In Silico Identification, Molecular Cloning And Verification Of A Novel Swine Gene Homologous To Human BCL10 Of Innate Immunity And Its Preliminary Expression Profiles In Pigs

Innate immune system is the first line of host defense that prevents host body from being infected with pathogen. As an important functional gene, BCL10 (B cell lymophoma-10) plays an essential role in the innate immune system. Results of cells transfection in vitro demonstrated that protein BCL10 activated the signal transduction pathway of NF-κB protein family and induced cell apoptosis. By activating the NF-κB signal transduction pathway, BCL10, which was expressed at physiologically normal level, promoted the development and maturation of B cells, increased the production of immune factors, and boosted the innate immunity of host body. Whereas, over expression of BCL10 could induce a series of secretion of immunosuppressive factors, and even lead to several diseases, such as lymphoma. To investigate the function of the novel swine BCL10 gene in pigs, Bioinformatics methods and Molecular Biology methods were used, and swine BCL10 gene was obtained by silico cloning, according to human BCL10 sequence. We systematically analyzed the function of swine BCL10 gene using Genomics methods, concluded the characteristics of the newly found gene and put up with some rules of protein expression, as follows:1. BCL10 was analyzed on the basis of Gene Ontology and homologous alignment. Human BCL10 gene was searched, data of pig Expressed Sequence Tag and swine genome were acquired, and pig homologous gene was discovered by silico cloning. The open reading frame (ORF) was searched, full-length cDNA was identified, and gene transcriptional regulation sequence was analyzed. Results suggested that the ORF contained 702 nucleotides, coding 233 aminos, and the 5'terminal was abundant with GC sequences. The first initiation codon of the largest ORF was ATG, followed by ACC. The ORF contained typical Kozak sequence, meeting the demand of eukaryotic cells transcription. The novel swine gene was submitted, and the accession number (EU088132) was available on GenBank. 2. Genetic evolutionary characteristics of protein BCL10 were analyzed, and functional domain was predicted. Results implied that secondary structure of protein BCL10 was helix mainly. The protein had no transmembrane domain, indicating that it was a nuclear protein. Moreover, the protein was verified to contain three exons, and locate on the chromosomeⅣ. Amino acids contained a CARD domain, indicating that protein BCL10 may be an important factor for cell apoptosis, which may offer a possible direction to further investigation.3. To determined the expression abundance of BCL10 in the seven tissues (heart, kidney, spleen, thymus, brain, liver and lymphonodi mandibulares), semi-quantitative PCR was applied. And single factor experiment and significance test of difference were carried out by software SPSS11.5. Results indicated that BCL10 mRNA was expressed at the highest level in spleen, followed by thymus, brain and lymphonodi mandibulares. Trace amount of mRNA was expressed in liver. None was detected in kidney. So the distributing characteristics of BCL10 in pig tissues were preliminary accounted.4. BCL10 fragment was ligated into eukaryotic expression vector pEGFP-C1 which contained a green fluorescence reporter gene, thus recombinant plasmid pEGFP- BCL10 was constructed. The recombinant plasmid and the control plasmid pEGFP-C1 were respectively transfected to PK-15 cells by Lipofectamine 2000 Regent. Transfected cells were both screened by optimum concentration G418 for 12 days, and positive transfected clones were selected. Positive cells were observed with inversion fluorescence microscope, and were further verified by RT-PCR. Meanwhile, prokaryotic expression system pET-BCL10 was constructed. Recombinant plasmid was transformed to BL21 (DE3), in which protein expression was detected at a high level. From the results above, conditions and profiles of BCL10 gene expression in vitro were acquired preliminary. And the findings may offer some references to the further revealing of BCL10 protein immunological properties, detection kit preparation and function exploration.