Cloning And Expression Of Swine Gene BCL-G_L And Preparation Of Its Polyclonal Antibody

Apotosis,named as programmed cell death or cell suicide,is an array of changes which are induced by many genes. It is a conservative pathway that exists extensively in all mammalian cells. Apoptosis is a necessary mechanism that regulates the development of organism. Once the regulation failed, sickness, abnormality or death may be induced. For the immune system, apoptosis is not only an indispensable segment in its development but also a manner in which the immune system functions. Many gene families are involved during the beginning and the regulation of apoptosis, in which BCL2 family is one of the gene families that link closely with apoptosis. BCL-G, also named as BCL2-like 14, belongs to the BCL2 family, was identified from a couple of organisms, such as human, mouse and cattle. It was proven to be a pro-apoptotic protein. The new gene BCL-G_L in swine was firstly cloned in silico and then was cloned by experiments according to Evolutionism and Gene Ontology. Then, swine BCL-G_L was cloned into prokaryotic expression vector pET-32a(+) and eukaryotic expressing vectors pEGFP-C1 and pcDNA3.1(+). The prokaryotic protein BCL-G_L was extracted for the preparation of its polyclonal antibody. A number of results were obtained, as follows:1. According to Evolutionism and Gene Ontology theory, Blast was made in swine EST database using human BCL-G sequence as a seed and the swine BCL-G sequence was obtained, and its characteristics were also analysed. The results show that the ORF of the gene contains 990 nucleotides and can be translated into 329 amino acids. There are couples of terminal codes in the upstream of ORF and there are polyA tails in the downstream of the ORF, the gene contains 6 exons and 5 introns.2. The amino acid sequence was blasted with the homologous proteins of other organisms. The results show that the similarity to the homologous protein of cattle is up to 75%, and the conservative domains BH2 and BH3 of BCL2 family were found in the protein.3. The primers was made according to the in silico sequence and the cloning sequence was obtained by RT-PCR from swine spleen. The cloning sequence was identified by sequencing and compared with the contig sequence.4. Swine BCL-G_L was cloned into prokaryotic expression vector pET-32a(+), and expressed in E.coli.BL21(DE3). The prokaryotic protein BCL-G_L was extracted to immunize the guinea pigs for preparing its polyclonal antibody. The titer of the polyclonal antibody was tested by ELISA; Swine BCL-G_L was cloned into eukaryotic expression vectors pEGFP-C1 and the reconstructed vectors were transfected into swine umbilical vein endothelial cells. Then, the specifity was tested by western blot assay.5. BH2 domain deficient, BH3 domain deficient and BH23 domains deficient BCL-G_L gene was cloned into eukaryotic expression vectors pEGFP-C1 or pcDNA3.1(+) in order to investigate the function of swine gene BCL -G_L.