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| Coding Sequence Cloning, Alternative Splicing and Expression Analysis of the CD4 Gene in Pigs (Sus scrofa) |
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Abstract Cluster of differentiation 4 (CD4) is a co-receptor and signal transduction molecule which is mainly expressed on the CD4+ cell membrane. CD4 molecule participates in the recognition of major histocompatibility complex (MHC) class Ⅱ molecules, and plays an important role in the development and differentiation of T lymphocyte subsets, and in the process during CD4+ cell antigen recognition. This study was to clone the coding sequence (CDS) of the CD4 gene and its spliceosome in pigs (Sus scrofa), and reveal the expression feature of CD4 mRNA in porcine tissues. Based on the mRNA sequence of the porcine CD4 gene (GenBank accession number: AY515292), the coding sequence of the CD4 gene was amplified in Large White pigs using reverse transcription-PCR (RT-PCR), and the expression profiling of CD4 alternative splicing was analyzed in different tissues. Meantime, the expression level of CD4 mRNA was analyzed in different tissues of Large White pigs, as well as in three immune tissues of Large White, Landrace and Songliao Black pigs by quantitative Real-time PCR (qRT-PCR). The results showed that two different CD4 coding sequences were identified (GenBank accession number: KC333253 and KC333254) by RT-PCR amplification, cloning and sequencing, and the complete type (1 375 bp) and splicing type (1 252 bp) CD4 encoding 457 and 397 amino acids, respectively; both the two CD4 coding sequences were expressed in porcine peripheral blood, thymus, lymph node and spleen, and the expression level of complete type CD4 was higher than splicing type. The qRT-PCR results showed that the expression level of the porcine CD4 gene in spleen was significantly higher than in liver, stomach, kidney, heart and muscle (P＜0.05); there was no significant difference in the CD4 mRNA level in the same immune tissue of Large White, Landrace and Songliao Black pigs (P＞0.05), and the expression level of the CD4 gene in thymus was significantly higher than that in spleen and lymph node among the three pig breeds (P＜0.05). This study provides a basis data for further investigating the structure and function of the porcine CD4 gene.
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Received: 29 December 2015
Published: 20 May 2016
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