【作者】 冯莹柱；

【导师】 王伯初； Heng Zhu；

【作者基本信息】 重庆大学 ， 生物医学工程， 2013， 博士

【摘要】 G蛋白偶联受体（G protein-coupled receptors, GPCR）作为最大的人膜蛋白受体家族，在细胞存活、维持细胞平衡、免疫监测、分子转运及神经调节等方面具有重要作用。因此目前全球市场70%以上的治疗药物都是针对GPCR,因此日益成为生物医药研究的重点、热点和难点。但是GPCR高度疏水的7次跨膜结构，灵活的构象仍然给体外研究其功能活性造成了巨大的困难，因此构建一种筛选GPCR功能活性的平台无疑对药物高通量筛选具有重要意义。目前新药筛选方法主要是在药物与某一种细胞GPCR相互作用后，借助荧光共振能量转移（fluorescence resonance energy transfer, FRET）等生物物理方法，检测细胞内分子间相互作用激发的化学荧光信号，从而发现具有特定疗效的药物。虽然这种在细胞水平的方法稳定、适于自动化，但是仍然存在无效信号或信号偏差导致筛选过程复杂化，成本高、集成度小、筛选效率低。因为病毒包膜上有许多规则排列的糖蛋白，如HSV-1的主要包膜糖蛋白gB（glycoprotein B, gB）、gC （glycoprotein C, gC）,所以本研究中提出采用HSV-1病毒为载体，将具有代表性的人膜蛋白CD4（单次跨膜蛋白的模式蛋白）、GPCR77（多次跨膜蛋白的模式蛋白）像gB、gC那样整合在球形的HSV-1包膜上，然后把病毒加载到芯片上。这种芯片将锚定各种GPCR，有望在一次芯片反应中筛选出具有活性的GPCR，为药物的高通量筛选搭建新的平台。本论文研究主要内容及结果如下：[1]制备gB:CD4、gB:GPCR77、gC:CD4、gC:GPCR77四种HSV-1重组病毒因为HSV-1是双链DNA，本文通过两种基因重组方法对HSV-1基因组编辑，表达外源天然膜蛋白或融合蛋白,通过病毒在细胞内的组装，制备gB:CD4、gB:GPCR77、gC:CD4、gC:GPCR77四种HSV-1重组病毒。gB:CD4、gB:GPCR77上的人膜蛋白CD4、GPCR77由HSV-1gB基因启动子表达，gC:CD4、gC:GPCR77上的CD4、GPCR77是CD4-gC和GPCR-gC的融合基因,由HSV-1gC基因启动子表达。该融合基因由CD4基因（去掉CD4跨膜区）、GPCR77基因（去掉最后一个跨膜区）分别与gC基因跨膜区和C-末端序列（32个氨基酸基因序列）融合而成。i.采用HSV-1病毒系统。在A.1.1细胞内，将克隆在PK△4B△gBss质粒上的CD4基因、GPCR77基因（包括基因全长编码序列、末端加进的14个氨基酸V5epitope tag序列及终止密码子TAG）分别重组到序列部分缺失的HSV-1基因组中gB基因的启动子下，制备人膜蛋白HSV-1重组病毒gB: CD4、gB:GPCR77。试验结果：a.通过免疫荧光试验（immunofluorescence assay, IFA），应用CD4、GPCR77荧光抗体对gB:CD4、gB: GPCR77感染的细胞表面染色，发现具有很好的荧光信号。gD荧光抗体在所有感染细胞表面都有荧光信号（阳性对照），但是野生型HSV-1（KOS）感染的细胞表面（负对照①）和没有病毒感染的细胞表面（负对照②）都没有检测到荧光信号。V₅荧光抗体对感染细胞内染色，发现都呈现了强的荧光信号，说明CD4、GPCR77在细胞内获得了正确表达,并且在内质网、高尔基体、细胞表面有翻译后修饰的动态处理过程。b.通过细胞免疫印迹试验（western blot, WB）和³⁵S-methionine放射性活性标记的免疫沉淀试验（immunoprecipitation, IP）分析，在胶图gD条带大小相近，确保各种感染病毒数量一致的条件下，gB:CD4、gB:GPCR77感染的HFT细胞出现了清晰的CD4、GPCR77条带,而KOS病毒（HSV-1野生型，负对照①）和K082病毒（HSV-1gB基因缺失,负对照②）感染的HFT细胞则没有相应分子量条带的出现，与IFA试验结果相吻合。c.通过流式细胞仪（flow cytometry, FCM），PE-conjugated的荧光抗体在HSV-1病毒表面成功检测到了CD4、GPCR77的荧光信号。结论：CD4、GPCR77分别整合到了gB:CD4、gB:GPCR77包膜，且在膜上整合的方向是正确的。ii.采用RED/ET Ecoli的细菌重组系统。通过两步将CD4-gC、GPCR77-gC融合基因同源重组进HSV-1基因组中，制备gC:CD4、gC:GPCR77重组病毒。结论：WB、IP、FCM生化试验证明，这种通过基因融合的方法把融合蛋白CD4-gC、 GPCR77-gC也整合到了HSV-1包膜，并且荧光信号比gB: CD4、 gB:GPCR77更强。[2]构建病毒芯片取4种不同芯片表面，设计不同病毒浓度梯度（2倍稀释）和对照组，然后将野生型HSV-1加载在芯片上，发现FAST玻片具有最好的检测水平。由于gD荧光抗体信号强弱对病毒浓度梯度具有依赖性，可以确定最佳的芯片病毒加载量为800,000个/spot。试验结果:通过Cy5标记的anti-CD4、anti-GPCR77抗体检测芯片上的信号检测，发现gB:CD4、gB:GPCR77、gC:CD4、gC: GPCR77四种重组病毒荧光信号显著，但是KOS（HSV-1野生型，负对照①）和K082（HSV-1gB基因缺失型,负对照②）则没有信号，再一次证明CD4、GPCR77整合到了相应的重组病毒上，但在芯片上发现Cy5-标记的C5a配体仅能够特异的识别gC:GPCR77，却很难识别gB: GPCR77。结论：CD4、GPCR77分别整合到了HSV-1包膜，但是GPCR77只在gC: GPCR77上保持完好的构象和活性，这为构建完整的GPCRs药物筛选芯片奠定了坚实的基础。病毒芯片的成功构建将对生物医药领域产生重大影响，将为活性药物筛选、分子配体的鉴定、以及膜蛋白结构功能研究建立一个很好的平台。更多还原

