中国中部地区间日疟原虫分离株Duffy血型结合蛋白Ⅱ区的克隆表达与初步鉴定

Cloning,expression and identification of Plasmodium vivax Duffy binding protein region Ⅱ of central China isolate

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 a Plasmodium vivax Duffy binding protein critical functional region Ⅱ（PvDBPⅡ） gene of central China isolate,and to express and identify the recombinant PvDBPⅡ protein in vitro.Methods PCR was performed to amplify PvDBPⅡ from P.vivax DNA of a central China isolate and the PCR product was inserted into pET28a（＋） vector.pET28a-PvDBPⅡ recombinant plasmid was constructed and transformed into E.coli host BL21（DE3＋）.IPTG was used to induce the recombinant PvDBPⅡ protein fused with His tag,and the protein was purified by His-NTA affinity chromatography.The recombinant protein was identified by SDS-PAGE and Western blot.Results The PCR product of PvDBPⅡ gene was about 1.1 kb,meeting the expectation of predicted fragment size.The recombinant pET28a-PvDBPⅡ plasmid was verified by sequencing that the insertion was correct both in direction and in frame,but with 4 non-synonymous mutations compared to reference P.vivax strain Sal-I.SDS-PAGE,and Western blot analysis showed that the recombinant PvDBPⅡ protein was about 44 kDa,and could be recognized by pooled sera from vivax malaria patients.Conclusion The PvDBPⅡ gene of central China isolate is successfully cloned,and recombinant PvDBPⅡ is expressed,thereby providing opportunity for further study on PvDBPⅡ.