| Introduction and objective:The second half of last century has witnessed the labeled immunoassay revolution in biomedical researches with new techniques emerging. Labeling immunoassay is an integrated discipline synchronizing label technology and immunoassays. These specific and sensitive techniques consist of chemiluminescence immunoassay (CLIA), electrochemiluminescent immunoassay (ECLIA), time-resolved fluoroimmunoassay (TRFIA) and enzymatic immunoassay (EIA). But the enzymes are easily inactivated to lower the sensitivity in EIA with the labeled molecules and hook effect. Because of the shorter fluorescence lifetime, the samples cannot be repetitively measured in CLIA. Not only are the maintenance and purchase of instrument costly, but also external circumstances can disturb the results in TRFIA. The separation of the labeled from free label needs to wash, so it is time to develop a new technique just like the amplified luminescent proximity homogenous assay(AlphaLISA) to overcome these limitations.The quantitative technique was derived from luminescent oxygen channeling immunoassay(LOCI) invented by Ullman EF in 1994. The homogenous assay combines the singlet oxygen, the laser technology and nano-scale microspheres with rapidly developing of PerkinElmer’s AlphaScreen(?) kits. It avoids the interference in serum and the wash step, so it could overcome the shortcomings of EIA, ECLIA, CLIA and AlphaScreen. It has been widely used and represented the trend of the analytical techniques. It was reported that the AlphaLISA has been used for detecting protein-protein interactions such as ER/SRC-1, OX40/OX40L, E6/E6AP and so on.Protein tyrosine kinase (PTK) can transfer a phosphate group from ATP to the Tyr residues in proteins. They can be divided into non-receptor tyrosine kinase and receptor tyrosine kinase(TPK), which play a role in cell differentiation, proliferation and growth. Tyrosine-Protein Kinase Lyn, JTK8, is the Src family of non-receptor tyrosine kinase. Lyn contains carboxy-terminal and amino-terminal domain SH3, SH2 domain) like Yes, Fyn, Lck and Fgr. The kinase mainly expressed in liver nerve tissues, hematopoietic cell and adipose tissues. It interacts with intracellular domain of receptor tyrosine kinase in signal transduction and plays an important part in normal cell growth, survival, adhesion, differentiation and migration. It is associated with simultaneous upgrades versus downgrades of pro-survival signal transduction. Its abnormal expression plays a pivotal role in the metastasis, growth and invasion of tumors. It is highly expression in Kaposi’s sarcoma cells and promotes metastasis in colorectal carcinoma by interacting with CD24. Its lacking inhibits the growth of hepatic metastatic tumor in breast carcinoma and Claudlin-2 may be down-regulated.Tyrosine-Protein Kinase Tec is the Tec family of non-receptor tyrosine kinase with PH, SH3, SH2 and kinase domains. Tec expressed in hematopoietic cells and its abnormal high expressions is related to proliferation of hepatic cellular cancer. The kinase locates in cytoplasm and plays an important role in activating T cells. It is a powerful tool in the development, proliferation, differentiation and apoptosis of immune cells. And it is interconnected with signaling molecule in cells and protein tyrosine kinase,such as Fyn, Lyn, P13K, Vav and PKB. Once the protein activated by Src-family kinase, Ras and Raf signal pathways will activate.The AlphaLISA has great advantages in detecting protein interactions and represents the trend of the immunolabeling technique. In this study, AlphaLISA kit with antibody-sandwich method for detecting Lyn-Tec interaction was developed.Methods:1. Construction and expression of recombinant plasmids:The Lyn, Tec genes were amplified by RT-PCR from total RNA in Hela cells and cloned into expression vector to construct plasmids (pcDNA3.1(-)-Lyn and pEnter-Tec). After transfecting them transiently into HEK293T cells, the products was purified by the BeaverBeadsTMis-tag Kit. Western blot was performed for detecting the proteins and the CCK-8 is used to measure cell proliferation of tyrosine-protein kinase Lyn.2. Preparation of anti-Tec polyclonal antibody:After transfecting the pEnter-Tec