利用套式PCR技术鉴别屋尘螨和粉尘螨主要变应原基因及其在尘螨疫苗研制中的应用

通过提取屋尘螨和粉尘螨疫苗研制用的原始种子和生产种子基因组DNA作模板,分别设计Der p1和Der f1各2套内外扩增引物并进行一步法套式PCR,根据扩增出目的片段来检测尘螨变应原Der p1和Der f1基因片段,并以培养基提取物为阴性对照和已通过形态学鉴别为纯屋尘螨、纯粉尘螨的基因组DNA为阳性对照。PCR产物经电泳后切胶回收并克隆到T载体上,进行DNA序列分析并在GenBank中进行同源性比较。结果显示,从屋尘螨和粉尘螨疫苗研制用的原始种子和生产种子分别扩增出含有屋尘螨和粉尘螨主要变应原Der p1和Der f1基因片段,扩增部分的序列与GenBank数据库里相应序列同源性为100%。从阴性对照提取物中未扩增得到目的基因片段,从阳性对照DNA提取物中能扩增得到目的变应原Der p1和Der f1基因片段。本研究首次应用套式PCR技术鉴别了屋尘螨和粉尘螨原始种子和生产种子,为尘螨疫苗研制以及工业化生产提供了一种有效的质量控制手段。

TO DISTINGUISH ALLERGEN GENES OF DERMATOPHAGOIDES PTERONYSSINUS FROM DERMATOPHAGOIDES FARINAE BY NESTED PCR AND ITS APPLICATION FOR THE VACCINE MANUFACTURE

In order to distinguish allergen genes of Dermatophagoides pteronyssinus from Dermatophagoides farinae, genomic DNA was separated from main species banks and working species banks for mite allergen vaccine manufacture and was used as templates for PCR amplification. The genomic DNA from culture materials without mite was also used as negative control and at the same time genomic DNA from D. pteronyssinus and D. farinaeas positive controls, respectively. The Der p1 and Der f1 DNA fragments were amplified by one step nested PCR. PCR products were cloned into pMD18-T vector and were sequenced. Sequences obtained were compared with GenBank data by BLAST. From main species banks and working species banks for mite allergen vaccine manufacture, 0.26 kb Der p1 and 0.3 kb Der f1 DNA fragments were amplified by one step PCR, which were consistent with the results from positive controls. No DNA fragment was amplified from negative control. Their DNA sequences were 100% identical with D. pteronyssinus and D. farinae from GenBank data respectively. The present method can distinguish D. pteronyssinus from D. farinae in the manufacture of mite allergen vaccine.