强直性脊柱炎相关抗原HLA—B2704基因克隆

目的 对ＨＬＡ－Ｂ２７０４的编码基因进行克隆。方法 通过酚提法获取基因组ＤＮＡ，用ＥｃｏＲⅠｇ／Ｌ琼脂糖凝胶分离目的基因片段，采用内闰素末端标记的寡核苷酸探针对重组体噬斑转膜原位杂交，筛选阳性克隆，并进一步克隆化，用Ｗｉｚａｒｄ λＤＮＡ纯化系统试剂盒撮以纯化λＤＮＡ。结果 经酚提法获取基因组ＤＮＡ浓度为０．。８ｇ／Ｌ，Ｄ２６０／Ｄ２８０为１．７５，经高盐洗脱和乙醇纯化法获取的ＤＮＡ浓度为０．１６

THE GENE CLONE OF ANKYLOSING SPONDYLITIS ASSOCIATED HLA-B2704

Objective To clone the genomic DNA of HLA-B2704. Methods Genomic DNA from HLA-B2704 positive patient with ankylosing spondylitis (AS) was isolated by using phenol extraction method, restricted with EcoR , and electrophoresed on 6 g/L agarose gel . A partial genomic DNA library was constructed with these size-selected DNA in the EcoR site of gt10 .The construction of genomic DNA libraries with gt10 EcoR vectors was carried out with ligation and packing system kit. After packing in vitro ,the recombinant gt10 were transfected to E.Coli C600 hflA.The recombinant gt10 was initially screened by in situ transmembrane hybridization using a radiolabelled-end oligonucleotide probe to identify the positive plaque containing HLA-B27 gene. Further cloning was repeated by the same procedure, and the gt10 recombinant lysate was purified by using Wizard DNA purifying system kit. ResultsWT5BZThe concentration of genomic DNA obtained by phenol extraction reached 0.8 g/L. The ratio of D260/D280 was 1.75. The concentration of 6.5 kb DNA purified by high salt electroelution and 70% ethanol precipitation reached 0.16 g/L,with the ratio of D260/D280 being 1.80. The recombinant phage plaques were colourless. By screening the library with a radiolabelled-end oligonucleotide probe, 3 positive plaques were isolated. The phages from the positive plaque were used to trabsfect E. Coli C600 hflA to prepare gt10 recombinant lysate. The lysate was purified by using Wizard DNA purifying system kit. Conclusions Through a series of complex procedures, a HLA-B2704 genomic DNA library was constructed as a preliminary work for establishing a transgenic rat model for HLA-B2704.