Establishment Of AlphaLISA-based Detection Of Interaction Between Insulin-like Growth Factor-1 Receptor And Its Binding Proteins

Background and Objective:Human exploration of the mysteries of life has not stopped since Hooke discovered cells, people began to start the study of life from the microscopic. The discovery of DNA is the milestone of exploring life, and it was predicted that if the human genome is measured, we will be able to understand life and to explain life. The Human Genome Project （human genome project, HGP） was first proposed by American scientists in 1985, and was officially launched by the United States, Britain, France, Germany, Japan and Chinese scientists in 1990, which is this budget of $ 3 billion. As of 2005, the sequencing of the human genome project has been completed. The Human Genome Project aims to study human genes to understand human beings, including a variety of physiological and pathological mechanisms. Although the human genome has been sequenced, it did’nt reach the deeper purpose. Humans began to further explore the proteins, the performer of life. International Human Proteome Project （HPP） is the second international Human Genome Project. Our commitment around the Human Liver Proteome expression profile, a modified spectrum and their interactions linkage map nine research tasks.Currently, methods exploring protein interactions include yeast two hybrid, phage display, co-immunoprecipitation, PμL1-down and so on. These methods are currently considered as standard techniques of protein interactions identified, but these methods are cumbersome procedures, spending more time, or influenced by many factors. The experiment hopes to discover new ways of detecting protein interactions with fast and accurate detection.There are many fast and accurate testing methods, and Labeled Immunoassays technology which is studied more is used widely and mostly. Labeled Immunoassays technology is fast detection, high specificity, high throughput detection and other advantages. In the hospital, there are more varieties of detecting methods using labeled immunoassays technology, such as detection of cancer protein AFP, detection of hepatitis B surface antigen and antibodies in the blood protein content of CEA and so on. Labeled Immunoassays technology is also used in medicine, such as to detect the expression of proteins, monoclonal antibodies detection. The most common techniques are the immune ELISA （Enzyme-linked immunosorbent assay, ELISA）, chemiluminescence immunoassay （Chemiluminescence immunoassay, CLIA）, electrochemiluminescence immunoassay （Electrochemiluminescent immunoassay, ECLIA） and time-resolved fluorescence immunoassay （Time-resolved fluoroimmunoassay, TRFIA）. Labeled Immunoassays technology also has its own advantages and disadvantages, such as EIA, a horseradish peroxidase-labeled easier inactivated antigen antibody also may be affected by steric hindrance, resulting in resulting in sensitivity is limited; CLIA method drawback is that factors too; CLIA method susceptible to interference from external environment it can only be used for non-open detection, the use of imported raw materials more expensive, the most important is that the sample can not be repeated measurements; TRFIA, ECLIA method is to improve the CLIA method, but the raw material is still high and is closed detection; more than technical procedures cumbersome and needs washing. Therefore, the use of many new theories and technologies labeled immunological techniques and should be shipped on. The one is the light chemiluminescence immunoassay （Amplified luminescent proximity homogenous assay, AlphaLISA） technology. The working principle of AlphaLISA is Antigen-antibody binding specificity and chemiluminescent light laser technology. Advantages of detection method using AlphaLISA for detecting protein interactions are using less samples, shorter time and large number of samples than the traditional method.Insulin-like growth factor receptor 1 （InsμLin like growth factor type1, IGF-1R） is total length of 1366 amino acids and its three-dimensional structure is composed of two IGF1R protein which is component of a and β segment after the action of the protease and forming a tetramer. IGF1R involved in several physiological reactions: the exocrine pancreas development, gynecomastia, male sex determination, immune response, brain development, insulin receptor signal transduction pathway, cell proliferation positive regulation, mitotic positive regulation, negative regulation of transcription factor activity, apoptosis and so on. IGF1R involved in multiple signaling pathways:AKT pathway, InsμLin