Preparation Of Tag Protein GST Nanobodies And Study The Association Between Rs11614913 And Rs2910164 Polymorphisms And ESCC Susceptibility

Sera of camelids contain an unique functional heavy(H)-chain antibodies(HCab). Cloning its variable region can obtain nanobodies, which are known as one of the smallest antigen-binding Ab fragments compared with the traditional antibodies. The beneficial properties, such as minimal size, higher stability and easy to express, make nanobodies wide development prospects in the diagnostic, protein affinity purification and development of antibody drugs. Glutathione S-Transferase(GST) is one of the most used tag protein. It can be used not only in the protein affinity purification feild, but also been applied to the detection of fusion protein and the interaction between proteins. Based on the advantages of nanobodies and the important functions of GST, we hope we can obtain the nanobodies against GST with independent intellectual property rights.In this study, we successfully expressed and purified the GST protein by prokaryotic expression system. The two humped camel were immunized and its blood was used as the material to build phage display libraries. Then panning and enrichment the specific antibodies by phage display technology. We used ELISA to obtain the specific positive clones binding with GST. Finally, GST pull-down method was used to verify the interaction function between the nanobodies and antigen GST.As a result, we successfully constructed the single domain heavy chain antibody library for GST protein. Screened and identified the nanobodies that could combine with GST eventually.Furthermore, we also conducted a study of genetic polymorphisms and cancer susceptibility. Esophageal cancer(EC) is one of the most common malignancies in the world. ESCC is the most prevalent pathological type of EC in China. Micro RNAs(mi RNA) mainly affect gene expression during the post-transcriptional process. Single nucleotide polymorphisms(SNPs) in mi RNA lead to the aberrant expression and structural alteration of mi RNA, and are hypothesized to be involved in tumorigenesis and cancer development. We conducted a population-based case-control study to evaluate the association between SNPs in mi RNAs and ESCC risk in 1400 ESCC cases and 2185 matched controls. Two SNPs including mi R-196a2 rs11614913, mi R-146 a rs2910164 were selected with database.As a result, we found that rs11614913 was significantly associated with an increased ESCC risk in 1400 cases and 2185 controls: The CC genotype significantly increased the risk of ESCC(OR=1.11, 95% CI:1.01-1.22, P=0.049). The association was more pronounced in non-drinkers in the recessive model(OR=1.13, 95% CI:1.01-1.27, P=0.029) and Homozygous model(OR=1.14, 95% CI:1.00-1.30, P=0.046). But to the mi RNA-146 a rs2910164(C/G), which has been proved to be associated with many other cancers, we have not observed any association between it and ESCC.