SPF大白猪和长白猪CIITA基因剪接突变体的鉴定分析及在不同组织中的表达模式

为了鉴定分析SPF大白猪和长白猪CIITA剪接突变体，研究其在猪不同组织中的表达模式。本研究以5周龄的8头SPF大白猪和7头SPF长白猪为研究对象，设计特异性引物对CIITA基因进行扩增、克隆和测序，对获得的序列分析其剪接突变体特征，利用在线软件对不同剪接突变体进行生物信息学分析，同时利用荧光定量PCR方法检测CIITA基因剪接突变体在2头SPF长白猪8种组织中的表达模式。结果显示，在SPF大白猪和长白猪CIITA基因CDS区存在3种不同的剪接突变体（CIITA-A、CIITA-B和CIITA-C），其CDS长分别为939、984和1 698 bp。CIITA-A和CIITA-B突变体由于Exon 11部分核苷酸缺失（40和199 bp）使终止密码子提前，从而致使编码CIITA蛋白的结构域受到了剪接的影响，缺失了末端富含亮氨酸的4个重复结构域，蛋白结构发生了明显的改变。进化树结果表明，针对CIITA，猪与绵羊和牛具有较近的亲缘关系，在不同猪品种中具有较高的保守性。同时，CIITA不同剪接突变体在猪8种组织中均有表达，且表达量趋于一致，均在脾或肺中表达量较高，其次是肾、脊髓、甲状腺、肝、胸腺，在心脏中表达最低。本研究从SPF大白猪和长白猪上共获得了3个CIITA剪接突变体，其中两个蛋白结构缺失了4个富含亮氨酸的重复结构域，且不同剪接突变体在猪不同组织中均有表达，表达量稍有差异，为后续CIITA基因在机体内的免疫调控功能研究奠定了理论基础。

Identification and Analysis of CIITA Gene Splicing Mutants in SPF Yorkshire and Landrace Pigs and Their Expression Patterns in Different Tissues

The study aimed to identify and analyze CIITA splicing mutants in SPF Yorkshire and Landrace pigs and study their expression patterns in different pig tissues. In this study, the CIITA gene was amplified using specific primers, cloned and sequenced in 8 SPF Yorkshire and 7 SPF Landrace pigs aged 5 weeks, and the splicing mutant characteristics of the obtained sequences were analyzed. The online software was used for bioinformatics analysis of different splicing mutants. The expression patterns of CIITA splicing mutants in 8 tissues of 2 SPF Landrace pigs were detected by fluorescence quantitative PCR method. The results showed that 3 different CIITA splicing mutants (CIITA-A, CIITA-B and CIITA-C) in SPF Yorkshire and Landrace pigs were identified with CDS lengths of 939, 984 and 1 698 bp, respectively. The deletion of partial nucleotides (40 and 199 bp) in Exon 11 in CIITA-A and CIITA-B mutants made the termination codon appeared in advance, which resulted in the splicing of the domain encoding CIITA protein and the deletion of 4 leucine rich repeat domains, the structure of the encoded protein had changed obviously. The results of phylogenetic tree showed that pigs had close relationship with sheep and cattle, and had higher conservation in different pig breeds for CIITA. Moreover, different splicing mutants of CIITA were expressed in 8 pig tissues, and the expression levels tended to be consistent, with high expression levels in spleen or lung, followed by kidney, spinal cord, thyroid, liver and thymus, and the lowest expression level in heart. A total of 3 CIITA splicing mutants were obtained in SPF Yorkshire and Landrace pigs, two of which deleted 4 leucine rich repeat domains. Different splicing mutants were expressed in different pig tissues with slightly different levels. The results of this study lay a theoretical foundation for the further studies on the immune regulation function of CIITA gene in the host.