Fluorescence Imaging Of ESCC Based On Labeling Of 7D12 By Expanding The Genetic Code And Photo-click

The targeted molecular probes related to nanobodies are widely used in molecular imaging research.When such probes adopt random labeling methods to introduce dyes,nuclides and other groups,it will cause problems such as products heterogeneity after labeling,low affinity to target,and low labeling efficiency.The expanding the genetic code technology can realize specific labeling of unnatural amino acids at specific sites of antibodies,thereby introducing luminescent groups,nuclides or nanoparticles required by molecular probes,which can avoid problems caused by random modification.In addition,unnatural amino acids with olefin groups can undergo a rapid biological cycloaddition with tetrazole compounds under photo-click.Therefore,this study used the expanding the genetic code technology and photo-click reaction to realize the site-specific fluorescent labeling of nanobody 7D12.In this study,the epidermal growth factor receptor(EGFR)specific nanobody 7D12 was selected as research object.Through the expanding the genetic code,nanobodies which were introduced the unnatural amino acids N-ε-acryllysine with olefin groups at 13 th or76th positions were expressed and purified in prokaryotic cells.Photo-click reaction conditions were optimized to enable the tetrazole compound to undergo a highly efficient cycloaddition reaction with the nanobodies to generate nanobdodies with fluorescent groups.The nanobody fluorescent probes were used in cell and in vivo imaging experiments to study their feasibility of being molecular probes for the diagnostic of esophageal squamous cell carcinoma.The main results are as follows:1.Completed the construction of prokaryotic expression plasmids: the recombinant plasmid p ET24a-7D12 expressing wild-type nanobody was constructed;the nanobody plasmids of experimental groups,which were the recombinant plasmids p ET24a-7D12-13 and p ET24a-7D12-76 whose amino acid codons at 13 th or 76 th positions were mutated to UAG were constructed;the nanobody plasmids of control group were also constructed,mutations of amino acid codons related to EGFR binding were performed on the basis of the experimental group plasmids to obtain p ET24a-7D12-mut,p ET24a-7D12-13-mut and p ET24a-7D12-76-mut plasmids.The above plasmids were verified by sequencing.2.Completed the expression,purification and affinity determination of nanobodies:N-ε-acryllysine,tetrazole compound T3 and cross-linked product Cy5-T3 were synthesized,purified and identified.N-ε-acryllysine was added to the culture medium to induce the expression of antibody proteins in the prokaryotic cells containing the above plasmids.Immobilized metal ion affinity chromatography and gel filtration chromatography were used to obtain 7D12 wild-type and 7D12-13-AcrK,7D12-13-AcrK-mut mutant proteins with purity greater than 90%.The affinities of the above proteins to EGFR were determined by indirect ELISA.The results revealed that the site-specific insertion of N-ε-acryllysine in 7D12 did not affect the binding ability of the antibody to EGFR.3.Obtained the fluorescent labeled nanobodies by photo-click reaction: the fluorescent labeling of antibodies 7D12-13-AcrK and 7D12-13-AcrK-mut was activated by 365 nm ultraviolet for 2 hours in HEPES solution containing compound T3.The SDS-PAGE in-gel detection showed that the bands of the above protein were fluorescent.The spectrum measurement results showed that the product of photo-click reaction had an ultraviolet absorption at 380 nm and a fluorescence emission band between 520～540 nm.4.Realized the fluorescence imaging of ESCC cell line ECa109 and its tumor-bearing mouse model in vivo and in vitro: in vitro imaging results showed that 12.5 n M of7D12-13-AcrK-T3 probe could realize the fluorescence imaging of ECa109 cells.No fluorescence signals were detected in cells in the EGFR negative group or in the control group.The in vivo results revealed that the 7D12-13-AcrK-T3 probe could target tumors quickly and accurately.The signal of tumors in the targeted group reached peak as early as3 hours post-injection,which could be restored to pre-injection level after 6 hours.Its highest tumor to background ratio(TBR)was 1.16(± 0.02),which was 2.8-fold the value of the non-targeted group.The results of isolated organs and tumors were consistent with the in vivo results.The imaging results of the 7D12-13-AcrK-T3-Cy5 probe showed that the signal of tumors reached peak at 24 hours,and its TBR was 0.88(± 0.01).This study optimized the labeling process of nanobody molecular probes,which could provide new ideas and means for synthesis of high-affinity,high-specificity and fast-targeting molecular probes,as well as for achieving efficient molecular imaging of esophageal squamous cell carcinoma.