The Biopanning, Expression And Application Of Anti-c-Myc Nanobodies

In cells, there is a class of genes or gene families. Under normal circumstances,they are in inhibitory, low expression and insufficient. When they come into some factors, the gene expression loss control and biological characteristics of normal cells take change, gradually transform to malignancy and ultimately result in cancerous.Such genes are called proto-oncogenes.Myc gene family, as a class of proto-oncogenes family, is divided into c-myc,n-myc and l-myc gene. The DNA binding protein is the myc expression product and in the nucleus, it can directly regulate the other genes’ transcription and promote cells proliferation or induce apoptosis. In which, activation of c-myc is related to the occurrence and metastasis of tumor.Quantum dots, also part of nanocrystals, is a new kind of nanoparticles made of II- VI or III- V elements. It can emit fluorescence by latter and be used for biological analysis. Affected by quantum effects, its excitation spectrum is broad and continuous and emission spectrum is narrow and symmetric. The emission wavelength can be changed according to adjust the size and composition of the quantum dots. Compared with the traditional organic fluorescent dye, it possesses the high fluorescence yield, well stability, anti-photo bleaching, long fluorescence lifetime and biocompatible, reducing toxic to organisms. Therefore, the quantum dots fluorescence technology has broad prospects in the field of biology.In this study, the phage display technology is used from alpaca immune library picking out nanobodies which can bind c-Myc and express, and the c-Myc nanobody express in e.coli. The recombinant c-Myc antibody marked by quantum dots used to detect the c-Myc protein expression in cells.the main research results are as follows:1、As alpaca myc protein immune library forexperimental materials,It selects 11 specifically binds to c-Myc protein clones by four rounds of selection and Compares the relative affinity.they are A13、A18、A25、A26、A31、A32、B34、C5、C24、C42、C46.2、Relatively high affinity A25、A26 clones are selected,then put into pET-25b(+)plasmid.It can obtaine Nanobodies after using the IPTG inductionin in E. coli expression system. SDS-PAGE electrophoresis shows that the recombinant protein molecule weight is approximately 20 kDa in line with expectation.3 、 The method of using the quantum dots ibaoji recombinant antibodies for intracellular nano target detection is validated. It prepares of SP2/0 cells climbing film and uses the quantum fluorescent technology to identify Nanobodies and detects the expression of c-Myc protein in the cell.The cells shows fluorescence after adding the Nanobodies and quantum dots,but donot shows fluorescence only adding the quantum dots.This study of Nanobodiesresults can be expectly used to detect c-Myc protein expression in cells and then applied to diagnostic cancer cells.