弓形虫MAPK1基因的克隆与原核表达∗

摘要

To clone the MAPK1 gene and construct the prokaryotic expression vector PET⁃28a⁃MAPK1, to transform the expression vector into E. coli BL21, to induce and identify recombinant protein, and to lay experimental foundation for further analysis of biological characteristics and im⁃mune protection of MAPK1 gene,the study was conducted. A pair of primers were designed accord⁃ing to the sequence of MAPK1 from GenBank. The MAPK1 gene was amplified by PCR. Using NCoⅠ and HindⅢ double enzyme, the primers were connected to the prokaryotic expression vector of PET⁃28a with the same double enzyme cut. Recombinant expression plasmid PET⁃28a⁃MAPK1 was constructed and transformed to the fabrication E.coli BL21 competent expressed by IPTG induc⁃tion. Expression product was purified with Ni2+ chelating affinity column, and the purified recombi⁃nant plasmid was analyzed by SDS-PAGE and Western blotting. Results showed that the cloned MAPK1 gene had 99�9% homology with gene sequence included in GenBank, The recombinantplasmids was identified by restriction enzyme and the desired size of 1 599 bp fragment was ob⁃tained, which was consistent with the expected results, indicating that recombinant plasmid PET⁃28a⁃MAPK1 was successfully constructed. Protein concentration of Ni2+ chelate column after purifi⁃cation was 0�26 mg/mL, indicating that the Ni2+ chelate column could be used as an immunogen. SDS-PAGE electrophoresis showed that molecular weight of fusion protein of PET⁃28a⁃MAPK1 was about 58 ku. Western⁃blotting showed that the fusion protein had good antigenicity. The expression plasmid was expressed in E.coli BL21, and the study lays foundation for further exploration of toxo⁃plasmosis diagnosis.%为获取具有生物学活性的弓形虫MAPK1蛋白，根据GenBank中编码MAPK1的已知序列设计并合成1对引物，用NCoⅠ和HindⅢ双酶切后，连接到同样双酶切的原核表达载体PET⁃28a上，构建重组表达质粒PET⁃28a⁃MAPK1并转化至E．coli BL21感受态，用IPTG诱导表达，表达产物用Ni2＋螯合柱亲和纯化，纯化后进行SDS－PAGE和 Western blotting分析。结果表明：表达质粒在E．coli BL21中得到表达，克隆的MAPK1基因与GenBank中收录的基因序列同源性为99�9％。对重组质粒进行酶切鉴定，获得1599 bp大小的目的基因片段，与预期结果相符，成功地构建了重组质粒PET⁃28a⁃MAPK1。Ni2＋螯合柱纯化后蛋白浓度为0�26 mg／mL，可用作免疫原，经SDS－PAGE电泳显示PET⁃28a⁃MAPK1融合蛋白的分子量约为58 ku。 Western blotting显示融合蛋白具有良好的抗原性。