Objective: To prokaryotic express and identify the recombinant plasmids containing the genes or gene segments which coding secreted or peripheral membrane protein of Schistosoma japonicum.
Methods: A number of 28 pET32 (+) previous constructed recombinant plasmids containing these genes or segments were transferred into E. coli BL21 (DE3) strain for culture via the CaCl2 transformation method, and then induced by IPTG for prokaryotic expression. Then, the bacteria were treated by broking the wall with ultrasonic, and the bacterial suspension, supernatant and precipitation were detected with 12% SDS-PAGE. The size and distribution of these target proteins were determined. Finally, the expression of the recombinant fusion protein was further identified by the protein microarray combined with anti-His tag fluorescent antibody.
Results: Under the inducing conditions of 0.1 mmol/L IPTG, 37 ℃, 200 r/min and 4 h, these fusion proteins were highly expressed in E. coli BL21 (DE3). The SDS-PAGE results showed all the 28 recombinant fusion proteins were expressed in different degrees, among which 2 were distributed in the supernatant, and the other 26 were distributed in precipitation, and the sizes of the fusion proteins were the same as that of prediction. The fluorescent signals of His antibody were successfully detected by confocal laser scanner.
Conclusions: A series of recombinant plasmids containing genes or gene segments that coding secreted protein and peripheral membrane protein of S. japonicum have been successfully expressed by using an efficient prokaryotic expression system of E. coli. It has established a foundation for the high throughput immunoscreening of the vaccine candidates or diagnostic antigens of S.japonicum.
［摘要］ 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pET32 (+) 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。.
Keywords: Peripheral membrane protein; Prokaryotic expression; Protein microarray; Schistosoma japonicum; Secretory protein.