【作者】 孟祥平；

【导师】 孙晗笑；

【作者基本信息】 暨南大学 ， 生物医药工程， 2009， 博士

【摘要】 艾滋病是由人类免疫缺陷病毒感染所导致的以全身免疫系统的严重损害为特征的,以患者易于发生机会性感染以及肿瘤为特征的临床综合征。全球约95%以上的艾滋病感染是由HIV-1造成的。自1981年美国报道首例艾滋病患者以来,全球已有超过2500万人死于艾滋病。近年来,科学家希望从细胞内天然的抗HIV分子的方向上为解决艾滋病的问题提供重要的途径。已有的研究表明,HIV-1病毒可以感染包括人类在内的许多哺乳类细胞,但却不能感染某些猴。2004年,Sodroski研究小组对这一事实进行研究,在灵长类细胞中发现了一种可以抗HIV-1的蛋白质,研究证明属于三结构域蛋白（tripartite motif,TRIM）家族的一个成员,定义为TRIM5α。新近发现猿猴细胞中的TRIM5α具有抗HIV-1的作用。从而使TRIM5α的研究引起广泛关注。但到目前为止,人们对于TRIM5α限制HIV-1复制的作用机制尚不完全清楚,许多问题还没有解决,大多停留在推测的水平。比如,TRIM5α的不同类型对不同物种病毒感染的抑制偏向原因是什么?它是否直接与HIV-1的核衣壳蛋白结合?TRIM5α还可能与其他什么蛋白结合?虽然人的TRIM5α也具有同样功能,但不如恒河猴的有效。然而,当有个别氨基酸改变时,人源的TRIM5α将具有和恒河猴TRIM5α相似水平的抗HIV-1功能。早期的临床试验证明异种蛋白应用于人体后,可引起机体免疫系统对该异种蛋白的免疫排斥反应,重复使用时甚至可导致病人严重的过敏性休克。此外,由于人抗异种动物的免疫应答反应的存在,异种蛋白在人体内往往被快速清除,其半衰期也较短。因此本实验室选择人源的TRIM5α（改构体）进行研究。以往关于TRIM5α的绝大多数研究,主要围绕在基因水平上进行的,例如,通过脂质体、病毒载体等结构将TRIM5α基因转运进入细胞使之大量表达,从而改变细胞的表型。但在实际操作过程中,往往存在这样那样的问题,如可操作性差,转运效率低,稳定性不好,生物毒性作用,而且并非所有细胞均能被转染等,这些均限制了其实际应用。使得目前关于TRIM5α的研究只能局限在细胞的水平上,严重阻碍了这一重要基因向临床治疗中的发展。如果能够体外得到大量的有生物学活性的TRIM5α蛋白,而且该蛋白又能穿过细胞膜,在细胞内发挥其本来的生物学活性,那么TRIM5α的应用范围将大大拓展。近年来,人们发现了一种蛋白质功能域,它们能够引领其所承载的生物大分子通过浆膜,并在细胞内积累,所以把它们称为蛋白质转导域（protein transductiondomain,PTD）。被PTD带入细胞的分子都还具有它们本来的活性。PTD的出现,使大分子蛋白质或多肽自由通透细胞膜成为了可能,疾病的一个新治疗平台——蛋白质治疗技术也成为了现实,加速了蛋白质工程技术的发展和临床上蛋白类药物的开发和利用。目前,已有数十种蛋白质被成功转导进入不同的细胞中,并被表现其相应的生物活性,还有部分蛋白被转导进入动物体内。随着其作用机理的阐明和这一技术的日益成熟,将被越来越广泛地应用到疾病治疗方法的研究以及未知蛋白功能的研究中。但体外获得的重组蛋白PTD-TRIM5α能否顺利穿过细胞膜进入到细胞内,外源性TRIM5α蛋白是否具有抑制HIV-1的作用?这些问题都值得我们去研究。并且目前国内外还未见报道。Matthew Stremlau等的研究表明,突变的TRIM5αH（R328-332）[即I→M（328）、G→Q（330）、R→P（332）]基因用逆转录病毒载体转染细胞后,具有较好的抑制HIV-1的作用。考虑到逆转录病毒载体的生物毒性作用（如致白血病等肿瘤）,操作复杂,难于调控等诸多不足。本实验室构建了该改构体（嵌合体）PTD融合的原核表达载体,并对该蛋白的表达、纯化和功能进行了初步的研究。目的:克隆人TRIM5α基因并对其编码序列进行分析。构建TRIM5α改构体TRIM5αH（R328-332）的原核表达载体pET28α。并通过表达获得重组的TRIM5α及TRIM5αH（R328-332）蛋白。并探讨重组蛋白体外抑制HIV-1的作用。方法:利用RT-PCR方法克隆人TRIM5α基因;通过与基因组序列对比分析基因组结构;用BioEdit,Genedoc和MEGA 4.1进行蛋白序列联配和进化分析。构建PTD-TRIM5α及其嵌合体PTD-TRIM5αH（R328-332）的原核表达载体（pET-28a）。转化大肠杆菌BL21（DE3）,诱导表达获得重组融合蛋白。重组蛋白再经过纯化,用SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）和Western blot分析重组蛋白。用免疫共沉淀和ELISA等方法探讨重组蛋白结合HIV-1Gag的能力,利用荧光显微镜证实PTD-TRlM5α和PTD-TRIM5αH（R328-332）具有高效的穿膜能力,从而为进一步研究重组蛋白细胞内功能奠定了基础。将HIV-1_ⅢB和Molt 4靶细胞混合培养,然后与不同浓度的药物作用后,用合胞体形成抑制实验检测药物对HIV-1_ⅢB的直接灭活作用;用HIV-1p24抗原检测试剂盒检测药物作用后细胞培养上清的p24抗原水平变化,以测定其体外抑制HIV-1的作用。结果:扩增了一个长1482bp的基因片断,序列分析表明其覆盖了完整编码框,编码由493个氨基酸组成的人TRIM5α基因。人TRIM5a蛋白和黑猩猩、大猩猩、猩猩、猕猴（恒河猴）TRIM5α蛋白的相似度分别为98.2%、96.8%、93.7%和87.6%。采用PCR和测序方法证实目的基因已正确地插入pET28a载体。用Western blot证实含有目的基因的重组PTD-TRIM5α和PTD-TRIM5αH（R328-332）成功实现了表达,采用镍柱亲和层析得到了纯度约90%的目的蛋白。PTD-TRIM5α和PTD-TRIM5αH（R328-332）经稀释和透析等处理后实现了复性。体外实验证实PTD—TRIM5α及PTD—TRIM5αH（R328-332）蛋白具有和HIV-1Gag结合的能力,利用荧光显微镜证实PTD-TRIM5αH（R328-332）具有高效的穿膜能力。测定二种蛋白能够抑制HIV-1_ⅢB介导的细胞融合的EC50分别为81.47μg/mL、7.55μg/mL;抑制p24抗原产生的EC50分别为62.04μg/mL、4.74μg/mL。结论:成功克隆人TRIM5α基因,成功构建了PTD-TRIM5α/pET28a及PTD-TRIM5αH（R328-332）/pET28a的大肠杆菌表达系统,并且表达、分离纯化出具有生物活性的PTD-TRIM5α及PTD-TRIM5αH（R328-332）蛋白。PTD—TRIM5α及PTD—TRIM5αH（R328-332）蛋白具有高效的穿膜能力,从而为进一步研究重组蛋白细胞内功能奠定了基础。PTD-TRIM5αH（R328-332）具有更好的抑制HIV-1的活性,PTD-TRIM5αH（R328-332）抑制HIV-1_ⅢB的效率比PTD-TRIM5α高10倍以上。PTD—TRIM5α及PTD—TRIM5αH（R328-332）蛋白和HIV-1Gag结合的能力和其抑制HIV-1的活性相关。更多还原

