| Atrazine is worldwide used triazine herbicides. In recent years, atrazine residue has been detected from groundwater, surface water and atmospheric deposition in many countries and regions. It has long retention time in the soil, which would lead to serious threat to food safety, interfere the endocrine and reproductive system of human body, and bring in negative influence to the global ecological environment. Therefore, real-time monitoring and evaluation analysis of the environmental atrazine residues would help to prevent and respond to the negative impact of atrazine.Current methods for monitoring and evaluating atrazine exposure are based on large instruments, which is time-consuming and costly. In comparison, immunology-based rapid detection of atrazine residue is more efficient, more convenient and more economic. The key point of using immunological methods is to obtain specific, high affinity antibodies.Phage display antibody library technologies are till now the most developed and widely used antibody library technology. This can bypass the hybridoma technology and even the immunization. Immune antibody libraries are construct from immune cells after immunized by antigen The lymphocytes being utilized for constructing antibody libraries have gone through the antigen selection and affinity maturation in vivo, thus we could produce antibodies with high specificity and high affinity screening from libraries with relatively smaller capacity (105-106).In this study, we constructed a phage display scFv antibody library by using the atrazine immunized mouse spleen. We screened the library with direct-coating (solid) and magnetic beads coating (semi-solid) methods respectively. The research and results were as following:1 Construct phage display immune antibody libraryTotal RNA was extracted from the atrazine immunized mouse spleen as template, and then cDNA was synthesized from mRNA using M-MLV reverse transcriptase. Heavy chain variable region gene (VH) and light chain variable region gene (VL) were amplified separately. After that, the VH, VL, and GS-linker were connected into scFv using overlapping PCR technology. Then the scFvs was cloned into T7Select(?) 10-3b vector. After the in vitro packaging and amplification by infecting the host bacteria BLT5403, a capacity of 1.0×105, titer of 2.0×1011 antibody library was constructed.2 Screen anti-atrazine scFv by direct coatingWe aminated the ELISA plate and used the active ester method to directly couple hydroxylation atrazine into the ELISA plate. After that, three rounds of panning of the antibody library were performed with "incubation - elution - amplification". By using this solid phase panning, specific phages were enriched by a 185-fold.16 phages were randomly picked from the eluted titer plates of the third cycle,9 phages were survived from the PCR verification. Sequence analysis showed that there are three phages having the same sequence. Randomly picking another three sequences and constructing expression vector. After the expression and purification, the 4 scFvs are assessed by ELISA and indirect competition ELISA,1 scFv was obtained with a high specificity and affinity.3 Screen anti-atrazine scFv by Magnetic microbeadsConjugate the carboxylated atrazine with the amino magnetic beads by using the active ester method. The antibody library was selected for four cycles of semi-solid phase panning, specific phages were enrichment of a 369-fold. Randomly picking up 47 phage plaques from the fourth round of the elution titer plate, the affinity and specificity of the phage display antibody were verified by phage ELISA and indirect competition ELISA. Another scFv which possessed high specificity and affinity to atrazine was obtained. |
PDF Full Text Request