pathway, apoptosis pathway, NF-kappa B pathway, mTOR pathway and so on. In tumors, IGF1R also play an important role. Maybe IGF1R involve tumor growth, invasion, resistance to apoptosis and metastasis. IGF1R protein has interaction with a variety of proteins such as Csk, SOCS1, GRB10, FAK and others involved in signaling pathways.Relevant reports, there are interactions between IGF1R and Csk, SOCS1. C-Src protein tyrosine kinase （C-Src tyrosine kinase, Csk） is a negative regulatory protein, the member of Src kinase family. Csk, non-receptor tyrosine kinase, need transmembrane receptors such as the need IGF1 receptor, EGF receptor, T cell receptor, or transmembrane adapter proteins such as CBP to help transfer completion signal. Speculate that functions of Csk are related to interaction with these proteins. SOCS1 is a member of the cytokine signaling （SOCS） family of proteins suppressor, and become focuses of cancer research such as SOCS1 expression, SOCS1 gene methylation, mutation and deletions decreased. SOCS1 has been played an important role in many cancers, such as colorectal cancer, breast cancer, liver plays an important role.IGF1R play different functions interacted with a variety of different proteins, so the study of different physiological and pathological are very important processes to study IGFR protein interaction. We hope that a new approach can be used to study of protein-protein interactions. method:Developping double antibody sandwich method using AlphaLISA to detect the interaction between IGF1R and Csk, SOCS1. 1. Preparation of raw materials IGF1R antibodyRNA extraction Hela cells obtained using the reverse transcription kit cDNA. Use Primer 5 software,β paragraph primer pMal-c2x vector map and gene sequences in GenBank IGF1R （NM₀01291858） design IGF1R protein in the upstream and downstream primers added six his tag and primer ends were added BamH I, EcoR I digestion sites and the protection of the base. Objective gene is obtained by PCR. The PCR product pMal-c2x vector obtained simultaneously with BamH I, EcoR I double digestion, digestion product was transferred to a connected TOP 10 competent in. Using enzyme digestion and sequencing recombinant plasmid is successful.The recombinant plasmid was successfully transferred into BL21 competent to achieve optimal expression conditions by varying the temperature and the concentration of inducer IPTG induction. According to the best expression conditions abundantly expressed. After collecting the induced bacteria were resuspended, repeated freezing and thawing, sonication, high-speed low-temperature centrifugation, the supernatant was expressed, expressed identified by SDS-PAGE Coomassie staining. The supernatant respectively, after AmyloseResin and nickel affinity chromatography column and purified the results identified by SDS-PAGE Coomassie staining, the pros and cons compare the two purification methods. And finally the purified protein, were sent to Guangzhou Rui Boao antibody preparation.2. Eukaryotic expression of IGF1R, Csk, SOCS1 and other proteins, and identification of interactionRNA extraction Hela cells obtained using the reverse transcription kit cDNA. Use Primer 5 software, pCNDA, pENTER the vector map and GenBank, β segment gene sequences were designed IGF1R protein, IGF1R full-length, IGF1R-α, Csk, SOCS1, FAK, IGF1, IGFBP3, GRB10, SHC1 primer and primer adding the corresponding ends of restriction sites and protected bases. Objective gene is obtained by PCR. After tapping recovery, the PCR product pCDNA, pENTER carrier obtained simultaneously with the double digested tapping digestion products recovered, digested the fragment with the carrier in accordance with 7:1 mass ratio via T4DNA after ligase, into to TOP 10 competent, the bacteria coated plate.12h after the pick to go monoclonal colonies, shake fungi recombinant plasmid. Using enzyme digestion and sequencing recombinant plasmid is successful.The success of the recombinant vector recombinant vector transfected by liposome transfected into 293T in eukaryotic expression by immunofluorescence localization Westernblot and identify whether the protein expression.References and ease of obtaining antibodies, we first select the test IGF1R interaction with SOCS1, IGF1R and the Csk.The recombinant successful IGF1R-β-pENTER respectively Csk-pENTER, SOCS1-pENTER 293T cells were co-transfected with the expression of the effect identified by Westernblot co. And by changing the IGF1R-β-pENTER and Csk-pENTER, the proportion of SOCS1-pENTER plasmid co-transfected with Csk reach IGF1R protein expression of, IGF1R and SOCS1 protein expression up to 1:1. Use COIP and fluorescence