尘螨1类变应原基因的DNA改组及生物信息学分析

DNA shuffling and bioinformatics analysis of the dust mites group 1 allergen genes,ProDer f1 and ProDer p1

目的构建尘螨1类变应原ProDer f 1和ProDer p 1基因的改组文库,并筛选出编码优势T细胞表位和低B细胞表位的嵌合基因。方法 PCR扩增ProDer f 1和ProDer p 1基因,等量(V/V)混合PCR产物,DNaseⅠ酶切200s,回收50～150bp的基因片段,进行3轮无引物PCR,以此为模板分别用ProDer f 1和ProDer p 1基因的特异性引物进行有引物PCR,切胶回收与ProDer f 1和ProDer p 1基因大小相当的基因,连接pUCm-T载体,并转化E.coli DH-5α,涂LB板(Amp+)37℃过夜培养,随机挑取单克隆菌落进行PCR验证,100个阳性克隆测序并进行序列比对及T细胞和B细胞表位预测。结果 ProDer f 1特异性引物扩增并筛选出10个改组明显的重组基因,生物信息学分析显示:与野生型变应原相比,6个重组子的T细胞表位增加,B细胞表位减少;而ProDer p 1特异性引物扩增的基因未发生明显交换。结论应用DNA shuffling技术成功构建尘螨变应原ProDer f 1基因的嵌合文库,并筛选出了编码优势T细胞表位、低B细胞表位的嵌合基因。

A chimeric gene library derived from the dust mites group 1 allergen,ProDer f 1 and ProDer p 1 gene,was constructed by using DNA shuffling method,and screened and analyzed by bioinformatics.ProDer f 1 and ProDer p 1 genes were amplified by PCR,respectively;the PCR products were mixed equally(v/v) and digested with DNase I for 200 sec.DNA fragments of 50 bp-150 bp were recovered and reassembled by primerless PCR for 3 rounds with ProDer f 1 specific primers.The reassembled product with correct size was inserted into pUCm-T vector and transformed into E.coli DH-5α.One hundred clones were picked out randomly,and sequenced and analyzed by multiple sequence alignment software DNAMAN.T helper(Th) cell epitopes and B cell epitopes were also predicted at NetMHCII 2.2 Server.As a result,ten recombinants with exchanges of DNA fragment were obtained.Compared with parent genes,six of these recombinants were characterized with increased Th cell epitopes and decreased B cell epitopes,according to their encoding amino acid sequences.However,there was no exchange occurred using ProDer p 1 primers.In brief,the chimeric gene library was successfully established using ProDer f1,and ProDer p1 as template,and chimeric proteins with increased T-cell epitopes and B-cell epitopes were also selected.