猪囊尾蚴抗原基因6H的克隆、表达及鉴定

目的为寻找猪囊尾蚴病新的免疫学候选诊断分子奠定基础。方法设计合成引物,用PCR法从cDNA文库中扩增出猪囊尾蚴抗原6H基因编码序列,将其克隆入PMD-18T载体,然后在原核表达载体PET-28a中亚克隆,用IPTG诱导表达,SDS-PAGE和Western blot观察表达结果。结果用PCR法扩增出一条大小约774bp的特异性片段,克隆质粒PMD-18T-6H和原核表达质粒PET-28a-6H作BamHⅠ和XhoⅠ双酶切和以重组质粒为模板进行PCR扩增,均可获得一条与PCR产物一致的DNA片段。诱导表达后,经SDS-PAGE可见一条约33kDa大小的融合蛋白条带,Western blot结果显示其可与猪囊尾蚴病人血清起反应。结论本实验成功地克隆了猪囊尾蚴抗原6H编码基因,并在原核细胞中进行了表达及鉴定,为进一步免疫诊断研究奠定了基础。

Molecular cloning, expression and identification of 6H gene from the cDNA library of cysticercosis cellulosae

Objective To probe a new candidate molecule for providing the basis for further study on immunodiognosis of cysticercosis. Methods Primers were designed and synthesized based on the 6H encoding sequence. The DNA fragment was amplified by PCR from the cDNA library of Cysticercosis cellulosae. The PCR products were cloned into PMD-18T vector, then the target gene was subcloned into PET-28a expression vector. IPTG was added to induce fusion expression and the expression was identified by SDS-PAGE and Western blotting. Results The PCR amplified product was 774bp in size. The size of PMD-18T-6H and PET-28a-6H digested with BamH I and Xho I was identical to the length of the PCR generated products. After inducible expression, one fusion protein band with about 33kDa in size was identified by SDS-PAGE. The fusion protein could be recognized by sera of the patients infected with cysticercosis. Conclusions The cDNA encoding antigen 6H gene of Cysticercus cellulosae has been cloned and expressed successfully,which provides the basis for further study on immunodiognosis of cysticercosis.