Preparation And Identification Of Monoclonal Antibodies To Resist Zhangfei Fusion Protein

Zhangfei is a basic region-leucine zipper(bZIP) protein. Its molecular mass is about 28.9ku. It is one of transcription facters participating regulation in endoplasmic reticulum stress and unfolded protein reaction. It is a novel cellular binding partner of activating transcription factor 4(ATF4). Unfolded protein is used to hint the genesis of stress reaction in endoplasmic reticulum generally. There is UPR in some tumour cells. UPR is very important in the physiology and pathological process of embryonic development and homo-disease. Family members of Leucine Zipper Protein such as Zhangfei, Luman have participated in ERS. Therefore, we have done researches, such as induction expression and depuration of Zhangfei protein, immune animal. We have prepared and identitied its McAb. The results as follow:1 We have sieved the best way of expressing Zhangfei Fusion Protein by induction of IPTG, Shaked cultivation at 37℃, Final concentration of IPTG: 3 mmol/L, the time: 7h.2 Comparing the two methods of depurating protein: Electroeluting and Glutathione Sepharose 4B; we obtain Zhangfei Fusion Protein that was higher purity by electroeluting.3 Using indirect ELISA to detect the valence of antibody in serum. The valence of antibody in mouse and rabbit immunized by Zhangfei Fusion Protein reach up to 1∶5×103 and 1∶3×106. Zhangfei protein has better immunogenicity.4 Using indirect ELISA to detect masc-clone, Cloning efficiency: 98.96%. Masc-Cloning efficiency: 30.18%. Subclone the positive cells by limiting dilution for fifth times to get identical cells, we got two positive hybridoma cells named ZFD1 and ZFD2.5 Valence of antibody resist Zhangfei Fusion Protein are 1∶2×105 and 1∶105 in abdominal dropsy. The antibody-serecting ability of the two hybridoma cells keep stable after serial subcultivation and resuscitation several times. The antibody-serecting ability of them keep stable after frozening and subcloning several times. The subtypes of monoclonal Antibodys are IgG1. Affinity constant of monoclonal Antibodys from ZFD1 and ZFD2 are 2.102×108 and 2.313×108.The monoclonal antibody is a foundation for detecting expression of Zhangfei protein in cell and tissue of animal. It offers rational for researching its regulations in geno- endocrine secretion.