福建省HIV-1CRF01_AE流行株的表型分析、基因序列特征、膜蛋白表达及感染性克隆构建的研究
发布时间：2018-09-10 11:33
【摘要】： 人类免疫缺陷病毒(Human Immunodeficiency virus,HIV),是逆转录病毒科慢病毒属灵长类慢病毒亚属中的一类病毒。据全国两次分子流行病学调查结果表明HIV CRF01_AE重组型株正在从南部及沿海省市向周围省市扩散,CRF01_AE重组型在全国所占比例也有所增加,在福建省HIV CRF01_AE重组型为主要的流行株。本研究首次在国内开展了HIV CRF01_AE流行株的表型分析、全长膜基因的特征和表达、全长基因的特征和感染性克隆构建的研究,为本型病毒的病原学、变异特征、疫苗、诊断方法和致病机理的研究提供了坚实的基础,本研究分为五个部分。 一.福建省HIV-1 CRF01_AE感染者的病毒分离及其生物学表型初步研究 1.成功分离4份HIV-1 CRF01_AE感染者的毒株,Fj200604在MT-2细胞诱导了典型的合胞体(SI),另外3个毒株没有形成明显的合胞体(NSI);分离株Fj200601-Fj200603利用CCR5辅助受体,Fj200604利用CXCR4辅助受体。 2.分离株的生物学表型与V3环序列变异密切相关,表明通过对V3环关键氨基酸的分析可以预测HIV-1辅助受体的使用情况。 二.福建省HIV-1 CRF01_AE亚型毒株全长gp120基因序列特征分析 1.基因系统树分析显示本研究所有的样本属于HIV-1 CRF01_AE重组型,遗传变异较大,组内基因距离为9.5±2.5 %。 2. V3环顶端四肽存在四种类型:GPGQ(72.24 %),GPGR(19.04 %),GPGH(4.76 %),GQGQ(4.76 %)。16份(76.19 %)样本可能使用CCR5作为辅助受体,1份(4.76 %)样本可能使用CCR5/CXCR4受体,4份样本(19.04 %)不能做出预测。 3. C1-C5区比较保守,而V1-V5环变化较大,其中V3相对保守。 4. N糖基化位点分析发现大多数位点较为保守,少数糖基化位点发生丢失和增加。 三.HIV膜蛋白gp120基因在果蝇S2细胞中的表达 1.构建了全长gp120基因和缺失V1/V2环的表达重组载体,在果蝇S2细胞中瞬时表达成功。 2.建立了稳定表达的筛选方法,筛选了稳定分泌表达全长gp120基因和缺失V1/V2环的重组果蝇S2细胞。 四.福建省13条HIV-1 CRF01_AE亚型毒株全长基因克隆及其序列特征分析 1.完成了13株HIV-1 CRF01_AE全基因组克隆,建立了HIV-1 CRF01_AE全基因组扩增平台。 2. 13条全基因组克隆测序提交至NCBI(美国国家生物信息数据库)的GenBank。 3.系统进化树分析表明所有13条全长基因序列与CRF01_AE参考株聚在一起,但可分成几个亚组分散在泰国分离株中。13条全长基因序列(去除超突变序列Fj061)env,gag,pol三个结构基因区的组内基因离散率均高于泰国分离株;全长基因序列Fj061证实为超突变序列。所克隆序列未出现基因水平的重组,对蛋白酶和逆转录酶的药物依然敏感。 五.福建省CRF01_AE重组型感染性克隆的构建和生物学活性初步鉴定 1.完成3个全长克隆的连接,293T细胞转染实验表明2个全长克隆(全长克隆A和嵌合全长克隆A-B)显示较好的活性。 2.全长克隆A和嵌合全长克隆A-B具有一定程度的感染PBMCs的能力和在PBMCs内进行有效复制的能力,而嵌合全长克隆A-B在PBMCs中的复制能力比全长克隆A更强些。 3.嵌合全长克隆A-B应用CCR5作为辅助受体,而全长克隆A未观察到细胞对辅助受体的利用情况。
[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.
【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R373
本文编号：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.
【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R373
【参考文献】
相关期刊论文 前10条
1 张东威,金宁一,王宏伟,张应玖,王莉馨,罗坤,李萍,于洪臣,殷震;HIV-1 gag-gp120嵌合基因重组杆状病毒载体的构建[J];白求恩医科大学学报;2001年02期
2 王秀清,金宁一,田梅,毛春生,郭军庆,顾万钧,佟明华,殷震;HIV-2env(gp120)基因在大肠杆菌中的表达[J];白求恩医科大学学报;1997年06期
3 王莉馨,金宁一,罗坤,秦云龙,王宏伟,郭志儒,殷震;HIV-1中国流行株gp120基因在痘苗病毒中的表达及其与核酸疫苗的实验免疫研究[J];高技术通讯;2001年02期
4 严延生,陈舸,郑兆双,翁育伟,郑健,颜苹苹,王惠榕,陈亮;福建省艾滋病流行态势及影响因素的分析[J];海峡预防医学杂志;2000年04期
5 王惠榕,严延生,陈亮,陈舸;HIV-1分离株FJ113生物学特征研究[J];海峡预防医学杂志;2002年06期
6 江文正,金宁一,李子健,张立树,韩文瑜;共表达HIV-1中国流行株gp120与IL-18重组鸡痘病毒的构建及其免疫原性观察[J];生物工程学报;2004年03期
7 金宁一,方厚华,郭志儒,罗坤,古长庆,殷震,邵一鸣,王虹;HIV-1 env基因在重组鸡痘病毒中的表达[J];中国生物制品学杂志;1999年04期
8 江文正,金宁一,李子健,金洪涛,王宏,韩文瑜;HIV-1表面糖蛋白gp120 DNA疫苗质粒的构建与表达[J];中国生物制品学杂志;2002年05期
9 江文正,金宁一,金洪涛;中国株HIV-1融合基因gag-gp120在大肠杆菌中的表达[J];中国生物制品学杂志;2002年06期
10 张应玖,金宁一,王宏伟,张东威,邵一鸣,殷霞,沈家骢;HIV-1-gp120在重组杆状病毒系统中的表达[J];微生物学杂志;2000年03期
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