Cloning Of Chicken Bcl10 Gene And Its Expression In Vitro

B cell lymphoma-10, an intracellular protein, is an apoptosis regulator. In the N-terminal of this protein, a caspase recruitment domain (CARD) is formed by six anti-parallel a-helixs (13-101 residues), which is quite like a death domain. Bcl10 can induce cell apoptosis, which has a highly homology with the region in E10 gene of the horse herpes virus-2. It can also mediate nuclear factor-κB (NF-κB) activation through taking part in lipopolysaccharide (LPS)/Toll-like receptors-4(TLR4) signaling after LPS stimulation. It is evidence that wild Bcl10 can activate NF-κB by Caspase-9 activation in vitro. Using knockout mice, scientists found that the mice can create normal number of immature B cells, but the B cell can't maturate due to the absence of Bcl10 impeding the activation of NF-κB. Meanwhile, it is also found that the occurrence and development of mucosa-associated lymphoid tissue (MALT) lymphoma was concerned with Bcl10 gene, researchers only discovered that the full-length Bcl10 has these features. Furthermore, researchers found that some protein complexes containing the Bcl10 protein play a role in activating NF-κB and some new functions of the Bcl10 gene was discovered later. The mouse Bcl10 gene was obtained in 1999 and the swine Bcl10 gene in 2008 by cloning. To investigate the function of the Bcl10 gene in chickens, a 959 bp cDNA was obtained by in silico cloning according to the Bcl10 sequence of human. After systematically analyzing the function of chicken Bcl10 gene, the prokaryotic and eukaryotic expression vectors were constructed, and the expression of the chicken Bcl10 gene in E coli and Vero cells were successfully achieved. The details are as follows:(1) A 959 bp cDNA was obtainde by in silico cloning according to human Bcl10 sequence. It is confirmed that the ORF of chicken Bcl10 gene contained 735 nucleotides, encoded 233 aminos, weighted 27.1 ku, and had typical Kozak sequence. The Bcl10 gene was located on the chromosome 8 of chicken genome, which included three exons; meanwhile the exons are in accord with GTAG rule. The protein encoding by BcllO doesn't contain transmembrane domain and it was likely a nuclear protein. In this study, the RNA from chicken spleen was obtained, and we found two bases difference between the sequence by PCR amplification and the one by in silico cloning, but the difference has no effect on the sequence of the corresponding amino acid. (2) The products was amplified by PCR and connected to pET-32a vector, which was named pET-Bcl10 and transformed to BL21 competent cells. The pET-Bcl10 plasmid was proved to be successfully constructed according to the determination of double digestion and sequencing. Positive bacteria contained pET-Bcl 10 plasmid was induced by IPTG. The band appeared in 47.5 ku by SDS-PAGE and the concentration of IPTG is 0.1,0.3,0.5mmol/L, the time after 6 hours and the temperature was 28℃. The result showed that Bel10 gene was expressed in E coli.(3) The pEGFP-Bcl10 was constructed by PCR and connected to pEGFP-C1 vector. And it is suggested that the pEGFP-Bcl10 plasmid was successfully constructed based on double digestion and sequencing. The pEGFP-Bcl10 vector and pEGFP-C1 vector were transfected to Vero cell line by lipofectin, and the fluorescence was obviously discovered in the cells by fluorescence microscope, which showed that the pEGFP-Bcl10 vector and pEGFP-C1 vector were successfully transferred to Vero cells. The cellular protein was obtained by Western Blot after a 60 h inoculation, a band in 60ku was found is the proteins get from the cell transferred pEGFP-Bcl10 vector and a band in 33ku is the product of cell transferred pEGFP-C1 vector. The results indicated that the Bel10 gene was successfully expressed in Vero cells.