【Abstract】 G protein-coupled receptors （GPCRs）, as the largest membrane protein receptorfamily and drug targets, play an important role in cell survive, maintaining cell balance,immune surveillance, molecular transhport and neural regulation. More than70%ofdrugs in the global market are for GPCR which are increasingly becoming the focus, hotand key point of biomedical research. However, seven transmembrane GPCR with highdegree of hydrophobicity and conformational flexibility caused enormous difficulties tostudy its function in vitro activity. Constraction of a platform for a screening GPCRfunctional activity is undoubtfully of great significance to high-throughput drugscreening.Current drug screening method is mainly built on a cellular level, which by meansof fluorescence resonance energy transfer biophysical techniques, through theinteraction of drugs with GPCR, detected the intracellular molecular interaction of theexcited chemical fluorescence signal, then screen out the drug with specific effect.Although this method is easy, stable, and suitable for automation, there are still havedefects that the invalid and bias signal will cause the filtering process complicated,expensive, low integration, and inefficiency for drug screening.Because virus membrane have many regularly arranged glycoproteins, such asHSV-1major envelope glycoprotein gB （glycoproteinB, gB）, gC （glycoprotein C, gC）,This rerearch proposed to use HSV-1as vector, integrate represented human membraneprotein CD4（single pass transmembrane protein） and GPCR77（multipletransmembrane protein） as similar form of gB or gC into the spherical shape of theHSV-1envelope. This virion chip will anchor various GPCRs, which is expected toscreen out functional GPCR in one chip reaction. It will provide a platform for highthroughput drug screening. The main research content and results of the thesis are asfollows:[1] Preparation of gB:CD4, gB:GPCR77, gC:CD4, gC:GPCR77,4kinds ofrecombinant HSV-1virionBecasue HSV-1DNA is double-strand, this research edite its genome by twogenetic recombinant methods, which express the exogenous natural membrane proteinor the chimeric protein for preparation of gB: CD4, gB: GPCR77, gC: CD4, gC:GPCR77,4kinds of recombinant HSV-1. CD4of gB: CD4and GPCR77of gB: GPCR77are expressed under HSV-1gB gene promoter, chimeric gene CD4-gC of gC:CD4and GPCR-gC of gC: GPCR77are expressed under HSV-1gC gene promoter.i. Adopt the HSV-1virus system. CD4gene and GPCR77gene were cloned intoPK△4B△gBss plasmid respectively （includes full length gene coding sequence, theend of the added V5tag sequence of14amino acids and the stop codon TAG）, thenhomolgously recombined into partial sequence deleted HSV-1genome, which is undergB gene promoter. It is for the preparation of the recombinant HSV-1, gB:CD4andgB:GPCR77in A.1.1cells. Experimental results: Through the immunofluorescenceassay （IFA） which used CD4, GPCR77fluorescent antibody for the infected cell surfacestaining, we found that it had a good fluorescence signal, namely, gD fluorescentantibody had good signal （positive control） in all infected cell surface. But the wild-type HSV-1（KOS） infected cells surface （negative control①） and not virus-infectedcell surface （negative control②） hadn’t detected any fluorescent signal. We used V5fluorescent