尘螨Ⅱ类变应原Der f2和Der p2的DNA改组及生物信息学分析

DNA shuffling and bioinformatics analysis of the dust mites group 2 allergen genes,Der f2 and Der p2

目的构建尘螨Ⅱ类变应原Der f2和Der p2嵌合基因文库,并进行生物信息学分析。方法用PCR分别扩增Der f2和Der p2,扩增产物等量混合后用DNaseⅠ酶解;回收50～100 bp的片段,连续3轮无引物PCR,以Der f2基因的特异性引物进行有引物PCR,扩增片段连接于pUCm-T载体并转化E.coli DH-5α,随机挑取60个单菌落测序,应用生物信息学进行序列比对分析和抗原表位预测(包括T和B细胞抗原表位)。结果获得的10个重组子其编码蛋白不仅呈现新的Th细胞表位,也减少了B细胞表位。结论成功获得Der f2和Der p2嵌合基因文库,为后期筛选和制备高免疫原性和低变应原性的高效尘螨哮喘疫苗奠定了基础。

Objective To construct and analyze a chimeric gene library of the dust mites group 2 allergen,Der f2 and Der p2 gene by DNA shuffling method and bioinformatics respectively.Methods Der f2 and Der p2 genes were amplified by polymerase chain reaction（PCR）,respectively;the PCR products were mixed equally（v/v） and digested with DNase Ⅰ.Fragments from 50 to 100 bp were purified,reassembled by PCR without primers for 3 times continously,and amplified with specific Der f2 primers at the last.The reassembled product of original size was inserted into pUCm-T vector and transformed into E.coli DH-5α.Sixty clones were randomly picked out,sequenced and aligned using DNAMAN software.The epitopes of T helper（Th） cell and B cell were deduced by NetMHCII 2.2 Server.Results The amino acid sequences deduced from 10 recombinants not only increased new Th cell epitopes but also decreased B cell epitopes as compared with original genes.Conclusions A chimeric gene library from Der f2 and Der p2 was successfully established which may provide the basis for selection of vaccine with high immunogenicity and low allergenicity applyed in asthma treatment.