Preparation Of Tag Protein MBP Nanobody And Study The Association Between MiR-4293 And MiR-608 Polymorphisms And ESCC Susceptibility

With the rapid development of functional genomics and proteomics, tag proteins and nanobodies are playing important roles, respectively. MBP( maltose binding protein)is a commonly used tag protein in modern molecular clone. At present, MBP is not only used for protein purification as it can easily increase the solubility of inclusion bodies, but also used for studying protein interaction with Pull-down and other experimental methods; Nanobody is widely used in many fields of biology and medicine for its good solubility, high stability and specificity. Based on the important functions of MBP and the advantages of nanobody, we hope we can obtain the nanobodies against MBP.In this study, we first immunized Camelus bactrianus with purified MBP antigen protein, established the immunity nanobody library with the material of Camelus bactrianus peripheral blood. Then we followed "absorption-elution-amplification" affinity screening route to panning and enrichment the specific antibodies by phage display technology, obtained the specific positive clones binding with MBP by ELISA method. Finally, we used Pull-down method to verify the interaction function between the nanobodies and antigen MBP.As a result, we successfully constructed the single domain heavy chain antibody library of Camelus bactrianus, eventually screened and identified the nanobodies that could combine with MBP.Furthermore,we also conducted a study of genetic polymorphisms and cancer susceptibility. At present, China has more than seven times incidence of esophageal cancer than the average global, ranking first in the world. More than 90% of esophageal cancer is esophageal squamous cell carcinoma(ESCC), and the genetic factors play an important role in ESCC. For the importance of microRNA and their polymorphisms, we had a online search on database and literature, and identified SNP rs12220909 G>C located in miR-4293 seed region and rs4919510 C>G located in miR-608 mature sequence but outside seed region to study the association of their polymorphisms and ESCC susceptibility by case-control method.As a result, we found that rs12220909 was associated with a decreased ESCC risk in 774 cases and 882 controls: The GC/CC genotypes significantly decreased the risk of ESCC(adjusted OR=0.77, 95% CI: 0.61-0.96). The C allele conferred 23% decrease in ESCC risk compared with the G allele in the allelic model(95% CI: 0.63-0.93). In the stratified analysis, we found smoking and drinking have different effects on rs12220909. But to the miR-608 rs4919510 C>G, which has been proved to be associated with many cancers, we have not observed association between it and ESCC in any models. Our results lay a good foundation to further explore the functional role of miR-4293 played in ESCC.