Construction And Validation Of HIV Entry Inhibitor Screening Model Based On FRET Technology

BackgroundAIDS.also known as acquired immunodeficiency syndrome,being induced by human immunodeficiency virus.The first case of AIDS appeared in the United Statesin in 1981.then in China in 1985 the first case of AIDS appeared.According to the latest data in 2015 July 21.World Health Organization(WHO)showed that global HIV infected persons in 2014 up to 36 million and 900 thousand people,which included 34 million 300 thousand adults and more than 17 million 400 thousand women.2 million 600 thousand teenagers,2 million deaths and about 2 million new infections,there are 1 million 200 thousand people died of AIDS.HIV has became one of the four most deadly pandemic diseases in the world,data from WHO showed that although 15 million 800 thousand people worldwide have been receiving antire troviral therapy(ART)from 2000 to 2015.there still are millions of AIDSpatients died every year.Until now we have not found a safe and effective HIV vaccine.China has become one of the world’s fastest growing countries in AIDS patients.AIDS is spreading from high-risk groups to the general population.Therefore,developing HIV therapy drugs has became the main strategy for diseases prevention and treatment.Now drugs approved by FDA(Food and Drug Administration)mainly include:nucleoside reverse transcriptase inhibitors,non nucleoside reverse transcriptase inhibitors and protease inhibitors,which can not only effectively inhibit the immune system damage and reduce the infection rate,but also relieve disease development speed,but they can not completely remove the virus.Moreover,serious side effects and virus resistant mutation were induced after long-term medication.There have been more and more patients cannot accept these persistent anti HIV drug treatment.Therefore,it is extremely urgent to research and develop effective and safe drugs.A new type of HIV entry inhibitors blocking HIV into human is a research hot spot at present.Human immunodeficiency virus entered the cell and organism mainly through interaction between the virus surface envelope glycoprotein gp120/gp41 with cell surface receptor CD4 and coreceptor CXCR4 or CCR5.In this process,entry inhibitors developed at present can be divided into three categories:the adhesion inhibitor blocking the connection of gp120 and CD4:auxiliary receptor inhibitors affecting on HIV receptor:and membrane fusion inhibitors affecting on the gp41.Fluorescence resonance energy transfer(FRET)is a kind of energy transfer phenomenon between two adjacent fluorescent molecules.Only when overlapping between the emission spectra of doner and absorption spectra of acceptor,moreover.distance between two molecules was less than 10 nm.there will be a non radioactive energy transfer.called FRET phenomenon.Due to the FRET efficiency highly depending on the distance between the donor and acceptor molecules,and particularly sensitization to receptor relative orientation changes of the transition dipole,this technology was firstly used for the determination of the interaction between molecules and receptors,which has been widely used for DNA hybridization,studying protein conformational changes,interactions between proteins and ligands in the life science field now.In view of the important role of gp120.CD4 and CXCR4 in HIV infection and in order to targetedly screen HIV entry inhibitors,we constructed gp120,CD4,and CXCR4 fluorescence expression vectors on the principle of FRET,then transfected them into cells,detected the fluorescence resonance energy transfer phenomenon among them using confocal microscopy to construct a HIV entry inhibitor screening model and further verify its reliability by using of CD4,CXCR4 specific antibodies or inhibitor.Methods1.The construction of fluorescent protein v ectorsPlasmid pEGFP-N3 expressing green fluorescent protein was selected as donors and plasmid pDsRed-Monomer-N1 expressing red fluorescent protein was selected as receptor,we used gene cloning techniques to insert three genes gp120,CD4,and CXCR4 into two plasmid vectors respectively to construct six recombinant fusion plasmids:gp120/pDsRed-N1.gp120/pEGFP-N3.CXCR4/pDsRed-N1,CXCR4/pEGFP-N3,CD4/pDsRed-N1、CD4/pEGFP-N3,then purpose gene fragments were verified by bacterial PCR and plasmid double enzyme cutting identification.nucleotide sequencing was used for verification