antibody for intracellular staining. They had shown strong fluorescencesignal, which indicated that either CD4or GPCR77can correctly expressed in infectedcell and there was dynamic process of post-translational modification in theendoplasmic reticulum, Golgi, and the cell surface. Further detection by cellimmunoblot （western blot, WB） and³⁵S-methionine radioactivity labeled immuneprecipitation, which used the similar bands of gD antibody on the gel to ensure the sameamount of various infected recombinant virus, antibody detection confirmed that CD4and GPCR77were highly expressed in human foreskin fibroblast cell （HFT）, whileKOS （wild type HSV-1, negative control①） and K082（gB gene minus, negativecontrol②） in infected cells did not detect the corresponding expression. The results ofthese assays were consisted with the IFA test. Flow cytometry applying PE-conjugatedfluorescent antibody had successfully detected CD4, GPCR77fluorescent signal on thegB: CD4and gB: GPCR77envelope. It is indicated that the orientation of them wascorrect.ii. Adopt RED/ET Ecoli recombination system. The chimeric gene CD4-gC andGPCR77-gC were homologously recombined into HSV-1genome by two stepsrespectively. Similar biochemical detection assays proved that, by means of genefusion, chimera CD4-gC and GPCR77-gC protein were integrated into HSV-1envelopeand the fluorescent signal was even stronger than gB:CD4and gB:GPCR77. [2] Construction of virion chipDesign different virus concentration gradient （2fold dilution） and the controlgroup on the4different chips’ surface, we found that FAST slide had the best detectionlevel. gD anti-antibody can capture all the recombinant virions on the chip. Becasue theintensity of the fluorescent signal is dependent on the virus concentration, this reseachcould obtain the optimal virus number anchored on the chip （800,000virions/sopt）.Experimental results: Cy-5labeling anti-CD4and anti-GPCR77antibody detect virionchip,4recombinant viruses of gB: CD4, gB: GPCR77, gC: CD4, gC: GPCR77werefound to have significant fluorescent signal, but KOS （negative control①） and K082（negative control②） had no fluorescent signal. It is demonstrated again that CD4andGPCR77were successfully integrated into the envelope of corresponding virus. Thisresearch further found that via the chip, Cy5-labeled functional ligand C5a canspecifically recoganize gC: GPCR77, but it is difficult to recoganize gB: GPCR77.Conclusion: CD4and GPCR77were integrated into the HSV-1envelope respectively,and only GPCR77of gC: GPCR77was still maintained intact conformation and activitycompared with gB: GPCR77. This has laid a solid foundation for constraction ofintegrated GPCRs drug screening chip.The success construction of virion chip will result in significant impact in thebiomedical field and will creat good plantform for the active drug screening,identification of molecular ligands, and study of membrane protein structure andfunction.更多还原