Molecular Characterization,Alternative Splicing And Subcellular Localization Of Porcine Sp110 Gene

Domestic and foreign researches found that Sp110 participated body’s specific immune response and antiviral response, a candidate gene that interrelated with the liability of human pulmonary tuberculosis, which act as transcriptional activator in the cell. Study about Sp110 on human is more, but not yet seen on porcine. In this study we take porcine Sp110 as research object, using a series of molecular biological methods such as cloning and sequencing to clone its c DNA sequences and splice variants; using Minigene technology to analyze its variation splices situation deeply; analyze its subcellular localition by liposome mediated transfection method; detect its tissue expression and inducible expression through Real time PCR. The results of this study were as followed:(1) The porcine Sp110’s cDNA sequence were cloned successfully and obtained 27 splice variants(GenBank accession Nos. KC811303-27、KF898045、JX280458), among these splice variants, the V26 is the longest and considered as reference(R) sequence, relative to which the others were formed for the sequences deficiency. The amplification full-length of R is 2016 bp with 11 bp 5’-UTR, 1950 bp CDS region and 55 bp 3’-UTR, expected to encode 649 amino acids. Compared with R, V、V1、V5、V6、V9、V14、V20 and V22 were deletion mutation, the others were all frameshift mutation which leading to early termination of trans lation.(2) Using exon 3/14/15 to build minigene vector, found 7 new splicing patterns(NV1-7) in this region. The NV1/5/6 were composed of partial sequences of exons 3 and 15, but with the different splice sites. NV2 was composed of complete exon 3 and 15. Both NV3 and 4 are composed of exons 3, 14, and 15: NV4 contained the complete sequence of these three exons, while NV3 has a 17 bp deletion in exon 14; NV7 was composed of the 5′ end of NV2 and the 3′ end of NV1, with 15 bp of intron 14 between them.(3) Subcellular localition result analysis shows that, only splice variants R and V located in the nuclear, while the others lost the nuclear localization ability. Further analyze the truncated mutants, indicated that the nuclear localization located in R’s 250-280 aa. Bioinformatics showed that 254-279 aa(KKGKKKKRCIWATPKRRHKKKCLPRE)is the nuclear localization sequence.(4) RT-qPCR detected that the splice variants R and V have the similar tissue expression profile: express in all tissues, and spleen tissue have highest expression quantity. Under the stimulation of Poly(I:C), R and V’s expression quantity increased with the similar reaction, and they had similar half-life. But the competitive RT-PCR analysis indicated that, among all tissues the expression quantity of R is higher than V’s. These results suggested that, the splice variants R and V have similar activity, and R is the main active form of the porcine Sp110.