Construction Of High Throughput Drug Screening Virion Chip

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.