析 1.完成了13株HIV-1 CRF01_AE全基因組克隆,建立了HIV-1 CRF01_AE全基因組擴增平臺。 2. 13條全基因組克隆測序提交至NCBI(美國國家生物信息數(shù)據(jù)庫)的GenBank。 3.系統(tǒng)進化樹分析表明所有13條全長基因序列與CRF01_AE參考株聚在一起,但可分成幾個亞組分散在泰國分離株中。13條全長基因序列(去除超突變序列Fj061)env,gag,pol三個結(jié)構(gòu)基因區(qū)的組內(nèi)基因離散率均高于泰國分離株;全長基因序列Fj061證實為超突變序列。所克隆序列未出現(xiàn)基因水平的重組,對蛋白酶和逆轉(zhuǎn)錄酶的藥物依然敏感。 五.福建省CRF01_AE重組型感染性克隆的構(gòu)建和生物學活性初步鑒定 1.完成3個全長克隆的連接,293T細胞轉(zhuǎn)染實驗表明2個全長克隆(全長克隆A和嵌合全長克隆A-B)顯示較好的活性。 2.全長克隆A和嵌合全長克隆A-B具有一定程度的感染PBMCs的能力和在PBMCs內(nèi)進行有效復制的能力,而嵌合全長克隆A-B在PBMCs中的復制能力比全長克隆A更強些。 3.嵌合全長克隆A-B應用CCR5作為輔助受體,而全長克隆A未觀察到細胞對輔助受體的利用情況。
[Abstract]:Human Immunodeficiency virus (HIV) is a kind of virus in the subgenus Lentiviridae of the Retrovirus family. According to the results of two national molecular epidemiological investigations, the recombinant strain of HIV CRF01_AE is spreading from the southern and coastal provinces to the surrounding provinces and cities, and the recombinant type of CRF01_AE accounts for a large proportion of the country. This study is the first to carry out the phenotypic analysis of HIV CRF01_AE epidemic strain in China, the characteristics and expression of the full-length membrane gene, the characteristics of the full-length gene and the construction of infectious clone, the etiology of the virus, mutation characteristics, vaccines, diagnostic methods and causes. The study of disease mechanism provides a solid foundation. This study is divided into five parts.
Isolation and biological phenotypes of HIV-1 CRF01_AE infected patients in Fujian
1. Four HIV-1 CRF01_AE strains were successfully isolated, Fj200604 induced typical syncytium (SI) in MT-2 cells, and the other three strains did not form obvious syncytium (NSI); Fj200601-Fj200603 used CCR5 coreceptor, Fj200604 used CXCR4 coreceptor.
2. The biological phenotype of the isolate is closely related to the variation of V3 loop sequence, which indicates that the use of HIV-1 coreceptor can be predicted by analyzing the key amino acids of V3 loop.
Two. Sequence analysis of full-length gp120 gene of HIV-1 CRF01_AE subtype strain in Fujian
1. Gene phylogenetic tree analysis showed that all the samples in this study belonged to the recombinant HIV-1 CRF01_AE, and the genetic variation was large. The intra-group gene distance was 9.5 + 2.5%.
2. There are four types of apical tetrapeptides in V3 ring: GPGQ (72.24%), GPGR (19.04%), GPGH (4.76%), GQGQ (4.76%). 16 (76.19%) samples may use CCR5 as a co-receptor, 1 (4.76%) samples may use CCR5/CXCR4 receptor, and 4 (19.04%) samples cannot predict.
3. the C1-C5 area is relatively conservative, while the V1-V5 ring is relatively large, and V3 is relatively conservative.
4. N-glycosylation site analysis showed that most of the sites were conservative, and a few of the glycosylation sites were lost and increased.
Expression of three.HIV membrane protein gp120 gene in Drosophila melanogaster S2 cells
1. The recombinant vector of gp120 gene and deleted V1/V2 loop was constructed and expressed successfully in Drosophila S2 cells.
2. Screening method of stable expression was established, and recombinant Drosophila S2 cells with stable expression of full-length gp120 gene and deletion of V1/V2 ring were screened.
Four. Cloning and sequence analysis of full-length gene of 13 HIV-1 CRF01_AE subtype strains isolated from Fujian Province
1. complete genome cloning of 13 HIV-1 CRF01_AE has been completed, and HIV-1 CRF01_AE whole genome amplification platform has been established.
2.13 complete genome cloning and sequencing were submitted to NCBI (GenBank.).
3. Phylogenetic tree analysis showed that all 13 full-length gene sequences were clustered with CRF01_AE reference strain, but could be divided into several subgroups and dispersed in Thai isolates. The cloned sequence was not recombined at gene level and was still sensitive to protease and reverse transcriptase.