【Abstract】 Aquired Immune Deficiency Syndrome（AIDS）,which is a collection of symptoms and infections resulting from the specific damage to the immune system in human,is caused by human immunodeficiency virus（HIV）.AIDS patients often have an increased risk of developing opportunistic infections and tumors.HIV-1 is the source of 95%HIV infections throughout the world.Since AIDS was first reported in the United States in 1981,there have been more than 2.5 million people died of AIDS throughout the world.In recent years,scientists hopes to solve the problem of AIDS in the direction of the natural anti-HIV molecules from the cells.Researches have shown that,HIV-1 virus can infect many mammals cells,including human cells,but not some monkey cells. Sodroski Group conduct a study on this fact,an anti-HIV-1 proteins was found in primate cells in 2004,TRIM5α,belonging to the tripartite motif（TRIM） protein family, is a cytoplasmic body protein with three domains.Recently a newly discovered function of TRIM5αisolated from Rhesus monkey is the potential restriction of HIV-1 and this exciting discovery has attracted widespread atention in the world.But so far,the mechanism of TRIM5αrestriction of HIV-1 replication is not yet entirely clear,many questions yet to be resolved,most of them stay at that level.What is the reason for bias of the different species of anti-virus of different types of TRIM5a?Although human’s TRIM5a also has the same function,but not the rhesus monkeys. However,when several amino acids change,the human TRIM5 alpha will have similar level of anti-HIV-1 as the TRIM5a rh.Early clinical trials prove that after heterologous protein used in the human body,the immune system can lead to the immune rejection of heterologous proteins,repeated use even can cause severe anaphylactic shock.In addition,because of the immune response of human anti- Heterogeneous animal. heterologous protein in the human body is often quick to be removed,and the half-life is shorter.Therefore our laboratory choose TRIM5α（mutant） come from Homo sapiens to research.But in the past,the majority of the researches about TRIM5αwere finished by gene transfection.For example,TRIM5αgene was conveyed into the cells by liposome or viral vector.But there are many problems during the operative procedure.Such as poor feasibility,low efficiency and stability,low