两组四种DNA-蛋白质靶向治疗艾滋病的复合药物的研究
摘要
HIV病毒是艾滋病的根本原因。它在艾滋病人体内以两种方式存在：既存在于被感染的CD4+细胞中，又同时以游离形式存在于血液循环中。清除这两种方式的HIV病毒，将从根本上治愈艾滋病。历史上，曾有过CD4-PE40(绿脓杆菌毒素Ⅱ-Ⅲ区段)的基因工程设计，试图达到上述目的，但鉴于大肠杆菌表达的CD4蛋白及作为人体异源蛋白的PE40具有强烈的免疫原性而归于失败。因此第一代针对gp120的免疫毒素(immunotoxin)，CD4-PE40缺少应用前景，有必要加以改进。本课题采用全新的策略，力求既要发挥毒素基因的生物学功能，又要摒弃其免疫原性等副作用。具体的策略如下：以由HIV／SIV受体／辅助受体与DNA结合蛋白组成的融合蛋白作为非病毒载体，所形成的非病毒载体可以与对细胞有强烈杀伤作用的PEIIImut(绿脓毒素第三区段的突变基因片段)的表达型重组体形成DNA-protein复合物并将其导入受HIV感染的细胞，毒素基因的表达型重组体分为两种，一种以CMV为启动子的非限制性表达重组体；另一种是以HIV 5’LTR为启动子的限制性表达重组体，该启动子可以被感染细胞中的TAT蛋白活化，从而诱导PEIIImut在导入细胞中的表达，杀灭该被感染的细胞，当毒素基因被错误地导入未被感染的细胞中的时候，由于该细胞中没有TAT蛋白，不能诱导毒素基因的表达，因此该重组体的表达对正常细胞没有杀伤作用，第二种重组体是一种双开关设计，可以保证其药物的安全性；第二，作为非病毒载体的融合蛋白与毒素基因重组体形成的复合物可以中和失活血液中游离的HIV病毒，防治病毒的再次感染；第三，融合蛋白与毒素基因重组体形成的复合物可以和新产生的位于细胞膜上的gp120相结合从而阻止被感染细胞和正常细胞之间的融合。这一设计，既避免了毒素蛋白的免疫原性，也避免了对任何其它类型的正常细胞的杀伤作用。
根据文献报道，本课题选取HIV的受体CD4分子和辅助受体CCR5与CXCR4与gp120特异结合的主要结构域，即CD4的V1区(100个氨基酸，第1位-第100位)、CXCR4的第二个胞外环区(XE，27个氨基酸)与CCR5的N末端(RN，30个氨基酸)的组合作为靶向部分。同时，还从GenBank中钓取编码一种人DNA结合蛋白(SON)的序列，选取其中一段编码72个氨基酸的DNA序列(788-859位)，该序列富含32个碱性氨基酸，与前述的靶向部分组成一组新型的可以编码融合蛋白的基因，即XRS(由XE，RN，SON三个功能域组成)，CD4-RN-SON(由CD4V1，RN，SON三个功能域组成)，CD4-XE-RN-SON(由CD4V1，XE，RN，SON四个功能域组成)，CD4V1SON(由CD4V1，SON两个功能域组成)。该组融合蛋白的C末端部分是富含碱性氨基酸的SON的多肽，富有正电荷，可与富有负电荷的PEⅢmut表达型DNA重组体形成正-负电的结合。N末端部分将携带整个DNA-蛋白质复合物与受HIV感染的细胞表面的gp120结合，HIV／SIV的配体与非病毒载体上的gp120的受体结合，又鉴于细胞的内化功能，进而使整个复合物进入该细胞。PEⅢmut蛋白的表达可以杀灭受HIV感染的细胞。与此同时，融合蛋白的N末端部分CD4V1或XR还将结合在血循环中存在的游离的HIV病毒，使之因中和而失去感染力。
将上述目的基因克隆到温度控制型表达载体CW111中，获得一组表达型重组体，这组表达型重组体只有XRS、CD4V1SON获得了很好的表达。通过温度诱导，对XRS高表达株进行大规模培养，提取包涵体，经(?)KTA系统纯化，大量制备目的蛋白。通过凝胶成像扫描系统分析，结果显示XRS蛋白的表达量占整个菌体蛋白的13％，目的蛋白CD4V1SON占细胞总蛋白的35％。同时本研究对XRS蛋白进行了理化性质的分析，波谱扫描分析了XRS蛋白的光谱特征；通过质谱技术测定了XRS蛋白的分子量为15703.44 Dalton；氨基酸序列分析N端15个氨基酸为MNVSEADDRYIXDRF，与相应的DNA编码一致；通过高压液相测定的蛋白纯度为93.18％，通过毛细管电泳测定的该蛋白的等电点为10.99。
构建了表达突变的强毒绿脓杆菌毒素第三功能结构域的重组体(PEⅢmut)作为杀伤部分，毒素基因的表达型重组体分为两种，一种以CMV为启动子的非限制性表达重组体pCMV-PEⅢ，这种重组体在导向作用下主要进入受HIV／SIV感染的细胞，其表达可以杀伤这类细胞。但极少数这样的重组体可能进入未受感染的正常细胞并表达出毒素蛋白质而杀灭正常细胞，造成毒副作用。这是因为在HIV／SIV的繁殖过程中，将有少数gp120的脱落，其中的极少数将可能粘贴在正常细胞的表面，在这种情况下，pCMV-PEⅢ有可能被非病毒载体误导入正常细胞。但是这种情况属于极少数的例外，因此，其毒副作用将是轻微的；另一种是以HIV5’LTR为启动子的限制性表达重组体pYL-PEⅢ，对于被HIV感染的细胞，大量TAT蛋白的产生可以激活HIV的HIV 5’LTR中的启动子，从而启动PEⅢmut的表达，进而杀灭该受感染细胞。对于为受感染的细胞，因无tat蛋白而不能驱动5’LTR中的启动子，PEⅢmut不表达，则不杀伤正常细胞，从而避免因正常细胞被毒素基因的错误导入而引起的伤害。由于HIV 5’LTR启动的毒素基因表达较低，本研究在pYL-PEⅢ毒素基因前加入Intron序列构建构建成pDYP-1质粒，以增强毒素基因在细胞内的表达。
将纯化所得的目的蛋白CD4V1SON、XRS分别或单独与毒素基因的表达型重组体在体外进行结合，形成DNA-蛋白质复合物(DNA-Protein complex)，即CD4V1SON／pDYP-1、XRSON／pDYP-1，同时与由pCMV-PEⅢmut与CD4V1SON、XRSON形成的复合物即CD4V1SON／pCMV-PEⅢmut、XRSON／pCMV-PEⅢmut以及XRSON+CD4V1SON／pDYP-1、XRSON+CD4V1SON／pCMV-PEⅢmut进行了比较。鉴于一般而言药物对HIV与SIV(猴免疫缺陷病毒)等效性，细胞活性试验是以SIV为对象进行的。用上述复合物处理被SIV感染的CEMX-174细胞，观察上述不同剂量的(1、2、4μg，以DNA计)复合物在单独或联合(如CD4V1SON／pDYP-1与XRSON／pDYP-1、CD4V1SON／pCMV-PEⅢmut与XRSON／pCMV-PEⅢmut)应用时对被感染的CEMX-174细胞的杀伤作用，对照组为不加任何处理因素的被SIV感染的CEMX-174细胞。同时还观察了上述复合物在单独或联合使用时对SIV感染正常细胞时的中和作用，对照组为SIV加正常的CEMX-174细胞。另一对照为单独使用非病毒载体CD4V1SON，XRSON；单独使用DNA重组体／pCMV-PEⅢmut，pDYP-1。
结果显示：(1)杀伤试验：全部对照组样品对受
感染细胞均
无杀伤作用。以DNA计，所使用1、2、4μg DNA剂量的XRS／pDYP-1对CEMX-174／SIV细胞的杀伤率约为27.91％、37.21％、50.54％，病毒滴度分别为1：320、1：320、1：160；三种剂量的XRS／pCMV-PEⅢmut对CEMX-174／SIV细胞的杀伤率约分别为18.45％、39.54％、68.53％，病毒滴度为1：320、1：320、1：80；三种剂量的XRS+CD4V1SON／pCMV-PEⅢmut对感染细胞的杀伤率为：12.97％、24.36％、43.64％，病毒滴度分别为1：80、1：80、1：40；三种剂量的XRS+CD4V1SON／pDYP-1对感染细胞的伤率分别为18.67％、28.47％和49.95％，病毒滴度分别为1：80、1：80、1：40。上述各种组合都表现出一定的剂量依赖关系。对照组的滴度为1：1280。
