| Nanobody is a region of the heavy chain variable region, which naturally occurring in camelidae, also known as single domain antibody(VHH). Although low molecular weight of nanobody about 15 k D, they have two significant differences compared with traditional antibody variable region: the FR2 areas of nanobody have four hydrophilic amino acid mutations from hydrophobic amino acid, an increase of the solubility in solution; nanobodie,s CDR1 and CDR3 hypervariable region is relatively long and bending arm formation by internal disulfide bond can target surface or concealed antigen binding site, improves the stability and antigen binding ability.Nanobodies has been widely used in many fields, such as in the food industry, which can neutralize the toxins, detection of microbial content in food to ensure safety; in scientific research, they can construct monovalent or multivalent nanobody complexes, improving antigen binding, and be modified with organicdyes or biomarkers using for molecular imaging, tracing cell structure in vivo and in vitro,too; in the disease diagnosis, nanobodies play an increasingly important role, such as dentification of pathogenic microrganisms in the clinical sample types; in tumor targeting, nanobody can co-delivery therapeutic drugs with nanobody to improve targeting effect. In addition, tumor markers in the diagnosis and treatment of tumor plays a crucial role. Therefore,screening the high specificity and good specificity tumor markers is the urgent problems to solve. With the discovery and study of nanobodies, it is providing convenience to screen and detecte tumor markers for early tumor diagnosis in clinical sample. In conclution, the development and application of nanobody providing great potential in tumor markers screening and tumor targeting antibody treatment drugs.With the development of genetic engineering, it is possible to make a large number of target proteins by different hosts. People can design and reform target protein gene sequence according to their own wishes and construct to gether with different functions of vector transforming into the host for fermentation production, such as prokaryotic expression system(Escherichia Coli) and eukaryotic expression system(Pichia pastoris and Saccharomyces cerevisiae) etc. The prokaryotic expression system has the advantages of simple operation, low cost, low genetic background effect, high expression of foreign protein, and is especially suitable for the production of small molecular protein with simple structure.In this experiment, three different affinity anti-CEACAM 6 nanobodies screening from the alpaca nanobody library(Nb15、Nb35 and Nb37)transformed into Escherichia coli TG1, and be induced by isopropyl thiogalactoside(IPTG). On the expression of IPTG concentration, temperature and induction time were optimized, the most suitable expression conditions be determined. Using affinity chromatography purified nanobodies and screening the optimal imidazole elution concentration to obtain the high purity nanobody protein. By detecting tumor cells surface antigen content using flow cytometry to verify nanobody binding activity, which dialyzed in PBS, and screen the CEACAM 6 high expression MCF-7 breast cancer cell lines. Finally the optimal nanobody Nb15-2 was screened by the tumor cells of MCF-7. At last, the nanobody Nb15-2 was purified by weak ion exchange chromatography and affinity chromatography for purification and optimization to obtain the purity of 99.6% of the nanobody protein, which can effectively improve the purity of the nanobody protein. In conclusion, these experimental results of nanobodies provide theoretical basis for nanobody industrial production, disease diagnosis and tumor targeted therapy. |
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