finally.2.Observation of fluorescence colocalization after cell transfection and qualitative detection of FRETThe human embryonic kidney HEK293T cells was cultured into 24-well plate,until the cell density reached 60-70%.constructed recombinant plasmids were transfected transiently using lipofetecation3000 kit,we transferred six single plasmids and co-transferred six pairs of donors and acceptors into 293T cells:gp120/pDsRed-N1 and gp120/pEGFP-N3.CXCR4/pDsRed-N1 and CXCR4/pEGFP-N3,CD4/pDsRed-N1 and CD4/pEGFP-N3.cells were cultured in cell culture box,then cell fluorescence expressions were observed and colocalization phenomenon were observed and taken photos via fluorescence microscope after 48 hours.These samples were placed in confocal microscopy for preliminary FRET detection.Firstly,green fluorescence images expressed in cells transfected with single donor plasmid were observated in EGFP channel excitated with 488 nm wavelength light,red fluorescence images expressed in cells transfected with single acceptor plasmid in DsRED channel excitated with 559 nm wavelength light.Secondly,green fluorescence images were obsevated in EGFP channel excitated with 488 nm wavelength light and red fluorescence images in DsRED channel excitated with 559 nm wavelength light in cells cotransfected with donor and acceptor plasmids.The fluorescence intensity and co-expression between two kinds of protein or doner and acceptor should be dectected and recorded.At last,we observated cells transfected with single donor and single acceptor and cotransfected samples in DsRED channel excitated with 488 nm wavelength excitation light.If the donor cells and acceptor cells are negative and cotransfected cell samples are positive,it is concluded that there may be a fluorescence resonance energy transfer between donor and acceptor.3.Verification of the proteins interaction by FRET technology sensitized emissionIn order to rule out FRET fluorescence cross color interference of the preliminary judgment method,we use the sensitized emission method to quantitatively detect the protein interaction in vivo.HEK293T cells were transfected with single donor and single acceptor,cotransfected with donor and acceptor,of which three cell samples as a sample group,thus we got six sample groups.At the same time a negative control group was set up:cells transfected with pDsRed-N1.pEGFP-N3,cotransfected with pDsRed-N1 and pEGFP-N3.After culturing 48 hours we did detect the protein interactions by sensitized emission method under confocal fluorescence microscopy,seven images were collected each sample,from which we choose three to six regions of interest(ROI).and each sample was repeated for three times.According to the original data in the experiment the corrected protein energy transfer efficiency E and protein distance R were calculated by FV10-ASW2.1 Viewer software.Results were expressed as mean ± standard deviation(mean±SD).4.Verification of the model reliability by specific inhibitors and antibodiesAccording to the last step results,CXCR4 specific inhibitor AMD3100 or CD4 specific antibody anti-CD4 IgG were added to the sample groups interacting with each other to verify whether the inhibitor could inhibit the interaction between proteins.We transfected six sample groups and a negative control group into HEK293T cells.We add 1 μM drugs into cells after culturing 46 hours.Then protein interactions were detected in the confocal fluorescence microscope using sensitized emission method after two hours’ recation.Finally,using FV10-ASW2.1 Viewer software the energy transfer rate and distance between donor and acceptor were calculated according to the experimental data.Results1.Fluorescent protein vectors were constructedCD4,CXCR4.gp120 genes were amplificated by polymerase chain reaction.they were double digested and cloned into two linear of plasmid vector pEGFP-N3 and pDsRed-Monomer-N1 respectively,then were transfected into E.coli DH5α and screened the nagetive monoclonal colony via the kanamycin resistance plate.After enriched culturing of bacteria did we identify them by PCR,extraction plasmid double enzyme digestion,DNA sequencing.Three methods all proved six fluorescent protein recombinant plasmid was constructed sucessfully.2.Fluorescence colocalization was observed after transfection and FRET was qualitively detedtedSix recombinant plasmids gp 120/pDsRed-N 1.gp 