(2)中和试验：2.3、4.6、9.2μg剂量的XRSON蛋白对SIV的保护作用为2.4％、10.33％、20.22％，病毒滴度分别为1：1280、1：1280、1：320；2.3、4.6、9.2μg剂量的XRSON+CD4V1SON蛋白对SIV的保护作用为-7.4％、4.8％、18.75％，病毒滴度分别为1：1280、1：320、1：80。1、2、4μgDNA剂量的XRSON／pDYP-1对SIV的保护作用为-4.3％、3.1％、10.33％，病毒滴度分别为1：1280、1：640、1：160。1、2、4μg DNA剂量的XRSON／pCMV-PEⅢmut对SIV的保护作用为0.5％、2.3％、7.3％，病毒滴度分别为1：320、1：80、1：20。1、2、4μg DNA剂量的XRSON+CD4V1SON／pDYP-1对SIV的保护作用为-6.4％、15.89％、22.5％，病毒滴度分别为1：1280、1：640、1：80。1、2、4μg DNA剂量的XRSON+CD4V1SON／pCMV-PEⅢmut保护作用为-2.3％、3.1％、21.94％，滴度分别为1：1280、1：640、1：80。上述各组药物与空白对照组相比，存活细胞数显著增多，即被SIV感染的细胞数显著减少。同一种药物对SIV的中和作用，具有剂量依赖关系。
细胞试验表明，本研究所设计的具有靶向性的融合蛋白可以有效地将毒素编码蛋白的基因的重组体导入被SIV感染的细胞中，并能表达出突变型毒素蛋白PEⅢmut。该毒素可以有效地杀伤被SIV感染的细胞。同时，本研究的数据还显示，靶向融合蛋白可以有效地中和游离状态的SIV，使被中和的病毒失去感染能力。需要强调的是中和作用和杀伤作用不是完全可分的，中和作用实际上既有靶向蛋白对病毒的中和作用，有些病毒仍然可以感染细胞。这些感染细胞仍然可以被蛋白-DNA复合物杀伤，但仍以中和作用为主。同样，在杀伤试验中，也有蛋白-DNA复合物对病毒的中和作用，但是以杀伤作用为主。
为了了解毒素基因重组体进入感染细胞的表达情况，需要一种直观而简便的观察方法。本课题利用重组技术将红色和绿色两种荧光蛋白基因替换毒素基因重组体中的毒素基因，构建了HIV 5’LTR启动的荧光蛋白基因重组体。其与靶向蛋白结合制成新的DNA-蛋白质复合物。希望通过观察荧光蛋白的表达情况来了解复合物中毒素基因表达。但是由于CEMX-174是一种悬浮细胞，这种细胞体积比较小，而且被HIV感染后细胞比较脆弱，因此这种转染难于观察。我们以荧光素酶基因代替荧光蛋白基因购见重组体并转染293细胞收到了很好的结果，其中贴壁细胞的转染结果明显好于悬浮细胞CEMX-174。表明启动子能够启动其下游的基因。同时我们将含有毒素基因的重组体通过脂质体转入到CEMX-174细胞中，流式细胞仪观察发现CMV启动的毒素基因的表达可以使被感染细胞凋亡，但是HIV5’LTR为启动子的组对细胞没有凋亡作用。这一结果说明，在CMV为启动子下，毒素基因得以表达，且强烈杀伤细胞。而HIV 5’LTR在受感染的细胞内不能启动毒素基因表达，因而毒素质粒对细胞无杀伤作用，以上两个试验表明毒素基因对细胞具有凋亡作用，但是HIV 5’LTR为启动子的毒素基因重组体只有在TAT蛋白的激活下才能发挥作用，在无TAT的作用下，即使5’HIV 5’LTR为启动子的毒素基因重组体由于误贴标签被错误地导入正常细胞，毒素基因也不表达，这样就可以实现双开关控制，保证药物的安全性。
本研究通过AKTA纯化的两种蛋白主要采用了反相柱和离子交换柱，反向柱纯化首先去掉了大量的变性剂，收集到的样品经冻干后很容易去掉有机溶剂乙睛。确定目的蛋白反向柱纯化在30％左右可以收集到目的峰，这样就可以尽量去除一些杂蛋白，有利于第二步阳离子交换柱纯化。通过280多次的小量摸索，尝试了多种离子柱、金属离子螯合柱、疏水柱以及分子筛等多种柱子，同时对各种缓冲系统和不同的pH值进行的探索，我们逐步探索出了一条纯化XRS，CD4V1SON蛋白的技术路线。收集的蛋白HPLC测定纯度达93.18％，基本达到了动物试验的要求。
由于动物试验需要大量的目的蛋白和毒素基因重组体，我们对目的蛋白和毒素基因重组体进行了大规模发酵和纯化，摸索出了蛋白和重组体大规模发酵和纯化的工艺以及各种影响因素。掌握了蛋白发酵和诱导表达所需要的条件，这些条件的获得为工业生产提供了重要的依据。毒素基因重组体的纯化主要采用了离子交换柱层析技术。探索出了一条快速、非有机的从细菌中提取高质量质粒DNA的方法。
目前动物试验的准备工作正在进行，这种药物在动物模型中的作用如何还需要作进一步的探索。但本研究为抗HIV／SIV提供了一种新途径。根据该设计原理，对编码靶向蛋白的基因加以变换，类似的方案还可以用于癌症、自身免疫性疾病等的治疗。
HIV is the unique pathogen of AIDS. There are two existing forms of HIV virus in AIDS patients: one existing in the infected CD4+ cells and another in the blood circulation as free HIV virus. AIDS patients can be cured if such two forms of HIV virus are cleared away. Historically, there was a engineered design regarding CD4-PE40 (Domain II-III of Pseudomonas aeruginosa, exotoxin) which was used to reach this aim [1-15]. But due to the very strong immunogenicity of CD4-PE40 protein resulted from engineered E.coli and PE40 as heteroprotein , this attempt leaded to failure. Yet as first-generation IT (immunotoxin) targeted to gp120, CD4-PE40 (chimeric immunotoxin using CD4 and enzymatic domains of Pseudomonas exotoxin A), showed limited promise in initial clinical testing, highlighting the need for improvement. [16]. The new strategy was utilized to both exert function of Pseudomonas exotoxin A and avoid side effect ,such as immunogenicity of heteroprotein . Detailed strategy was as follows: Fusion proteins composed of receptor and coreceptor and DNA-binding Protein were used as noviral carrier which can combine with PEIIImut expression recombinant to form protein-DNA complexes and transect PEIIImut DNA recombinant into cells infected by HIV. There are two kinds of toxin gene recombinant. One is unrestricted recombinant pCMV-PEIIImut which uses CMV as promotor. Another is restricted recombinant pYL-PEIIImut and pDYP-1 (Domain III mutant of Pseudomonas aeruginosa exotoxin) which uses HIV-5'LTR as promotor. PEIIImut can be activated by TAR protein which exists in cells infected by HIV virus because promotor of PEIIImut DNA recombinant is 5'LTR. PEIII mutant protein expression can kill the cells infected by HIV in a targeting way. When PEIIImut DNA