Guo Ping, Dai Wuxing, Liu Shuojie, Yang Ping, Cheng Jizhong, Liang Liang, Chen Zhihao, Gao Hong
Department of Biochemistry and Molecular Biology, School of Basic Medical Science, Tongii Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
J Huazhong Univ Sci Technolog Med Sci. 2006;26(5):493-6. doi: 10.1007/s11596-006-0501-z.
In order to construct a eukaryotic co-expression plasmid containing membrane-anchored Sjcl4FABP and Sjc26GST genes and identify their expression in vitro, Sj14 and Sj26 genes were obtained by RT-PCR with total RNA of Schistosoma japonicum adult worms as the template and cloned into eukaryotic expression plasmid pVAC to construct recombinant plasmids pVAC-Sj14 and pVAC-Sj26. Then a 23 amino-acid signal peptide of human interleukin-2 (IL-2) upstream Sj14 or Sj26 gene and a membrane-anchored sequence containing 32 amino-acids of carboxyl-terminal of human placental alkaline phosphatase (PLAP) downstream were amplified by PCR as the template of plasmid pVAC-Sj14 or pVAC-Sj26 only to get two gene fragments including Sj14 gene and Sj26 gene. The two modified genes were altogether cloned into a eukaryotic co-expression plasmid pIRES, resulting in another new recombinant plasmid pIRES-Sj26-Sj14. The expression of Sj14 and Sj26 genes was detected by RT-PCR and indirect immunofluorescent assays (IFA) when the plasmid pIRES-Sj26-Sj14 was transfected into eukaryotic Hela cells. Restriction enzyme analysis, PCR and sequencing results revealed that the recombinant plasmids pVAC-Sj14, pVAC-Sj26 and plRES-Sj26-Sj14 were successfully constructed and the expression of modified Sj14 and Sj26 genes could be detected by RT-PCR and IFA. A bivalent membrane-anchored DNA vaccine encoding Sj14 and Sj26 genes was acquired and expressed proteins were proved to be mostly anchored in cellular membranes.
为构建含膜锚定日本血吸虫14kD脂肪酸结合蛋白（Sj14FABP）和26kD谷胱甘肽-S-转移酶（Sjc26GST）基因的真核共表达质粒并鉴定其体外表达情况，以日本血吸虫成虫总RNA为模板，通过RT-PCR获得Sj14和Sj26基因，并克隆至真核表达质粒pVAC中构建重组质粒pVAC-Sj14和pVAC-Sj26。然后以质粒pVAC-Sj14或pVAC-Sj26为模板，通过PCR扩增人白细胞介素-2（IL-2）的23个氨基酸信号肽置于Sj14或Sj26基因上游，以及人胎盘碱性磷酸酶（PLAP）羧基末端含32个氨基酸的膜锚定序列置于下游，从而获得包含Sj14基因和Sj26基因的两个基因片段。将这两个修饰基因共克隆至真核共表达质粒pIRES中，得到另一个新的重组质粒pIRES-Sj26-Sj14。当将质粒pIRES-Sj26-Sj14转染至真核Hela细胞时，通过RT-PCR和间接免疫荧光分析（IFA）检测Sj14和Sj26基因的表达。限制性内切酶分析、PCR及测序结果显示，重组质粒pVAC-Sj14、pVAC-Sj26和pIRES-Sj26-Sj14成功构建，且修饰后的Sj14和Sj26基因的表达可通过RT-PCR和IFA检测到。获得了一种编码Sj14和Sj26基因的双价膜锚定DNA疫苗，且表达的蛋白大多锚定在细胞膜上。
