Preparation Of Monoclonal Antibody Against Molt-inhibiting Hormone Of Eriocheir Sinensis And Its Expression In Its Optic Nerve

Eriocheir sinensis,commonly known as crabs,are arthropod doors,crustaceans,decapoda,is one of China’s five major economic crabs.For crustaceans,only through the continuous molting to complete its growth and development,but the molting is a very complex physiological process,not only by the nervous system regulation,but also by the endocrine system.Molt inhibiting hormone(MIH)of Eriocheir sinensis belongs to neuropeptide hormones,which plays an important role in the molting of crustaceans,and regulates the molting process with molt hormone(MH)together.The study of MIH not only further understand its impact on the molting mechanism,but also put forward the theoretical guidance of production practice.In this experiment,MIH prokaryotic expression vector was successfully constructed by gene recombination technique and expressed in Escherichia coli BL 21(DE3).By ultrasonically breaking the expressed cells,we purified supernatant and inclusion bodies after centrifugation separately,It was found that prokaryotic expression of the molting inhibitory hormone recombinant protein in the form of inclusion bodies.The inclusion bodies was dissolved with 8M urea and purification by nickel column affinity chromatography.Using the protein to immunizing mice,after immunizations,the titer of the antiserum was more than1:10000,which indicating that the mouse is suitable for the experiment of cell fusion.In the experiment of cell fusion,positive hybridoma cells were detected by indirect ELISA.There are 13 positive wells were detected,including 6 strong positive wells.Subclones were screened by limiting dilution to screen monoclonal hybridoma cell lines which can stable hereditary.A monoclonal hybridoma cell line was successfully screened by 3subclones.The titer of the monoclonal hybridoma cell line was 1: 51200 detection by indirect ELISA.The monoclonal antibody was prepared in a large amount of mouse ascites by this strain,the titer of the monoclonal antibody was 1:102400,and the concentration was 2.8mg/mL.The results of Western blot showed that the prepared MIH monoclonal antibody could bind to MIH in vivo.Application of immunofluorescence technique,the MIH monoclonal antibody was usedto study the expression of MIH in the optic nerve of the eye socket.The results showed that the cell types 1,2,3 and 4 were involved in the secretion of MIH.