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目的原核表达和鉴定含有编码日本血吸虫分泌蛋白、外膜蛋白基因的重组质粒,为高通量筛选日本血吸虫病疫苗或诊断抗原奠定基础。方法将前期构建的28个含有分泌蛋白、外膜蛋白编码基因序列的p ET32(+)重组质粒通过化学法转到大肠杆菌(E.coli)BL21(DE3)菌株中进行培养,加入异丙基-β-D-硫代半乳糖苷(IPTG)进行诱导表达。对诱导表达后的细菌进行超声破壁,以12%SDS-PAGE分别对菌液、上清及沉淀进行检测,判断目的蛋白的大小和表达分布情况。利用蛋白芯片结合抗His标签荧光抗体对重组融合蛋白表达情况做进一步鉴定。结果在IPTG终浓度0.1 mmol/L、37℃、200 r/min和4 h的诱导条件下,含有编码分泌蛋白、外膜蛋白序列的重组质粒能够在E.coli BL21(DE3)中高效表达。SDS-PAGE显示28个融合蛋白具有不同程度的表达,其中2个分布在上清中,26个分布在沉淀中,条带大小与预测大小一致。蛋白芯片成功检测到His抗体荧光信号,进一步证实了目的蛋白的表达情况。结论利用E.coli成功表达了一批编码日本血吸虫分泌蛋白、外膜蛋白的融合蛋白,为后续疫苗和免疫诊断研究奠定了基础。
Objective To express and identify prokaryotic recombinant plasmids containing the gene encoding secreted proteins and outer membrane proteins of Schistosoma japonicum and establish the basis for high-throughput screening of schistosomiasis japonica vaccine or diagnostic antigen. Methods 28 pET32 (+) recombinant plasmids containing secreted protein and outer membrane protein coding sequence were transformed into E. coli BL21 (DE3) strain by chemical method. Isopropyl -β-D-thiogalactoside (IPTG). The induced bacteria were sonicated and the bacterial liquid, supernatant and sediment were detected by 12% SDS-PAGE respectively to determine the size and expression of the target protein. Using protein chip combined with anti-His tag fluorescent antibody to further evaluate the expression of recombinant fusion protein. Results Recombinant plasmids containing secreted protein and outer membrane protein sequences were highly expressed in E. coli BL21 (DE3) under the induction of IPTG at the concentration of 0.1 mmol / L, 37 ℃, 200 r / min and 4 h. SDS-PAGE showed 28 fusion proteins with different degrees of expression, of which two were in the supernatant, 26 were distributed in the pellet, and the size of the band was consistent with the predicted size. Protein chip successfully detected His antibody fluorescence signal, further confirmed the expression of the target protein. Conclusion A series of fusion proteins coding for secreted proteins and outer membrane proteins of Schistosoma japonicum were successfully expressed by E.coli, which laid the foundation for the follow-up vaccine and immunodiagnosis research.