The Expression Of Human Bid Peptides In E.coli And Identification Of Its Polyclonal Antibodies

Apoptosis, described is programmed cell death, were described in hepatocytes by Kerr in 1972. The term "apoptosis" refers to the "falling off" of petals from flowers, or leaves from trees, for the betterment of the organism as a whole. The name is meant to reflect the widespread importance of regulated cell death in homeostasis is an active process characterized by cell shrinkage, nuclear and cytoplasmic condensation, chromatin fragmentation and phagocytosis of dying cells.It is now clear that the mitochondria play a critical role in the regulation of both apoptotic and necrotic cell death. Mitochondrial permeabilization and release of intermembrane space proteins are important features of both models of cell death. The release of caspase activating proteins during early apoptosis is regulated primarily by the Bcl-2 family of proteins.BCL-2 family proteins can generally be subdivided into three classes on the basis of their functions and the number of BCL-2 homology (BH) domains present: the anti-apoptotic members such as BCL-2 and BCL-xL that have four BH domains (BH1 to BH4), the pro-apoptotic members such as BAK and BAX that possess three BH domains (BH1 to BH3), and the'BH3-only'pro-apoptotic members , which include Bim, Bmf, Bik, Bad, Bid, Puma, Noxa and Hrk, mediate many developmentally programmed and induced cytotoxic signals.such as Bid and Bim that share homology only within the BH3 domain.Bid, BH3-interacting domain death agonist, which is mainly involved in the regulation of apoptosis at the mitochondrial level. Bid is one of the best studied BH3-only molecules in terms of its activation at the periphery and its subsequent actions at the mitochondria. While the pro-death activity is the first function defined for Bid, Bid is proven to have alternative functions promoting survival and proliferation. These properties of Bid render it to be a unique and fascinating molecule to be investigated to understand how the survival and death processes could be integrated in a cell.The anti-Bid polyclonal antibodies are essential for evaluating function of Bid in progress of apoptosis. The anti-Bid antibodies are expensive, so we developed the anti-Bid polyclonal antibodies which from rabbits immunized with GST-Bid fusion proteins for detection of Bid in western blot or ELISA.The research purpose is to construct a recombinant vectors pGEX-6p-1 which contain the gene encoding the human Bid polypeptide. These vectors were transformed into BL21 strain, the GST-Bid fusion proteins would be highly induced expression by IPTG. Purified Bid polypeptide was used to immunize the rabbit to produce large volumes of polyclonal anti-Bid sera.The pGEX plasmids are designed for inducible, highlevel intracellular expression of genes or gene fragments as fusions with Schistosoma japonicum GST. Fusion proteins are easily purified from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B contained in the GST Purification Modules. Cleavage of the desired protein from GST is achieved using a site-specific protease whose recognition sequence is located immediately upstream from the multiple cloning site on the pGEX plasmids. Fusion proteins can be detected using a colorimetric assay or immunoassay provided in the GST Detection Module.The genes of coding Bid peptides was obtained by PCR method and subsequently cloned into the vector of pGEX-6P-1. The recombinant vector was identified with digestion by BamHI and XhoI. The released gene fragments were indicated at the site of 4900bp(vectors) and 147bp( gene fragments), respectively. The sequenced results shown that we had got the coding gene of human Bid peptides. The peptides includes the twice-copy of amino acids'KALDEVKTAFPRDMENDK'for enhancement its immunogenicity.The GST-Bid fusion proteins were induced to express in BL21 by IPTG and purified with affinity chromatography. The concentration of GST-Bid fusion proteins reached 1.17μg/μl. The purified proteins was used to immunize rabbits(600μg/rabbit ) to produce anti-Bid polyclonal antiserum. The specificify and sensitivity of antiserum was tested by Western blot. The results shown that the antiserum with dilution to 1: 100from immunized rabbits can recognize the Bid.We have successfully got the anti-Bid polyclonal antibodies which can be used to detecting the Bid in apoptosis.