Preparation Of Anti-TEM-1 Nanobody And Preliminary Development Of Kit

β-Lactamase can decomposeβ-Lactam antibiotics,so they will be illegally added to dairy products to degrade antibiotics.In recent years,illegal addition in dairy productsβ-Lactamase are emerging in endlessly,so develop a kit that can detectβ-Lactamase is very meaningful.TEM-1 is the most numerous and commonβ-Lactamase in China.Nanobody is a kind of antibody with high stability,specificity,easy expression and production,which is widely used in food science,biomedicine and other fields.In this study,we successfully constructed a display library of anti-TEM-1nanobodies through phage display technology,cloned and expressed the anti-TEM-1nanobody sequence obtained through biopanning and identification,and developed a P-ELISA Kit for TEM-1.The main research results are as follows:1.Construction and biopanning of nanobody display library.According to the codon usage preference of the Escherichia coli expression system,the TEM-1 gene sequence was optimized,and the optimized sequence was constructed into the p ET-25b（+）for expression.After immunizing alpacas with the recombinant protein TEM-1 four times,we built a library（its capacity of 1.20×10⁸cfu）.By colony PCR identification,the insertion rate of VHH was 100%.After M13KO7 assisted phage rescue,obtained phage display nanobody library of a titer of 6.80×10¹³cfu/m L.Ten unique sequences of nanobodies were obtained by biopanning.2.Prokaryotic expression and purification of anti-TEM-1 nanobody.The gene sequences of 10 unique nanobodies were cloned into the p ET-25b（+）,these vectors were transformed into the Escherichia coli Rosetta（DE3）competent cell.The nanobodies were expressed by induce with IPTG,and identified with SDS-PAGE.Among them,Nb9 exhibits good binding activity,and its specificity is good,which will be used for subsequent experiments.3.Preliminary development of TEM-1 detection kit.Using Nb9 as the capture antibody,after exclusion and pairing screening of antibody pairs,Phage8 was finally determined as the detection antibody,and a P-ELISA method for TEM-1 detection was established.Its linear range is 7.8～250 ng/m L,with a minimum detection limit of5.1 ng/m L.Applied in milk detection,the recovery rate range is 83.1%～93.4%.