recombinants were mis-transfected into normal cells, they can't be expressed due to TAT protein absence in normal cells. The safety can be ensured by dual-key control. In the meantime, the N-terminal part of the fusion protein will also combine the free HIV virus existing in the blood stream, making it lose infectivity because of neutralization. Thirdly, this complexes can prevent cell-cell fusion by binding to gp120 on the surface of infected cells. Such a design could avoid the immunogenicity and cell toxicity to kill any other kind of unifected cells.The reported key binding regions of CD4, CXCR4 and CCR5 used as receptor and coreceptor by HIV are V1 domain of CD4 containing 100 amino acid, the second extracellular loop of CXCR4 containing 27 amino acids and the N terminal region of CCR5 containing 30 amino acids as targeting protein. DNA-binding protein, SON spanning 72 amino acids which is rich in the positive amino acids, was chosen. The targeting protein and the DNA-binding protein were engineered to be fusion proteins, shch as XRSON(composed of XE-RN-SON), CD4Vl-RN-SON(composed of CD4V1-RN-SON), CD4V1-XE-RN-SON(composed of CD4V1-XE-RN-SON), CD4V1SON(composed of CD4V1-SON). N-terminal of Fusion proteins was the receptor or coreceptors of HIV, such as CD4, CXCR4 and CCR5 that can bind Env gp120 molecule on the surface of infected cells by HIV and the primary free HIV virus. C-terminal was DNA-binding peptide from SON gene which is rich in positive amino acids and binds PEⅢmut DNA recombinant via charge interaction. N-terminal can carry the whole DNA-protein complex into cells infected by HIV. PEⅢmut DNA recombinant plasmid can express PEⅢprotein which can kill the cells infected by HIV. In the meantime, receptor or coreceptors of HIV can bind the Env gp 120 of free HIV virus in the blood stream, making virus lose infectivity by neutralization.
Coding fusion genes were inserted into expression vector, pCW111 whose promoter is controlled by temperature. Only XRSON and CD4V1 SON were expressed well. After the induction and harvest of the engineered bacteria, the fusion proteins were isolated and purified by AKTA. The expression rate of fusion protein XRSON was 13％. The expression rate of CD4V1SON was 35％. Physiochemical property of XRS was identified. High-resolution MALD-TOF evaluation revealed that the molecular weight of XRS was 15703.44.15. Amino acid sequencing of N-terminal of XRSON was MNVSEADDRYIXDRE Capillary electrophoresis revealed that the Isolectric Point of XRS was 10.99. The constitutes of amino acid of XRS protein was the same as design. Purity of XRS revealed by HPLC was 93.18％.
Two kinds of recombinants (pCMV-PEⅢmut and pDYP-1,) expressing the mutant format of domainⅢof Pseudomonas aeruginosa exotoxin were used to kill cells infected by HIV/SIV. Their promoters are CMV promoter as unrestricted recombinant and HIV-1 5'LTR as restricted recombinant, respectively. The tat protein expression in infected cells by HIV can activate 5'LTR promoter, so that the toxin gene PEⅢmut will be especially expressed in cells infected, Though pDYP-1, recombinants was sent into in normal cells, PEⅢmut protein will not be expressed without the existence of tat protein. Cells will not be killed accordingly. To enhance tat expression level in infected cells, 5'LTR-Intron-PEⅢmut recombinant pDYP-1 were constructed by inserting IntronⅡsequence under stream of 5'LTR sequence. DNA-protein complexes as drugs were formed by mixing fusion protein XRS with recombinants (pCMV-PEⅢand pDYP-1, respectively).