transfection rate,biological toxicity.So at present,the researches about TRIM5αare executed only in cells.the application of the important gene-TRIM5αis restricted.If we can get quantity of active protein TRIM5αin vitro,meanwhile,if the protein can permeate plasma membrane spontaneously,the application range of TRIM5αwill be expanded greatly.In the recent years,people found a kind of protein functional domain,they can guide the biomacromolecule to permeate the plasma membrane and accumulate in the cells,so they were called protein transduction domain（PTD）.Those molecules carried into the cells hold their native activity.They can do their native work.So PTD is very charming because the character.It is possible for the macromolecule protein or polypeptide to permeate cytoplasmic membrane because of the PTD,meanwhile,another therapy platform that is to say treatment technic by protein becomes ture.PTD accelerates the protein engineering and the exploitation of protein medicine.At present, many kinds of protein have been carried into the different cells or the animals,they hold their native activity.PTD will be applicated to find new therapy method and new function of protein.But in vitro,does the fusion protein TRIM5αhave good biologic activity?Can the exogenous TRIM5a protein Antivirals against HIV-1 in vitro? These questions are very valuable for us,however,there is no report about that till to now.Matthew Stremlau’s research of had shown that mutated TRIM5 a H（R328-332）[I→M（328）,G→Q（330）,R→P（332）]transfected by by retroviral vectors had a better function of Antivirals against HIV-1 than TRIM5α.Our laboratory has constructed the prokaryotic expression vector with PTD and conducted a preliminary study on the expression,purification and function of the protein.Objective:Cloning,sequence analysis and constructing the molecular phylogenetic tree of human TRIM5αgene of innate immunity.To construct a prokaryotic expression vector carrying PTD-TRIM5αand TRIM5αH（R328-332） gene,To express them in E.coli.and to explore the function of Antivirals against HIV-1 in vitro of new recombinated proteins.Methods:RT-PCR was employed to clone the human TRIM5αgene.Genomic structure was analyzed by comparing it with human genomic sequence.BioEdit,Genedoc and MEGA4.1 were used for alignment and phylogenetic analysis.PTD-TRIM5α/ pET28a and TRIM5αH（R328-332）/pET28a were constructed and were cloned into the expression vector pET 28a（+）,transformed to E.coli BL21（DE3） strain,of PTD-TRIM5αand PTD- TRIM5αH（R328-332） proteins were induced and expressed,purified with Ni²⁺ chromatography.The expression and purification of PTD-TRIM5αand PTD-TRIM5αH（R328-332） were analyzed by SDS-PAGE and Western blot.Dilution