co-localization method for identifying IGF1R and Csk, the interaction of SOCS1.3. the initial establishment of IGF1R and Csk, SOCS1 interaction detection reaction system and the reaction conditions fumbleUsing chemiluminescence detection system has established a relatively complete pairing AFP AFP antigen and antibody, build light chemiluminescence immunoassay detection system. Cyano sodium boron chemical attachment method microspheres 1 receptor antibody is connected to AFP, AFP 2 biotinylated antibody. By changing the ratio of AFP receptor antibodies with the microspheres 1 is connected, the amount of biotinylated antibody using different buffer conditions, the most suitable reaction conditions screening.According to a preferred reaction system was established to establish interaction detection method based on AlphaLISA of IGF1R and channel proteins. The IGF1R receptor antibody is linked to the microspheres, and SOCS1 will Csk antibody biotinylated antibody detection samples were co-transfected 293 T cell lysates expressing IGF1R and Csk protein, co-transfected with SOCS1 protein expression of IGF1R 293T cells Lysates. The negative control, the connection receptor microspheres IGF1R antibody to the receptor antibodies connection IL4R microspheres remaining ingredients unchanged.4. using AlphaLISA detecting the interaction between IGF1R and SOCS1 to detect cell linesAccording to the above method, test Hap3, HFF-1, IGF1R-P transfected 293T cells and co-transfected with SOCS1 IGF1R-P cells. First, cells were collected for each cell line, and then use the buffer cells were collected and total protein concentrations of regulatory proteins. AlphaLISA also used to detect the same amount of total protein, the difference between the fluorescence signal, result:Successfully IGFlR-β-pMal-c2x recombinant prokaryotic expression vector was successfully constructed and successfully expressed in E. coli. By Ni affinity chromatography, purified monoclonal antibodies produced raw materials. Eukaryotic successfully constructed IGF1R-pCDNA 4.0, IGF1R-β-pENTER, IGF1R-a-pCDNA 3.1, Csk-pENTER, SOCS1-pENTER, IGF1-pENTER, IGFBP3-pENTER, FAK-pENTER, GRB10-pENTER, SHC1-pENTER etc. the expression vector and expressed in the 293T.The IGF1R-β-pENTER and Csk-pENTER were co-transfected expression, IGF1R-β-pENTER with SOCS1-pENTER were co-transfected expression success. By COIP and colocalization identified IGF1R and Csk, the interaction of SOCS1. Through the exploration AFP response patterns, and explore light chemiluminescence immunoassay detection technology, found that use of superior standard buffer PBS buffer, biotinylated antibodies using the most appropriate concentration of 0.125μg/hole. Connection quality ratio receptor antibody microspheres is 20:3, at a concentration of microspheres receptor 2.5μg/hole. Through interaction with Csk IGF1R established inspection system to detect, it can be found along with protein concentration, fluorescence value increased, and the process linear, R2=0.9968. Detection IGF1R interaction with SOCS1 can be found as protein concentration, fluorescence value increased, and the process linear, R2= 0.9983.Use established detection IGF1R and SOCS1 interaction AlphaLISA detection Hap3, HFF, IGF1R-β transfected 293T cells and co-transfected with IGF1R-β and SOCS1 293T cells, we found IGF1R-β transfected 293T cells and co-transfected with IGFIR-β and SOCS1 and Hap3 in 293T cells, detection of IGF1R and SOCS1 strong interaction, and HFF in IGF1R and SOCS1 weak interaction. IGF1R and SOCS1 different interaction strength in different cell lines, according to the intensity of the role of small to achieve:Hep3 cell> IGFIR-β co-transfected 293T cells with SOCS1> single turn and then IGF1R-β 293T cells> HFF cells, HCT116 cell> LOVO cells> 7721> Thpl cells, in conclusion:These results indicate that this study protein interactions detected light chemiluminescence immunoassay detection method has been successfully able to detect the interaction of IGF1R and Csk, IGF1R and SOCS1 between. This method can be used to detect other cell interactions, for example Hep3, HFF and other cell lines.This detection method advantages as compared to other methods are:1) the use of fewer samples:samples used to 600ng AlphaLISA about 1μL of the cell lysate.2) detection time less:AlphaLISA detection time of 30min to 2h, generally 30min result could be detected.3) fewer steps:AlphaLISA step only step that is added to the sample and the detector.4) test sample more:AlphaLISA disposable can detect 96 holes, sample testing more.5) higher detection sensitivity:AlphaLISA null cells can be detected in weak interactions.