用PCR－RFLP分析血清学和细胞学确定的DLA－DRB1基因单倍型及其分离

AN ANALYSIS OF SEROLOGICALLY AND CELLULARLY DE-FINED DLA HAPOLTYPES OF DLA-DRB1 AND THEIR SEGREGA-TION BY PCR-RFLP

用ＰＣＲ－ＲＦＬＰ方法研究ＤＬＡⅡ类基因，包括１１只分别为８个不同ＤＬＡ单倍型的纯合子参考狗，和两个犬家族各８只狗。用引物对ＨＬＡ－ＤＲＢ－ａｍＰＡ／Ｂ扩增其基因组ＤＮＡ，获得２７４ｂｐ扩增产物。该产物被３２个能识别ＤＬＡ－ＤＲＢ区等位基因变异的、不同的内切酶酶解，只有ＨａｅⅡ、ＨｈａＩ、ＨｌｎｆＩ、ＭｓｐＩ、ＲｓａＩ和Ｓａｕ９６Ｉ酶解后分别显示出高度的多态性，被归类为９个不同的ＲＦＬＰ带型，分别与所研究的ＤＬＡ单倍型及８个细胞学确定的ＤＬＡ－Ｄ特异性相关联。对两个犬家族的分离研究证实了ＲＦＬＰ带型的分型价值。因此上述引物对和内切酶构成了一个实用的分型系统，能对ＤＬＡＤＲＢ１基因进行ＰＣＲ－ＲＰＬＰ分型。

The polymerase chain reaction based restriction fragment length polymorphism(PCR-RFLP) method was used to study dog leukocyte antigen(DLA)class Ⅱ gene. Genomic DNAfrom 11DLA homozygous reference dogs representing 8 different haplotypes and 2 families witha total of 16 animals were amplified for DRB1 with the oligonucleotide primer pair(HLA-DRB-AMP-A/B)and 274bp amplified products vvere obtained. Amplified DNA was digested with 32different restriction endonucleases,which eould recognize allelic variations within DLA-DRB, butonly with HaeⅢ,Hha Ⅰ,Hinf Ⅰ,MspⅠ,Rsa Ⅰ and Sau96 Ⅰ high polymorphism was revealedand 9 distinct RFLP patterns were obtained, which could be correlate to the DLA haplotypesand DLA-D specificities. The segregation patterns of different DLA haplotypes could be demon-strated in two families. We conclude that PCR-RFLP typing utilizing above mentioned primerpair and endonucleases is a valuable tcol for DLA class Ⅲ genotyping in the dog.