Gene Recombination And Prokaryotic Expression Of Human Mammaglobin And Preparation Of Its Monoclonal Antibodies

Human mammaglobin(hMAM), a novel breast-specific protein, is proven to be restrictively expressed in the adult benign and malignant mammary gland, so it can be used as a specific breast tumor marker. Many studies have shown that hMAM is a promising marker for breast cancer, especially in early diagnosis for this disease.Objective:To construct a prokaryotic expression vector of hMAM, establish its protein purification scheme, and prepare monoclonal antibodies by hybridoma method, so as to found a basis for early diagnosis of breast cancer and nursing intervention for finding breast cancer in early stage.Methods:1. hMAM cDNA from breast cancer tissue was amplified by RT-PCR, and then cloned into pGEM-T vector for sequencing. After the plasmid pGEM-T/hMAM was digested by Kpnâ… and Hindâ…¢, the purified fragment was subcloned into same enzymes cut expression vector pQE40. After the recombinant was transformed into E.coli M15 [pREP4], the bacteria was induced by IPTG. The recombinant protein was identified by SDS-PAGE.2.The E.coli M15[pREP4] harboring the recombinant pQE40-hMAM was fermented with IPTG induction, and lysated with supersonic wave. The collected inclusion body from the E.coli was treated sequencely by washing, denature and dissolved in urea, Ni2+ chelating affinity chromatography, dialysis renature, and anion-exchange chromatography. Bradford method was used to detect the concentration of purified fusion protein. The purity of the obtained fusion protein was measured by HPLC.3. Balb/c mice were immunized with purified fusion hMAM. Hybridoma technique, ELISA test and immunohistochemical method were used to prepare and screen the hybridoma cells which secreting monoclonal antibodies against hMAM. The subclasses and isotypes of the antibodies were identified by Mouse Monoclonal Antibody Isotyping...