新等位基因人类白细胞抗原B＊15：133的鉴定及其原核表达

背景：国际上至2010-04共报告人类白细胞抗原4633个等位基因,中国自2000年至少发现200个新的等位基因,由于资金和时间有限,大部分新等位基因未作血清分型、家系研究以及进一步的功能性研究。目的：在健康志愿者人群中发现新等位基因,并对其进行家系研究,同时构建重链胞外区多肽的原核细胞表达载体,鉴定其在原核细胞中是否表达。方法：利用PCR-SSO和PCR-SBT技术,对人群的人类白细胞抗原进行筛选。使用血清学和SBT技术对拥有新等位基因的供者进行家系分析。用分子克隆的方法构建表达载体转染原核细胞,运用Western-blot鉴定是否表达。结果与结论：发现一个新等位基因与人类白细胞抗原B＊15：18：01在第419位相差一个核苷酸,C-〉T,即Codon116位TCC-〉TTC,导致氨基酸由丝氨酸改变成苯丙氨酸。最终确定命名为人类白细胞抗原B＊15：133。血清学表达B71（70）抗原。发现此供者的新等位基因来自于母亲。表达载体pET30a（＋）-B＊15：133在原核细胞中表达的多肽可被抗HIS标签单克隆抗体特异性识别。结果表明,使用分子生物学技术在大样本的筛查下发现了新等位基因人类白细胞抗原B＊15：133,利用分子克隆的方法成功构建表达载体pET30a（＋）-B＊15：133,并在原核细胞中有大量高纯度蛋白表达。

Identification of a human new allele human leucocyte antigen-B＊15：133 and its prokaryotic expression

BACKGROUND： In total 4 633 alleles have been reported in world up to April 2010. More than 200 new alleles have been found in China, most of which need to be further studied. OBJECTIVE： Serologic and genetic studies were performed to confirm the identity of a new human leucocyte antigen （HLA） allele within the Chinese population to construct an expression vector containing the heavy chain of HLA-B＊15：133 in a prokaryotic system and to identify its activity. METHODS： During routine typing work, genomic DNA was typed by PCR-SSO and PCR-SBT. An ambiguous result was found and identified as a novel allele by Serologic and DNA sequence analysis. The extra-membrane gene fragment of HLA-B＊15：133 （digested with BamHⅠand HindⅢ） was inserted into vector pET 30a（＋）. The expression vector pET30a（＋）-B＊15：133 was transfected into the BL21（DE3） cells, and western-blotting was used to identify its expression. RESULTS AND CONCLUSION： A new allele which had one nucleotide substitution at position 419 from C to T （condon116 TCC-TTC） was identified, resulting in an amino acid change from Serine （S） to Phenylalanine （F）. Serologic specificity is B71 （70） genetic and serological analysis indicated that the novel HLA-B allele of the donor was inherited from his mother. The restriction endonuclease digestions and PCR suggested the expression vector pET 30a（＋）-B＊15：133 was constructed successfully. A Western-blot identified the extra-membrane polypeptide chain of HLA-B＊15：133. The expression vector pET30a（＋）-B＊15：133 can express the extra-membrane polypeptide chain of HLA-B＊15：133 in BL21（DE3）.