transiently into 293T cells, the Tec fusion protein was purified. The synthesis was injected into New Zea-land rabbits to prepare polyclonal antibody, which was identified by the indirect ELISA and Western-blot assays.3. Coupling of anti-His antibody to acceptor beads:Add 30μg anti-His antibody to micro spin column and use a centrifuge at 8,000rpm for 6min six times with 200μl pH8.00.13mol/L PBS to concentrate and wash antibody. Supplement 200μg acceptor beads,1.25μl 10%Tween-20,10μl PBS containing 25mg/ml NaBH3CN and make up to 200μl with PBS. After antibody was directly coupled to the aldehyde groups of beads at 37℃ for 48h, 10μl 65mg/ml CMO solution was added and incubated for 1 h at 37℃ to block non-conjugated sites. Following washed four times, the reagents were resuspended at 5mg/ml with pH7.40.13mol/L PBS including 0.05%Proclin-300 by ultra-centrifugation.4. Biotinylation of anti-Tec antibody:30μg anti-Tec antibody was added to micro spin column and used a centrifuge at 8,000rpm for 6min six times with 200μl pH9.5 0.1mol/L carbonate buffer including 0.1%NaN3 to purify and concentrate anti-Tec antibody. The solution was added into 3μg Biotin N-hydroxysuccinimide ester with 2μl DMSO and incubated at room temperature for 4h. Following molecules was washed four times, the reagents were resuspended at 0.5mg/ml with pH7.40.13mol/L PBS containing 0.05%NaN3 by ultra-centrifugation.5. Preparation of cells sample:10μg recombinant plasmid was transfected transiently into HepG2 cells in culture dish. After 48 hours, cells was collected and washed two times with cold sodium phosphate buffer repeatedly, lml cell lysis buffer with 1%(v/v) Triton X-100, lmmol/L PMSF was added to the solution and incubated at 4℃ for lOmin. Then, the solution was centrifuged for 30min at 14,000 rpm.6. AlphaLISA detection assay:A certain amount of cells sample, antibody-acceptor beads and biotinylated anti-Tec antibody were mixed in 96-well Optiplates and incubated with shaking at at room temperature for 30min. Supplement 175μl streptavidin-donor beads and make up to 235μl per well at the absence of light. Following these reagents were performed with shaking at 37℃ for 30min, the fluorescence intensity was measured with the 2300 EnSpireTM AlphaPLUS Multilabel Plate Reader.7. Co-immunoprecipitation for detecting the interaction in vivo:After transient expression for 48h, cells was collected and washed three times with ice-cold PBS. lml cell lysis buffer with PMSF was added on ice for 15min and centrifuged at 4℃ 14,000rpm for 30min. Cell lysates were incubated with 2mg anti-Flag antibody for one night at 4℃ and incubated with Protein G beads at 4℃ for 1h. Following washed 3 times with lysis buffer, the reactants were analyzed by 10%SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto acetic nitrocellulose sheets.NC membrane were blocked in 5%skimmed milk powder, incubated with anti-Flag or anti-His antibody, and performed with ECL.Results:The results of sequencing and PCR showed that eukaryotic expression plasmid of human tyrosine-protein kinase lyn, Tec were constructed successfully. After transfecting the recombinant plasmid transiently into 293T cells, Western blot showed that the fusion proteins were expressed in 293T cells and purified by BeaverBeadsTMHis-tag. Anti-Tec polyclonal antibody was prepared by injecting into New Zea-land rabbits. According to the indirect ELISA, the titer of the anti-Tec antibody was about 1:256,000. Western blotting showed that the purified polyclonal antibody had a specific affinity for natural 70kDa Tec kinase in HepG2 cells. In this study, the AlphaLISA kits for detecting Lyn-Tec interaction were developed with antibody-sandwich method. The co-immunoprecipitation assays was used to be confirmed Lyn-Tec interaction in vivo.Conclusion:The results demonstrate that human tyrosine-protein kinase Lyn and Tec are expressed in HEK293T cells and the products are purified by BeaverBeadsTM His-tag successfully. Anti-Tec polyclonal antibody was prepared and the purified polyclonal antibody had a specific affinity for natural Tec kinase. The AlphaLISA reagent for Lyn-Tec we developed meets the demands for based medicine research. |
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