猪囊尾蚴cDNA文库的筛选与重组蛋白的初步鉴定

目的从猪囊尾蚴cDNA文库中免疫学筛选阳性蛋白的编码基因,以期获得囊尾蚴病新的免疫学诊断候选分子。方法免疫学筛选cDNA文库,用PCR法扩增出猪囊尾蚴候选蛋白基因编码序列,T载体连接,鉴定后将其亚克隆入pET28a(+)载体中,经酶切、测序证实正确后,转化入大肠杆菌BL21并用IPTG对该重组菌诱导表达,SDS-PAGE和Western blot方法观察表达结果。结果通过cDNA文库免疫筛选、测序和EXPASY软件分析,其一个684bp ORF DNA序列为新基因(命名为Ts1),录入号为EU009656;将Ts1基因克隆入pET28a(+)载体中诱导表达,可得到一分子量约28.0kDa融合蛋白,并能被囊尾蚴病人血清识别。结论本试验成功筛选、克隆出一个新囊尾蚴编码基因,并在原核细胞中表达,为进一步验证其免疫诊断价值奠定了基础。

Screening of cDNA library of cysticercus cellulosae and ptrlimianry identification of the recombinant protein

To screen the genes encoding the positive proteins of cysticercus cellulosae in order to discover the novel immuno-diagnostic candidate molecules,the cDNA library was screened ,the coding sequence of the candidate protein genes of cysticercus cellulosae were amplified with PCR,cloned into PMD-Tvector and then subcloned into prokaryotic expression vector pET28a(+). After identification by endonuclease digestion and sequencing,the recombinant gene was tansformed to E. coli BL21 and expressed following induction with IPTG. The expressed products were identified by SDS-PAGE and Western blotting. It was found that 10 positive clones were obtained after screening of cDNA library of cysticercus cellulosae,in which the screened Ts1 clone was demonstrated to be a novel gene of cysticercus cellulosae with 684 bps and one open reading frame(ORF)(GenBank accession No EU009656). A fusion protein with molecular mass of 28 kDa was obtained,when the ts1 gene was cloned into vector and expressed after induction,and this protein could be specifically recognized by sera of patients with cysticercosis. It is apparent that a novel coding gene of cysticercus cellulosae was successivelly screened and it may provide the basis for the further studies on the immunodiagnosis of this infection.