DNA-protein complexes were formed, such as CD4V1SON/pDYP-1、XRSON/pDYP-1 which were compared with CD4V1SON/pCMV-PEⅢmut、XRSON/pCMV-PEⅢmut, XRSON+CD4V1SON/pDYP-1、XRSON+CD4V1SON /pCMV-PEⅢmut after toxin DNA recombinants, CD4V1 and XRS were purified. Cell activity experiment had been done with SIV in terms of the similar relationship of the HIV and SIV(simian immunodeficiency virus )in biological behavior. The killing effects of these complexes on the cells infected by SIV were observed respectively and combinationally after CEMX-174 cells was treated with varied doses of complexes(1, 2, 4μg, calculated in DNA). cells in the control group were uninfected by HIV. The neutralizing effects of the complexes on the free SIV were observed respectively and combinationally after CEMX-174 cells was treated with varied doses of complexes(1, 2, 4μg, calculated in DNA) for 48 hours. The normal ceils were mixed with SIV virus as contral. After 7 days, The neutralizing effects of the complexes were significant.
The results showed that the killing rates of the complexes in single and combined manner in different dosages(1, 2, 4μg, calculated in DNA) were as follows: the killing rates of XRS/pDYP-1 were 27.91％、37.21％、50.54％, respectively; the killing rate of control cells infected by SIV without complex was 0. Virus titers were 1: 320、1: 320、1: 160, respectively; the titer of control cells infected by SIV without complex was 1: 1280. XRS/pCMV-PEⅢmut were 12.97％、24.36％、43.64％, respectively; the killing rate of control was 0. Virus titers were 1: 320、1: 320、1: 80, respectively; the titer of the control was 1: 1280. XRS+CD4V1/pCMV-PEⅢmut were 12.97％、24.36％、43.64％, respectively; the killing rate of the control was 0. Virus titers were 1: 80、1: 80、1: 40 respectively; the titer of the control was 1: 1280. XRS+CD4V1/pDYP-1 were 18.67％、28.47％and 49.95％, respectively; the killing rate of the control was 0. Virus titers were 1: 80、1: 80、1: 40, respectively; the titer of the control was 1: 1280. The cytotoxicity is dependent on the concentration of complex.
On the other hand, the protecting effects of the complexes on the host cells in neutralization assay are as follows: the rates in XRSON (2.3, 4.6, 9.2ug) were 2.4％、10.33％、20.22％, respectively; the protecting rate of the control was zero. Virus titers were 1: 1280、1: 1280、1: 320, respectively; the titer of the control was 1: 1280. XRSON +CD4V1 were-7.4％、4.8％、18.75％, respectively; the protecting rate of the control was zero. Virus titers were 1: 1280、1: 320、1: 80, respectively; the titer of control was 1: 1280. XRSON/pDYP-1 (1, 2, 4μg, calculated in DNA) were-4.3％、3.1％、10.33％, respectively; the protecting rate of control was 0. Virus titers were 1: 1280、1: 640、1: 160, respectively; the titer of control was 1: 1280. XRSON/pCMV-PEⅢmut were 0.5％、2.3％、7.3％, respectively; the protecting rate of control was 0. Virus titers were 1: 320、1: 80、1: 20, respectively; the titer of control was 1: 1280. XRSON+CD4V1/pDYP-1 were-6.4％、15.89％、22.5％, respectively; the protecting rate of the control was 0. Virus titers were 1: 1280、1: 640、1: 80, respectively; the titer of control was 1: 1280. XRSON+CD4V1/pCMV-PEⅢmut were-2.3％、3.1％、21.94％, respectively; Protecting rate of the control was 0. Virus titers were 1: 1280、1: 640、1: 80, respectively; the titer of the control was 1: 1280. The protection is dependent on the concentration of drugs.
According to the above results, we concluded that the recombinants can effectively express the target toxin protein which could kill the cells infected by SIV after the fusion proteins effectively mediate the recombinants into the target cells. Moreover, the data showed that the fusion proteins could neutralize the free SIV and lead to the markedly decrease of the infectivity of SIV in different treatment groups. We must emphasize the fact that the killing effect of the complexes didn't absolutely separate from neutralizing effects of the complexes. Some cells can be infected by SIV virus in the neutralizing effect of the complexes. But the neutralizing effect of the complexes is significant. To the contrary, the neutralizing effect of the complexes involves in the killing effect. But the killing effect is significant.
To understand the expression of toxin gene in infected cells, two new recombinants was constructed by replacing the toxin gene with the red and green fluorescence protein gene in pCMV-PEⅢmut plasmid and pDYP-1 plasmid. The recombinant and the nonviral vector targeting protein were mixed to produce new complexs. As CEMX-174 cells is so small and tender a kind of suspended cells that the fluorescence protein is difficult to be deserved. One new recombinants was constructed by replacing the toxin gene with the luciferase gene in pCMV-PEⅢmut plasmid and was transfected into 293 cells and CEMX-174 cells. Transfection efficiency is enhanced significantly in 293 cells than in CEMX-174 cells. Those datas elucidated that toxin gene can be activated under the stream of CMV and LTR promoter. In the mean time, FACS showed that PEⅢmut can trigger apoptosis under the stream of CMV when injected into CEMX-174 cells through lipofectAmine TM Reagent. But PEⅢmut can't trigger apoptosis under the stream of LTR promoter when injected into CEMX-174 cells through lipofectAmine TM, resulting from the absence of TAR. When toxin gene recombinants were transfected into normal cells, the effect of killing was significant in the group of pCMV-PEⅢmut plasmid, pDYP-1 plasmid group did't have such effect. Such two Test above showed that the effect of toxin gene under the stream of LTR promoter should be activated by TAR protein. When PEⅢmut DNA recombinants were mis-transfected into normal cells, they can't be expressed due to TAT protein absence in normal cells. The safety can be ensured by dual-key control.
