ctxB和ureB抗原表位的融合表达及其免疫学活性

为研制新型有效的幽门螺杆菌疫苗，设计了一个由ctxB(去除信号肽部分)和ureB抗原表位融合的新型分子。PCR扩增霍乱弧菌的ctxB基因，插入到质粒pEl32a上，再将化学合成的ureB抗原表位序列(编码的氨基酸为CHHLDKSIKEDVQFAQSRI）连接到ctxB下游，融合基因命名为ctube。将含有融合基因的pET32a转化到大肠杆菌BL21(DE3)，诱导表达后SDS-PAGE测定融合蛋白ctube的分子质量，ELISA法检测ctube对神经节苷脂GM1的结合活性，Western blotting检测对兔抗幽门螺杆菌(Hp)多抗的结合活性。序列分析结果表明，ctube基因全长387bp，编码129个氨基酸，其中ctxB的密码子与GenBank上的序列同源性达到99％。转化后的大肠杆菌BL21（DE3)经IPTG诱导表达后，产生了一个分子质量为2．5万的蛋白，经肠激酶酶切后的产物为ctube。ELISA试验和West Blotting表明，ctube与神经节苷脂性GM1、兔抗Hp多抗均具有很好的结合活性，有希望成为一个具有抗幽门螺杆菌感染的新的活性分子。

The Fusion Expression and Immunoactivity Analysis of ctxB and Epitope of ureB from Helicobacter Pylori

In order to develop a new vaccine of Helicobacter pylori, the ctxB is created from Vibrio cholerae by PCR and inserted into pET32a vector. An artificially synthesized cDNA of ureB epitope, which code the amino acids CHHLDKSIKEDVQFADSRI, is linked to the 3' end of ctxB. The fusion gene of ctxB and epitope of ureB is named ctube. The vector pET32a which include the ctube is be transformed to E. coli BL21 (DE3) . The molecular weight of ctube is be determined by SDS-PAGE, the immunoactivity of expressed fusion protein ctube is identified by ELISA with GMj and western blotting with rabbit antiserum. The result of sequencing of ctube shows that the length of ctube is 387bp. In comparison with the reported corresponding sequences, the homology of nucleotide of the ctxB is 99% . Measured by SDS-PAGE, the molecular weight of TrxA-ctube is about 25 ku and the cleaved product ctube is about 14.3 ku. Isolated with Ni+ affinity chromatography and Sepadex G-50, the purified ctube is detected the activity of binding to GM1 and antibody from rabbit serum. The product ctube may be a new molecular which can act as the vaccine against the infection of Helicobacter pylori .