CAJ | 학술논문

运用优化的扩增和克隆测序技术，建立了人类白细胞抗原( HLA-B)基因的高分辨率分型方法。针对HLA-B基因保守区序列设计引物进行等位基因扩增，基于质粒不相容原理将杂合型等位基因有效克隆入质粒DNA中，经细菌培养后进行Sanger测序，根据测序结果经ClustalX2软件分析和IMTG/HLA数据库的BLAST比对即可完成HLA-B基因的高分辨率分型。利用建立的方法对7例临床样本进行了HLA-B基因分型，并且与第三方直接碱基序列分析基因分型技术( PCR-SBT)进行比对，结果完全一致。本方法无需专业分型软件，准确度高，成本低；采用通用引物进行等位基因的扩增和测序，无需传统方法中繁琐的引物设计和过程优化，实现了HLA-B基因的高分辨率分型。
운용우화적확증화극륭측서기술，건립료인류백세포항원( HLA-B)기인적고분변솔분형방법。침대HLA-B기인보수구서렬설계인물진행등위기인확증，기우질립불상용원리장잡합형등위기인유효극륭입질립DNA중，경세균배양후진행Sanger측서，근거측서결과경ClustalX2연건분석화IMTG/HLA수거고적BLAST비대즉가완성HLA-B기인적고분변솔분형。이용건립적방법대7례림상양본진행료HLA-B기인분형，병차여제삼방직접감기서렬분석기인분형기술( PCR-SBT)진행비대，결과완전일치。본방법무수전업분형연건，준학도고，성본저；채용통용인물진행등위기인적확증화측서，무수전통방법중번쇄적인물설계화과정우화，실현료HLA-B기인적고분변솔분형。
Ahigh-resolutionmethodforhumanleukocyteantigen-B(HLA-B)genotypingwasestablished based on the optimized polymerase chain reaction, cloning and sequencing technology. The exon2 and exon3 of HLA-B gene were amplified with primers based on the HLA-B gene sequence. These produced heterozygous alleles were effectively cloned into plasmid DNA based on the principle of plasmid incompatibility, and were followed by bacterial culture. Then Sanger sequencing was carried out and after analyzing the result by software ClustalX2 and IMTG/HLA database comparison, the HLA-B genotype of the samples was achieved. Seven clinical samples were detected, and the results were consistent with those of PCR-SBT genotyping method. The method was cost-effective, high-resolution and it did not require technical software. The use of universal primers simplified the cumbersome design and optimization process of specific primers.