Reverse phase column and cation exchange chromatography were selected to purify two kinds of proteins. Proteins purified was natured by dialysis after freeze-dry. Interest protein was obtained at 30％B buffer during Reverse phase column purification. Other proteins were cleaned away. Probational columns involve Gel column, Ion column, affinity column and so on. Some buffers and pH range were used. We establish a successful process to purify XRS and CD4V1SON after 280 times tryout. HPLC mensuration showed that purify of obtained protein is 93.18％. As a result of a large needs of target protein and toxin gene recombinants, they were fermented and purified in large-scale. The process and factors of large-scale fermentation and production were obtained. Large-scale production in industry is possible on the basis of all techniques. Anion exchange chromatographys was selected to purify toxin gene recombinants. The process of rapid purification of toxin gene recombinants was obtained.
We are ready for animal test. Effects of protein-DNA-complexes in animal model are exploring further. Such kind of protocols used in this study is a novel strategy for AIDS therapy, and it is very likely to be extended for the efficient therapy of other diseases only if the targeting protein as the nonviral vector could targeted to other suitable receptor on the cell surface of cancer or autoimmune diseases.
根据文献报道，本课题选取HIV的受体CD4分子和辅助受体CCR5与CXCR4与gp120特异结合的主要结构域，即CD4的V1区(100个氨基酸，第1位-第100位)、CXCR4的第二个胞外环区(XE，27个氨基酸)与CCR5的N末端(RN，30个氨基酸)的组合作为靶向部分。同时，还从GenBank中钓取编码一种人DNA结合蛋白(SON)的序列，选取其中一段编码72个氨基酸的DNA序列(788-859位)，该序列富含32个碱性氨基酸，与前述的靶向部分组成一组新型的可以编码融合蛋白的基因，即XRS(由XE，RN，SON三个功能域组成)，CD4-RN-SON(由CD4V1，RN，SON三个功能域组成)，CD4-XE-RN-SON(由CD4V1，XE，RN，SON四个功能域组成)，CD4V1SON(由CD4V1，SON两个功能域组成)。该组融合蛋白的C末端部分是富含碱性氨基酸的SON的多肽，富有正电荷，可与富有负电荷的PEⅢmut表达型DNA重组体形成正-负电的结合。N末端部分将携带整个DNA-蛋白质复合物与受HIV感染的细胞表面的gp120结合，HIV／SIV的配体与非病毒载体上的gp120的受体结合，又鉴于细胞的内化功能，进而使整个复合物进入该细胞。PEⅢmut蛋白的表达可以杀灭受HIV感染的细胞。与此同时，融合蛋白的N末端部分CD4V1或XR还将结合在血循环中存在的游离的HIV病毒，使之因中和而失去感染力。
将上述目的基因克隆到温度控制型表达载体CW111中，获得一组表达型重组体，这组表达型重组体只有XRS、CD4V1SON获得了很好的表达。通过温度诱导，对XRS高表达株进行大规模培养，提取包涵体，经(?)KTA系统纯化，大量制备目的蛋白。通过凝胶成像扫描系统分析，结果显示XRS蛋白的表达量占整个菌体蛋白的13％，目的蛋白CD4V1SON占细胞总蛋白的35％。同时本研究对XRS蛋白进行了理化性质的分析，波谱扫描分析了XRS蛋白的光谱特征；通过质谱技术测定了XRS蛋白的分子量为15703.44 Dalton；氨基酸序列分析N端15个氨基酸为MNVSEADDRYIXDRF，与相应的DNA编码一致；通过高压液相测定的蛋白纯度为93.18％，通过毛细管电泳测定的该蛋白的等电点为10.99。
构建了表达突变的强毒绿脓杆菌毒素第三功能结构域的重组体(PEⅢmut)作为杀伤部分，毒素基因的表达型重组体分为两种，一种以CMV为启动子的非限制性表达重组体pCMV-PEⅢ，这种重组体在导向作用下主要进入受HIV／SIV感染的细胞，其表达可以杀伤这类细胞。但极少数这样的重组体可能进入未受感染的正常细胞并表达出毒素蛋白质而杀灭正常细胞，造成毒副作用。这是因为在HIV／SIV的繁殖过程中，将有少数gp120的脱落，其中的极少数将可能粘贴在正常细胞的表面，在这种情况下，pCMV-PEⅢ有可能被非病毒载体误导入正常细胞。但是这种情况属于极少数的例外，因此，其毒副作用将是轻微的；另一种是以HIV5’LTR为启动子的限制性表达重组体pYL-PEⅢ，对于被HIV感染的细胞，大量TAT蛋白的产生可以激活HIV的HIV 5’LTR中的启动子，从而启动PEⅢmut的表达，进而杀灭该受感染细胞。对于为受感染的细胞，因无tat蛋白而不能驱动5’LTR中的启动子，PEⅢmut不表达，则不杀伤正常细胞，从而避免因正常细胞被毒素基因的错误导入而引起的伤害。由于HIV 5’LTR启动的毒素基因表达较低，本研究在pYL-PEⅢ毒素基因前加入Intron序列构建构建成pDYP-1质粒，以增强毒素基因在细胞内的表达。
将纯化所得的目的蛋白CD4V1SON、XRS分别或单独与毒素基因的表达型重组体在体外进行结合，形成DNA-蛋白质复合物(DNA-Protein complex)，即CD4V1SON／pDYP-1、XRSON／pDYP-1，同时与由pCMV-PEⅢmut与CD4V1SON、XRSON形成的复合物即CD4V1SON／pCMV-PEⅢmut、XRSON／pCMV-PEⅢmut以及XRSON+CD4V1SON／pDYP-1、XRSON+CD4V1SON／pCMV-PEⅢmut进行了比较。鉴于一般而言药物对HIV与SIV(猴免疫缺陷病毒)等效性，细胞活性试验是以SIV为对象进行的。用上述复合物处理被SIV感染的CEMX-174细胞，观察上述不同剂量的(1、2、4μg，以DNA计)复合物在单独或联合(如CD4V1SON／pDYP-1与XRSON／pDYP-1、CD4V1SON／pCMV-PEⅢmut与XRSON／pCMV-PEⅢmut)应用时对被感染的CEMX-174细胞的杀伤作用，对照组为不加任何处理因素的被SIV感染的CEMX-174细胞。同时还观察了上述复合物在单独或联合使用时对SIV感染正常细胞时的中和作用，对照组为SIV加正常的CEMX-174细胞。另一对照为单独使用非病毒载体CD4V1SON，XRSON；单独使用DNA重组体／pCMV-PEⅢmut，pDYP-1。
结果显示：(1)杀伤试验：全部对照组样品对受
感染细胞均
无杀伤作用。以DNA计，所使用1、2、4μg DNA剂量的XRS／pDYP-1对CEMX-174／SIV细胞的杀伤率约为27.91％、37.21％、50.54％，病毒滴度分别为1：320、1：320、1：160；三种剂量的XRS／pCMV-PEⅢmut对CEMX-174／SIV细胞的杀伤率约分别为18.45％、39.54％、68.53％，病毒滴度为1：320、1：320、1：80；三种剂量的XRS+CD4V1SON／pCMV-PEⅢmut对感染细胞的杀伤率为：12.97％、24.36％、43.64％，病毒滴度分别为1：80、1：80、1：40；三种剂量的XRS+CD4V1SON／pDYP-1对感染细胞的伤率分别为18.67％、28.47％和49.95％，病毒滴度分别为1：80、1：80、1：40。上述各种组合都表现出一定的剂量依赖关系。对照组的滴度为1：1280。
