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目的克隆中国中部地区间日疟原虫分离株Duffy血型结合蛋白Ⅱ区基因(PvDBPⅡ),体外表达和鉴定重组PvDBPⅡ蛋白。方法PCR法从间日疟患者血液DNA样品中扩增PvDBPⅡ基因,将产物插入到原核表达载体pET28a(+)中,构建pET28a PvDBPⅡ重组表达质粒,转化大肠埃希菌(E.coli)BL21(DE3+),异丙基βD硫代吡喃半乳糖苷诱导表达带有His标签的重组蛋白,采用镍柱亲和层析纯化重组蛋白,相应表达物分别采用十二烷基磺酸钠聚丙烯酰胺凝胶电泳和Western blot进行分析鉴定。结果PCR扩增的PvDBPII基因为1.1 kb,重组pET28a PvDBPⅡ质粒经测序验证,其插入片段序列与GenBank参考序列相似性为99%,转化E.coli所表达的重组蛋白分子量约为44 kDa,且能被间日疟患者血清特异性识别。结论成功克隆了PvDBPⅡ基因,表达了重组PvDBPII蛋白,为进一步研究基于PvDBPⅡ的红内期间日疟疫苗奠定了基础。
Objective To clone Duffy blood group Ⅱ domain gene (PvDBPⅡ) of Plasmodium vivax in central China and express and identify recombinant PvDBP Ⅱ protein in vitro. Methods The gene of PvDBP Ⅱ was amplified by PCR from blood DNA samples of Plasmodium vivax and inserted into the prokaryotic expression vector pET28a (+) to construct the recombinant plasmid pET28a PvDBP Ⅱ. The recombinant plasmid was transformed into E. coli BL21 (DE3 + ), Isopropyl βD thiogalactopyranoside induced the expression of His-tagged recombinant protein using nickel affinity chromatography purification of the recombinant protein, the corresponding expression were sodium dodecyl sulfate polyacrylamide coagulation Gel electrophoresis and Western blot analysis identified. Results The gene of PvDBPII amplified by PCR was 1.1 kb. The recombinant plasmid pET28a PvDBP Ⅱ was verified by sequencing. The inserted fragment was 99% identical to the reference sequence of GenBank and the molecular weight of the recombinant protein was about 44 kDa Serum specific identification of vivax malaria patients. Conclusion The successful cloning of PvDBPII gene and expression of recombinant PvDBPII protein laid the foundation for the further study of PvDBPII-based red malaria vaccine.