and dislysis were used to renaturated the new recombinated proteinsThe interaction between PTD-TRIM5α/PTD-TRIM5αH（R328-332） and HIV-1Gag was detected by co-immunoprecipitation,His pull down and ELISA.Molt4 cell line was used to test the transmembrane capability of PTD-TRIM5α/PTD-TRIM5αH（R328-332） by fluorescence microscope.HIV-1_ⅢB mixed with target cell,then stained cells were treated with serially diluted drugs,anti-HIV-1 activity was oberserve by syncytial formation inhibition assay; viral duplication inhibition rateis was evaluated by measuring the levels of p24 antigen in culture supernatant by quantitative enzyme-linked immunosorbent assay（ELISA） kit.Result:A DNA fragment of 1482bp was cloned and sequencing analysis indicated that it carried an entire open reading frame and encoded a protein of 493 amino acid residues. The deduced amino acid sequence of human TRIM5αshowed similarities of 98.2%, 96.8%,93.7%,87.6%with those from Pan troglodytes,Gorilla gorilla,Pongo pygmaeus, Macaca mulatta,respectively.PTD-TRIM5/pET28a and PTD-TRIM5αH（R328-332）/pET28a were constructed and were cloned into the expression vector pET 28a （+）.The expression and purification of PTD-TRIM5αand PTD-TRIM5αH（R328- 332） were confirmed by SDS-PAGE and Western blot.The new recombinated proteins in inclusion body was renaturated by dilution and dislysis.The study demonstrates that the human PTD-TRIM5αand PTD-TRIM5αH（R328-332） interacts with HIV-1 Gag in vitro.High transmembrane effectiveness of PTD-TRIM5αand PTD-TRIM5αH（R328-332） on Molt4 cells were verified by fluorescence microscope The date demonstrated that PTD-TRIM5αand PTD-TRIM5αH（R328-332） can inhibit the formation of syncytia,and the EC50 of PTD-TRIM5αand PTD-TRIM5αH（R328-332） against the formation of syncytia of Molt4 induced by HIV-1_ⅢB were 81.47μg/mL,7.55μg/mL resperctively.The EC50 of PTD-TRIM5αand PTD-TRIM5αH（R328-332） against p24 were 62.04μg/mL,4.74μg/mL resperctively.Conclusions: The human TRIM5αgene was cloned successfully.E.Coli.expression system of PTD-TRIM5 a and PTD-TRIM5 a H（R328-332） was constructed successfully and bioacitive PTD-TRIM5 a and PTD-TRIM5 a H（R328-332） proteins were obtained by expressoin,separation and purification.High transmembrane effectiveness of PTD-TRIM5αand PTD-TRIM5αH（R328-332） on Molt4 cells were verified by fluorescence microscope which offering a initial feasibility of the designed proposal,and the study demonstrates that TRIM5α/TRIM5αH（R328-332） interacts with HIV-1 Gag,PTD-TRIM5αH（R328-332） has more than10 times higher anti- HIV-1_ⅢB activities than TRIM5α.in vitro.The anti- HIV-1_ⅢB activities of the PTD-TRIM5αand PTD-TRIM5αH（R328-332） are connected with virus capsid（Gag） binding of the two proteins.更多还原