Recombinant And Prokaryotic Expression Of ?-Enolase From Three Species And Generation Of Monoclonal Antibody Specific Against ?-Enolase

Enolase is highly conserve during the evolution process, as one of the most abundant glycolytic enzymes in the cytosol as is known to all. It catalyzes the conversion between 2-phosphoglycerate and phosphoenolpyruvate during glycolysis and gluconeogenesis, respectively. Apart from its control of energy production,?-enolase takes part in the rheumatoid arthritis morbidity process as autoantigen. The antibody in rheumatoid arthritis serum to cycle citrullinated peptide and citrullinated enolase form in serum relate to the positive reaction with anti-citrullinated protein. Though the auto-antibody to ?-enolase exsit extensively in rheumatoid arthritis, whether a-enolase is one of the pathogenesis of rheumatoid arthritis is unknown. This thesis focus on generation the monoclonal antibody specificity against enolase.In this study, enolase in Escherichia coli, Porphyromonas gingivalis and human beings are obtained by polymerase chain reaction amplification, then recombinant expression plasmid were constructed and expressed in Escherichia coli by molecular cloning technique. The three reconstructed proteins expressed soluble and high-efficiently. Mice were immunized by these three antigens in the same way. The specificity of three antibody against enolase in serum were detected by indirect ELISA. The hybridoma technique was used to generate the monoclonal antibody. The results show as follow:1. Enolase genes come from three different sources were obtained from Escherichia coli, Porphyromonas gingivalis and human beings by PCR amplification. The recombinant plasmids were constructed and expressed in E. coli BL21 (DE3). After analyzed by SDS-PAGE, the soluble, high-efficiency expressions were detected. Compare with the inducible temperature, inducible time, IPTG concentration, the optimal express condition is as follows:37?,0.5 mM IPTG,6 h.2. After inducing the E. coli BL21 (DE3) which contain the recombinant pET-eno at the optimal condition, the inocula were collected after culture for 6 hours when the expression is maximum. The supernatant was obtained by the high pressure cracker and samples were loaded to SDS-PAGE. The precool potassium chloride in 4? was adopt to coloration the objective strap, then the strap was cut and pulverize into pasty. Those Balb/c mice were injected multiple sites subcutaneous by these pasty after diluted with normal saline to the 0.1 mg/mL. The mice were immuned every 2 weeks,4 times in all. The tail vein blood was suck up before every injection. Indirect ELISA was used to detect the titer serum, which mice serum has the highest titer was chose to generate for the monoclonal antibody.3. Generation of monoclonal antibody:The spleen cells of the immuned mice were fused with a myeloma cell line during logarithmic phase. The number of these two cells ratio is about 1:5. After fused for 12 hours, the HAT selective medium was added.10 or 15 days later, double-antibody sandwich ELISA was used to screen positive clone. One strain of monoclonal antibody with enolase specificity was obtained.