(2)中和试验：2.3、4.6、9.2μg剂量的XRSON蛋白对SIV的保护作用为2.4％、10.33％、20.22％，病毒滴度分别为1：1280、1：1280、1：320；2.3、4.6、9.2μg剂量的XRSON+CD4V1SON蛋白对SIV的保护作用为-7.4％、4.8％、18.75％，病毒滴度分别为1：1280、1：320、1：80。1、2、4μgDNA剂量的XRSON／pDYP-1对SIV的保护作用为-4.3％、3.1％、10.33％，病毒滴度分别为1：1280、1：640、1：160。1、2、4μg DNA剂量的XRSON／pCMV-PEⅢmut对SIV的保护作用为0.5％、2.3％、7.3％，病毒滴度分别为1：320、1：80、1：20。1、2、4μg DNA剂量的XRSON+CD4V1SON／pDYP-1对SIV的保护作用为-6.4％、15.89％、22.5％，病毒滴度分别为1：1280、1：640、1：80。1、2、4μg DNA剂量的XRSON+CD4V1SON／pCMV-PEⅢmut保护作用为-2.3％、3.1％、21.94％，滴度分别为1：1280、1：640、1：80。上述各组药物与空白对照组相比，存活细胞数显著增多，即被SIV感染的细胞数显著减少。同一种药物对SIV的中和作用，具有剂量依赖关系。
细胞试验表明，本研究所设计的具有靶向性的融合蛋白可以有效地将毒素编码蛋白的基因的重组体导入被SIV感染的细胞中，并能表达出突变型毒素蛋白PEⅢmut。该毒素可以有效地杀伤被SIV感染的细胞。同时，本研究的数据还显示，靶向融合蛋白可以有效地中和游离状态的SIV，使被中和的病毒失去感染能力。需要强调的是中和作用和杀伤作用不是完全可分的，中和作用实际上既有靶向蛋白对病毒的中和作用，有些病毒仍然可以感染细胞。这些感染细胞仍然可以被蛋白-DNA复合物杀伤，但仍以中和作用为主。同样，在杀伤试验中，也有蛋白-DNA复合物对病毒的中和作用，但是以杀伤作用为主。
为了了解毒素基因重组体进入感染细胞的表达情况，需要一种直观而简便的观察方法。本课题利用重组技术将红色和绿色两种荧光蛋白基因替换毒素基因重组体中的毒素基因，构建了HIV 5’LTR启动的荧光蛋白基因重组体。其与靶向蛋白结合制成新的DNA-蛋白质复合物。希望通过观察荧光蛋白的表达情况来了解复合物中毒素基因表达。但是由于CEMX-174是一种悬浮细胞，这种细胞体积比较小，而且被HIV感染后细胞比较脆弱，因此这种转染难于观察。我们以荧光素酶基因代替荧光蛋白基因购见重组体并转染293细胞收到了很好的结果，其中贴壁细胞的转染结果明显好于悬浮细胞CEMX-174。表明启动子能够启动其下游的基因。同时我们将含有毒素基因的重组体通过脂质体转入到CEMX-174细胞中，流式细胞仪观察发现CMV启动的毒素基因的表达可以使被感染细胞凋亡，但是HIV5’LTR为启动子的组对细胞没有凋亡作用。这一结果说明，在CMV为启动子下，毒素基因得以表达，且强烈杀伤细胞。而HIV 5’LTR在受感染的细胞内不能启动毒素基因表达，因而毒素质粒对细胞无杀伤作用，以上两个试验表明毒素基因对细胞具有凋亡作用，但是HIV 5’LTR为启动子的毒素基因重组体只有在TAT蛋白的激活下才能发挥作用，在无TAT的作用下，即使5’HIV 5’LTR为启动子的毒素基因重组体由于误贴标签被错误地导入正常细胞，毒素基因也不表达，这样就可以实现双开关控制，保证药物的安全性。
本研究通过AKTA纯化的两种蛋白主要采用了反相柱和离子交换柱，反向柱纯化首先去掉了大量的变性剂，收集到的样品经冻干后很容易去掉有机溶剂乙睛。确定目的蛋白反向柱纯化在30％左右可以收集到目的峰，这样就可以尽量去除一些杂蛋白，有利于第二步阳离子交换柱纯化。通过280多次的小量摸索，尝试了多种离子柱、金属离子螯合柱、疏水柱以及分子筛等多种柱子，同时对各种缓冲系统和不同的pH值进行的探索，我们逐步探索出了一条纯化XRS，CD4V1SON蛋白的技术路线。收集的蛋白HPLC测定纯度达93.18％，基本达到了动物试验的要求。
由于动物试验需要大量的目的蛋白和毒素基因重组体，我们对目的蛋白和毒素基因重组体进行了大规模发酵和纯化，摸索出了蛋白和重组体大规模发酵和纯化的工艺以及各种影响因素。掌握了蛋白发酵和诱导表达所需要的条件，这些条件的获得为工业生产提供了重要的依据。毒素基因重组体的纯化主要采用了离子交换柱层析技术。探索出了一条快速、非有机的从细菌中提取高质量质粒DNA的方法。
目前动物试验的准备工作正在进行，这种药物在动物模型中的作用如何还需要作进一步的探索。但本研究为抗HIV／SIV提供了一种新途径。根据该设计原理，对编码靶向蛋白的基因加以变换，类似的方案还可以用于癌症、自身免疫性疾病等的治疗。
HIV is the unique pathogen of AIDS. There are two existing forms of HIV virus in AIDS patients: one existing in the infected CD4+ cells and another in the blood circulation as free HIV virus. AIDS patients can be cured if such two forms of HIV virus are cleared away. Historically, there was a engineered design regarding CD4-PE40 (Domain II-III of Pseudomonas aeruginosa, exotoxin) which was used to reach this aim [1-15]. But due to the very strong immunogenicity of CD4-PE40 protein resulted from engineered E.coli and PE40 as heteroprotein , this attempt leaded to failure. Yet as first-generation IT (immunotoxin) targeted to gp120, CD4-PE40 (chimeric immunotoxin using CD4 and enzymatic domains of Pseudomonas exotoxin A), showed limited promise in initial clinical testing, highlighting the need for improvement. [16]. The new strategy was utilized to both exert function of Pseudomonas exotoxin A and avoid side effect ,such as immunogenicity of heteroprotein . Detailed strategy was as follows: Fusion proteins composed of receptor and coreceptor and DNA-binding Protein were used as noviral carrier which can combine with PEIIImut expression recombinant to form protein-DNA complexes and transect PEIIImut DNA recombinant into cells infected by HIV. There are two kinds of toxin gene recombinant. One is unrestricted recombinant pCMV-PEIIImut which uses CMV as promotor. Another is restricted recombinant pYL-PEIIImut and pDYP-1 (Domain III mutant of Pseudomonas aeruginosa exotoxin) which uses HIV-5'LTR as promotor. PEIIImut can be activated by TAR protein which exists in cells infected by HIV virus because promotor of PEIIImut DNA recombinant is 5'LTR. PEIII mutant protein expression can kill the cells infected by HIV in a targeting way. When PEIIImut DNA recombinants were mis-transfected into normal cells, they can't be expressed due to TAT protein absence in normal cells. The safety can be ensured by dual-key control. In the meantime, the N-terminal part of the fusion protein will also combine the free HIV virus existing in the blood stream, making it lose infectivity because of neutralization. Thirdly, this complexes can prevent cell-cell fusion by binding to gp120 on the surface of infected cells. Such a design could