120/pEGFP-N3.CXCR4/pDsRed-N1.CXCR4/pEGFP-N3,CD4/pDsRed-N1,CD4/pEGFP-N3 were transiently transfected into HEK293T cells.Cell fluorescence expression and colocalization phenomenon were evaluated via the fluorescence microscope with red and green filters after culturing 48 hours,we found that the transfection efficiency of plasmid with green fluorescent protein was higher than that of plasmid with red fluorescent protein.Cells after transfection glowed normally and fluorescence expressed in the cytoplasm,the nucleus and even the whole cell.However.fluorescence co-localization in the co-transferred cells concentrated expressed in the cytoplasm and around the cell membrane.The samples were placed in the laser scanning confocal microscope to detect FRET,preliminary results:single donor cells in EGFP channel with 488 nm excitation light displayed green fluorescence image,single acceptor cells in DsRED channel with 559 nm excitation light displayed red fluorescence image,co-transfected cells displayed green fluorescence image in EGFP channel with 488 nm excitation light and displayed red fluorescence image in DsRED channel with 559 nm excitation light at the same time.Then cells were observed in DsRED channel with 559 nm excitation light.we found that six groups of samples appeared strong or weak red fluorescent image campared with the control samples without occurrence of this phenomenon.We preliminary proofed that there existed fluorescence resonance energy transfer among gp120,CXCR4,CD4 proteins.3.FRET technology verified the interaction among proteinThe normal fluorescence plasmids were transfected into cells,then seven imageswere collected via the laser scanning confocal microscope each group.which consisted of three samples.The results were analysised and calculated by sensitized Emission software.Experimental results showed that compared to negative control group A,FRET efficiency of experimental group B-G were between 0.450～0.669.distances between proteins are less than 10 nm.The results confirmed that the gp120,CXCR4,and CD4 existed interaction and FRET phenomenon between each others.4.Sensitization emission method screened drug inhibitorsCXCR4 specific inhibitor AMD3100 or CD4 specific antibody anti-CD4 IgG were added in the transfected cell groups,then the FRET efficiency and interaction distance between proteins were detected via sensitized emission method and FV10-ASW2.1 software.Experimental results showed that after adding inhibitors the energy transfer rate between proteins reduced to 0.102～0.195,which had significant difference(P<0.05)compared with the group without treatment;the distance between proteins after adding inhibitors also increased slightly,which proved the protein interaction between each others can be inhibited by the inhibitors.The model is stable and reliable and can be used for further screening of HIV entry inhibitors.Acorrding to the statistical analysis,we did the single sample t-test in each group.The results showed statistical significance(P<0.05)and there existed interaction among protein gp120,CXCR4,CD4,paired t-test were did for six groups before and after medication.The results explained significant difference(P<0.05)between untreated group and treatment group,therefore inhibitors had inhibitory effects on protein interactions between each others.Conclusion1.gp120.CXCR4.CD4 three exogenous genes and plasmid pEGFP-N3.pDsRed-Monomer-N1 were sucessfully constructed.we got six new recombinant plasmid vectors:gp120/pDsRed-N1,gp 120/pEGFP-N3.CXCR4/pDsRed-N1,CXCR4/pEGFP-N3,CD4/pDsRed-N1,CD4/pEGFP-N3.2.Six vectors expressed fluorescence after transfecting into HEK 293T.Six of the paired combinations of recombinant plasmid vectors in the cell can be colocated expression.Three chanels FRET detection preliminary examined that three protein gp120.CXCR4,CD4 have fluorescence resonance energy transfer phenomenon between each others.3.The FRET technology sensitized emission method offered quantitative detection.which provided further confirmation of the interaction between three proteins gp120.CXCR4,CD4.HIV entry inhibitor screening model was successfully constructed.4.The interactions between gp120.CXCR4,or CD4 were inhibited detecting via sensitized emission measurement after CXCR4 inhibitors AMD3100 or CD4 specific antibody anti-CD4 IgG were added.The results confirmed that the model is stable and reliable and could be further used for screening of HIV inhibitors.