Cloning And Bioinformatics Analysis Of Swine Leukocyte Antigens Class Ⅰ And Class Ⅱ Genes In Guizhou Minipigs And Establishment Of PCR-SSP Typing Method

In porcine，the MHC is usually called swine leukocyte antigen（SLA），whichincluded three major gene cluster（sclass I, class II and class III）and been mapped to thecentromere of chromosome7spanning. The MHC is a gene-dense region containing anumber of important immune-related genes，They are highly polymorphic and associatewith disease resistance and economic traits which encode a series of membrane-boundcell surface glycoproteins and play very important roles in immune system. As improvingof xenotransplantation, the study in providing the organ grafts of pigs for human is paidmore popular attention，because the lack of organs from human and the similarphysiological characteristics between human and porcine. The pigs are usuallyconsidered as an ideal animal for human xenotransplantation, not only the similarity insize and physiology between pig and human, but also the high structural homology ofmajor histocompatibility complex (MHC). In recent years, a mount of SLA alleles andhaplotypes of different herds in the worldwide have been characterized, which aresystematic named by the SLA Nomenclature Committee of the International Society forAnimal Genetics (ISAG) and available in the Immune Polymorphism Database. Guizhouminipigs is a precious rare species which maintained as a closed colony from Guizhouprovince in China. It has been characterized as a valuable resource that is excellentlysuited for laboratory animals with small body size， early sexual maturation andpurebreeding. The research about SLA is later than H-2, RT1and HLA, and althoughplenty of minpigs species in our country, this work have not took on prosperity. Pigs withwell characterized SLA haplotypes that are homozygous in their SLA loci are invaluablefor the study of vaccines, production performance and immune responses to allo-orxenografts.In this study, we have characterized three SLA class I genes (SLA-1, SLA-3, SLA-2)and four SLA class Ⅱ genes（SLA-DRA, SLA-DRB1, SLA-DQA, SLA-DQB1）of22founder Guizhou minipigs using sequence-based typing method. Objective genes areamplified by RT-PCR, purified products were cloned into pMD18-T vectors andbi-directional sequenced, and then compared with the SLA allele sequences in GenBankdatabase and phylogenetic trees were constructed by the neighbor-joining method usingthe MEGA4program. The research results are following：1. There are four novel alleles found on SLA-1locus of Guizhou miniature pigs, and given temporary names: SLA-1*gz01(accession: JQ361648), SLA-1*gz02(accession:JQ361649), SLA-1*gz03(accession: JQ361650), SLA-1*gz04(accession: JQ361651). Inthe SLA-1phylogenetic tree, the novel alleles of SLA-1*gz01and SLA-1*gz04areclassified to a same cluster of SLA-1*11allele group. SLA-1*gz02is classified to thecluster of SLA-1*08allele group. SLA-1*gz03is classified to the cluster of SLA-1*04allele group.2. There are five alleles found on SLA-2locus, and four novel alleles are giventemporary names: SLA-2*gz01(accession: JQ361652), SLA-2*gz02(accession:JQ361653), SLA-2*gz03(accession: JQ361654) and SLA-2*gz04(accession: JQ361655),another allele that has been reported is SLA-B*m(Accession: AF464049.2). In the SLA-2phylogenetic tree, SLA-2*gz01allele is classified to a cluster of SLA-2*05allele groupand closely related to SLA-2*0502. SLA-2*gz02allele is classified to a cluster ofSLA-2*08allele group. SLA-2*gz03allele is classified to a cluster of SLA-2*11allelegroup. SLA-2*gz04allele has a closely related to SLA-2*02allele group.3. There are four alleles found on SLA-3locus, and three novel alleles are giventemporary names: SLA-3*gz01(accession: JQ361656), SLA-3*gz02(accession:JQ361657) and SLA-3*gz03(accession：JQ361658), another allele that has been reportedis SLA-3*0302(Accession: EU432097.1). In the SLA-3phylogenetic tree, SLA-3*gz01and SLA-3*gz03alleles are classified to a cluster of SLA-3*04allele group.SLA-3*gz02allele is classified to a cluster of SLA-3*05allele group.4. There are two alleles were found on SLA-DRA locus, one novel allele is giventemporary names: SLA-DRA*gz01(accession: JQ361659), another allele that has beenreported is SLA-DRA*010103(Accession: EU432070.1). SLA-DRA*gz01allele has aclosely related to SLA-DRA*010101allele.5. There are two alleles are found on SLA-DRB1locus, four on SLA-DQA locus,four on SLA-DQB1locus, all of which have been reported：SLA-DRB1*0901(Accession:EU432073.1), SLA-DQA*yn(Accession: AY102475.1), SLA-DQA*0301(Accession:EU722913.1), SLA-DQA*0101(Accession: DQ883218.1), SLA-DQB1*0801(Accession:DQ303219.1),SLA-DQB1*113C3(Accession:AY135574.1),SLA-DQB1*0901(Accession: EU432063.1), SLA-DQB1*0601(Accession: DQ883219.1), additional oneSLA-DRB1allele (Accession: AK347078.1) and one SLA-DQA allele (Accession:AK230483.1) exist in the GenBank but name absent, for the sake of convenience,temporarily named as SLA-DRB1*gz01and SLA-DQA*gz01.6. Two SLA classⅠhaplotypes and two classⅡ haplotypes were obtained from the founder Guizhou minipigs. Two SLA classⅠhaplotypes and one classⅡ haplotypes arenovel, temporarily named and designated as Hp-gz01.00(SLA-1*gz01, SLA-3*gz01,SLA-2*gz01), Hp-gz02.00(SLA-1*gz02, SLA-3*gz01, SLA-2*B*m) and Hp-gz00.01(SLA-DRA*gz01, SLA-DRB1*gz01, SLA-DQA*0101, SLA-DQB1*0601), and anotherone SLA classⅡ haplotype previously published which officially named Hp-00.14(SLA-DRA*010103, SLA-DRB1*0901, SLA-DQA*0301, SLA-DQB1*0801).7. Based on25alleles sequences information, we developed a simple methodimplemented to SLA-typing for unknown offsprings of Guizhou minipigs, relying ondesigned25sequence specific primers. According this PCR-SSP typing methods, wewere able to rapidly conduct SLA typing for Guizhou breeding stock and identify fourSLA haplotypes present in the herd.