avoid the immunogenicity and cell toxicity to kill any other kind of unifected cells.The reported key binding regions of CD4, CXCR4 and CCR5 used as receptor and coreceptor by HIV are V1 domain of CD4 containing 100 amino acid, the second extracellular loop of CXCR4 containing 27 amino acids and the N terminal region of CCR5 containing 30 amino acids as targeting protein. DNA-binding protein, SON spanning 72 amino acids which is rich in the positive amino acids, was chosen. The targeting protein and the DNA-binding protein were engineered to be fusion proteins, shch as XRSON(composed of XE-RN-SON), CD4Vl-RN-SON(composed of CD4V1-RN-SON), CD4V1-XE-RN-SON(composed of CD4V1-XE-RN-SON), CD4V1SON(composed of CD4V1-SON). N-terminal of Fusion proteins was the receptor or coreceptors of HIV, such as CD4, CXCR4 and CCR5 that can bind Env gp120 molecule on the surface of infected cells by HIV and the primary free HIV virus. C-terminal was DNA-binding peptide from SON gene which is rich in positive amino acids and binds PEⅢmut DNA recombinant via charge interaction. N-terminal can carry the whole DNA-protein complex into cells infected by HIV. PEⅢmut DNA recombinant plasmid can express PEⅢprotein which can kill the cells infected by HIV. In the meantime, receptor or coreceptors of HIV can bind the Env gp 120 of free HIV virus in the blood stream, making virus lose infectivity by neutralization.
Coding fusion genes were inserted into expression vector, pCW111 whose promoter is controlled by temperature. Only XRSON and CD4V1 SON were expressed well. After the induction and harvest of the engineered bacteria, the fusion proteins were isolated and purified by AKTA. The expression rate of fusion protein XRSON was 13％. The expression rate of CD4V1SON was 35％. Physiochemical property of XRS was identified. High-resolution MALD-TOF evaluation revealed that the molecular weight of XRS was 15703.44.15. Amino acid sequencing of N-terminal of XRSON was MNVSEADDRYIXDRE Capillary electrophoresis revealed that the Isolectric Point of XRS was 10.99. The constitutes of amino acid of XRS protein was the same as design. Purity of XRS revealed by HPLC was 93.18％.
Two kinds of recombinants (pCMV-PEⅢmut and pDYP-1,) expressing the mutant format of domainⅢof Pseudomonas aeruginosa exotoxin were used to kill cells infected by HIV/SIV. Their promoters are CMV promoter as unrestricted recombinant and HIV-1 5'LTR as restricted recombinant, respectively. The tat protein expression in infected cells by HIV can activate 5'LTR promoter, so that the toxin gene PEⅢmut will be especially expressed in cells infected, Though pDYP-1, recombinants was sent into in normal cells, PEⅢmut protein will not be expressed without the existence of tat protein. Cells will not be killed accordingly. To enhance tat expression level in infected cells, 5'LTR-Intron-PEⅢmut recombinant pDYP-1 were constructed by inserting IntronⅡsequence under stream of 5'LTR sequence. DNA-protein complexes as drugs were formed by mixing fusion protein XRS with recombinants (pCMV-PEⅢand pDYP-1, respectively).
DNA-protein complexes were formed, such as CD4V1SON/pDYP-1、XRSON/pDYP-1 which were compared with CD4V1SON/pCMV-PEⅢmut、XRSON/pCMV-PEⅢmut, XRSON+CD4V1SON/pDYP-1、XRSON+CD4V1SON /pCMV-PEⅢmut after toxin DNA recombinants, CD4V1 and XRS were purified. Cell activity experiment had been done with SIV in terms of the similar relationship of the HIV and SIV(simian immunodeficiency virus )in biological behavior. The killing effects of these complexes on the cells infected by SIV were observed respectively and combinationally after CEMX-174 cells was treated with varied doses of complexes(1, 2, 4μg, calculated in DNA). cells in the control group were uninfected by HIV. The neutralizing effects of the complexes on the free SIV were observed respectively and combinationally after CEMX-174 cells was treated with varied doses of complexes(1, 2, 4μg, calculated in DNA) for 48 hours. The normal ceils were mixed with SIV virus as contral. After 7 days, The neutralizing effects of the complexes were significant.