Five. Construction of recombinant infectious clone of CRF01_AE in Fujian province and preliminary identification of its biological activity.
1. The results of 293T cell transfection showed that two full-length clones (full-length clone A and chimeric full-length clone A-B) showed good activity.
2. Full-length clone A and chimeric full-length clone A-B have the ability to infect PBMCs to a certain extent and replicate effectively in PBMCs, while the replication ability of chimeric full-length clone A-B in PBMCs is stronger than that of full-length clone A.
3. Chimeric full-length clone A-B used CCR5 as a coreceptor, while full-length clone A did not observe the use of coreceptors.
【學位授予單位】：福建醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R373
本文編號：2234315
[Abstract]:Human Immunodeficiency virus (HIV) is a kind of virus in the subgenus Lentiviridae of the Retrovirus family. According to the results of two national molecular epidemiological investigations, the recombinant strain of HIV CRF01_AE is spreading from the southern and coastal provinces to the surrounding provinces and cities, and the recombinant type of CRF01_AE accounts for a large proportion of the country. This study is the first to carry out the phenotypic analysis of HIV CRF01_AE epidemic strain in China, the characteristics and expression of the full-length membrane gene, the characteristics of the full-length gene and the construction of infectious clone, the etiology of the virus, mutation characteristics, vaccines, diagnostic methods and causes. The study of disease mechanism provides a solid foundation. This study is divided into five parts.
Isolation and biological phenotypes of HIV-1 CRF01_AE infected patients in Fujian
1. Four HIV-1 CRF01_AE strains were successfully isolated, Fj200604 induced typical syncytium (SI) in MT-2 cells, and the other three strains did not form obvious syncytium (NSI); Fj200601-Fj200603 used CCR5 coreceptor, Fj200604 used CXCR4 coreceptor.
2. The biological phenotype of the isolate is closely related to the variation of V3 loop sequence, which indicates that the use of HIV-1 coreceptor can be predicted by analyzing the key amino acids of V3 loop.
Two. Sequence analysis of full-length gp120 gene of HIV-1 CRF01_AE subtype strain in Fujian
1. Gene phylogenetic tree analysis showed that all the samples in this study belonged to the recombinant HIV-1 CRF01_AE, and the genetic variation was large. The intra-group gene distance was 9.5 + 2.5%.
2. There are four types of apical tetrapeptides in V3 ring: GPGQ (72.24%), GPGR (19.04%), GPGH (4.76%), GQGQ (4.76%). 16 (76.19%) samples may use CCR5 as a co-receptor, 1 (4.76%) samples may use CCR5/CXCR4 receptor, and 4 (19.04%) samples cannot predict.
3. the C1-C5 area is relatively conservative, while the V1-V5 ring is relatively large, and V3 is relatively conservative.
4. N-glycosylation site analysis showed that most of the sites were conservative, and a few of the glycosylation sites were lost and increased.
Expression of three.HIV membrane protein gp120 gene in Drosophila melanogaster S2 cells
1. The recombinant vector of gp120 gene and deleted V1/V2 loop was constructed and expressed successfully in Drosophila S2 cells.
2. Screening method of stable expression was established, and recombinant Drosophila S2 cells with stable expression of full-length gp120 gene and deletion of V1/V2 ring were screened.
Four. Cloning and sequence analysis of full-length gene of 13 HIV-1 CRF01_AE subtype strains isolated from Fujian Province
1. complete genome cloning of 13 HIV-1 CRF01_AE has been completed, and HIV-1 CRF01_AE whole genome amplification platform has been established.
2.13 complete genome cloning and sequencing were submitted to NCBI (GenBank.).
3. Phylogenetic tree analysis showed that all 13 full-length gene sequences were clustered with CRF01_AE reference strain, but could be divided into several subgroups and dispersed in Thai isolates. The cloned sequence was not recombined at gene level and was still sensitive to protease and reverse transcriptase.
Five. Construction of recombinant infectious clone of CRF01_AE in Fujian province and preliminary identification of its biological activity.
1. The results of 293T cell transfection showed that two full-length clones (full-length clone A and chimeric full-length clone A-B) showed good activity.
2. Full-length clone A and chimeric full-length clone A-B have the ability to infect PBMCs to a certain extent and replicate effectively in PBMCs, while the replication ability of chimeric full-length clone A-B in PBMCs is stronger than that of full-length clone A.
3. Chimeric full-length clone A-B used CCR5 as a coreceptor, while full-length clone A did not observe the use of coreceptors.
【學位授予單位】：福建醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R373
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