The results showed that the killing rates of the complexes in single and combined manner in different dosages(1, 2, 4μg, calculated in DNA) were as follows: the killing rates of XRS/pDYP-1 were 27.91％、37.21％、50.54％, respectively; the killing rate of control cells infected by SIV without complex was 0. Virus titers were 1: 320、1: 320、1: 160, respectively; the titer of control cells infected by SIV without complex was 1: 1280. XRS/pCMV-PEⅢmut were 12.97％、24.36％、43.64％, respectively; the killing rate of control was 0. Virus titers were 1: 320、1: 320、1: 80, respectively; the titer of the control was 1: 1280. XRS+CD4V1/pCMV-PEⅢmut were 12.97％、24.36％、43.64％, respectively; the killing rate of the control was 0. Virus titers were 1: 80、1: 80、1: 40 respectively; the titer of the control was 1: 1280. XRS+CD4V1/pDYP-1 were 18.67％、28.47％and 49.95％, respectively; the killing rate of the control was 0. Virus titers were 1: 80、1: 80、1: 40, respectively; the titer of the control was 1: 1280. The cytotoxicity is dependent on the concentration of complex.
On the other hand, the protecting effects of the complexes on the host cells in neutralization assay are as follows: the rates in XRSON (2.3, 4.6, 9.2ug) were 2.4％、10.33％、20.22％, respectively; the protecting rate of the control was zero. Virus titers were 1: 1280、1: 1280、1: 320, respectively; the titer of the control was 1: 1280. XRSON +CD4V1 were-7.4％、4.8％、18.75％, respectively; the protecting rate of the control was zero. Virus titers were 1: 1280、1: 320、1: 80, respectively; the titer of control was 1: 1280. XRSON/pDYP-1 (1, 2, 4μg, calculated in DNA) were-4.3％、3.1％、10.33％, respectively; the protecting rate of control was 0. Virus titers were 1: 1280、1: 640、1: 160, respectively; the titer of control was 1: 1280. XRSON/pCMV-PEⅢmut were 0.5％、2.3％、7.3％, respectively; the protecting rate of control was 0. Virus titers were 1: 320、1: 80、1: 20, respectively; the titer of control was 1: 1280. XRSON+CD4V1/pDYP-1 were-6.4％、15.89％、22.5％, respectively; the protecting rate of the control was 0. Virus titers were 1: 1280、1: 640、1: 80, respectively; the titer of control was 1: 1280. XRSON+CD4V1/pCMV-PEⅢmut were-2.3％、3.1％、21.94％, respectively; Protecting rate of the control was 0. Virus titers were 1: 1280、1: 640、1: 80, respectively; the titer of the control was 1: 1280. The protection is dependent on the concentration of drugs.
According to the above results, we concluded that the recombinants can effectively express the target toxin protein which could kill the cells infected by SIV after the fusion proteins effectively mediate the recombinants into the target cells. Moreover, the data showed that the fusion proteins could neutralize the free SIV and lead to the markedly decrease of the infectivity of SIV in different treatment groups. We must emphasize the fact that the killing effect of the complexes didn't absolutely separate from neutralizing effects of the complexes. Some cells can be infected by SIV virus in the neutralizing effect of the complexes. But the neutralizing effect of the complexes is significant. To the contrary, the neutralizing effect of the complexes involves in the killing effect. But the killing effect is significant.
To understand the expression of toxin gene in infected cells, two new recombinants was constructed by replacing the toxin gene with the red and green fluorescence protein gene in pCMV-PEⅢmut plasmid and pDYP-1 plasmid. The recombinant and the nonviral vector targeting protein were mixed to produce new complexs. As CEMX-174 cells is so small and tender a kind of suspended cells that the fluorescence protein is difficult to be deserved. One new recombinants was constructed by replacing the toxin gene with the luciferase gene in pCMV-PEⅢmut plasmid and was transfected into 293 cells and CEMX-174 cells. Transfection efficiency is enhanced significantly in 293 cells than in CEMX-174 cells. Those datas elucidated that toxin gene can be activated under the stream of CMV and LTR promoter. In the mean time, FACS showed that PEⅢmut can trigger apoptosis under the stream of CMV when injected into CEMX-174 cells through lipofectAmine TM Reagent. But PEⅢmut can't trigger apoptosis under the stream of LTR promoter when injected into CEMX-174 cells through lipofectAmine TM, resulting from the absence of TAR. When toxin gene recombinants were transfected into normal cells, the effect of killing was significant in the group of pCMV-PEⅢmut plasmid, pDYP-1 plasmid group did't have such effect. Such two Test above showed that the effect of toxin gene under the stream of LTR promoter should be activated by TAR protein. When PEⅢmut DNA recombinants were mis-transfected into normal cells, they can't be expressed due to TAT protein absence in normal cells. The safety can be ensured by dual-key control.
Reverse phase column and cation exchange chromatography were selected to purify two kinds of proteins. Proteins purified was natured by dialysis after freeze-dry. Interest protein was obtained at 30％B buffer during Reverse phase column purification. Other proteins were cleaned away. Probational columns involve Gel column, Ion column, affinity column and so on. Some buffers and pH range were used. We establish a successful process to purify XRS and CD4V1SON after 280 times tryout. HPLC mensuration showed that purify of obtained protein is 93.18％. As a result of a large needs of target protein and toxin gene recombinants, they were fermented and purified in large-scale. The process and factors of large-scale fermentation and production were obtained. Large-scale production in industry is possible on the basis of all techniques. Anion exchange chromatographys was selected to purify toxin gene recombinants. The process of rapid purification of toxin gene recombinants was obtained.
We are ready for animal test. Effects of protein-DNA-complexes in animal model are exploring further. Such kind of protocols used in this study is a novel strategy for AIDS therapy, and it is very likely to be extended for the efficient therapy of other diseases only if the targeting protein as the nonviral vector could targeted to other suitable receptor on the cell surface of cancer or autoimmune diseases.
引文
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