恶性疟原虫海南株var2csa基因的克隆、表达及免疫识别研究

Cloning,expression and characterization of HN isolate of Plasmodium falciparum var2csa genes and the encoded proteins

目的克隆、表达恶性疟原虫海南株HN(2)var2csa基因DBL4、DBL5、DBL6区,通过ELISA方法比较其与硫酸软骨素A(CSA)的亲和能力差异,以及人体对恶性疟原虫海南株HN(1)、(2)VAR2CSA不同DBL区的免疫应答差异。方法根据DBL区序列设计3对引物,PCR扩增目的片段,并与pMD18-T克隆载体连接。经PCR、酶切、测序鉴定正确后克隆至表达载体pET-22b,通过转化、诱导、纯化表达重组蛋白质,SDS-PAGE和Western blot检测。通过ELISA方法进行重组蛋白质与CSA的粘附实验以及与患者血清的免疫识别实验。结果酶切及DNA测序结果均表明表达载体构建成功,表达并纯化3个DBL区重组蛋白质,分子量与预计大小相同,Western blott检测目的蛋白质具有免疫反应性,ELISA显示不同蛋白浓度条件下DBL5区均明显比其它两个DBL区有更高的OD405值,并且DBL5区被妊娠相关疟疾病人血清识别水平显著高。结论 var2csa基因3个DBL区重组蛋白质表达成功,DBL5区与CSA的粘附能力较强,人体对DBL5区的免疫应答反应可能在抗病免疫过程中起到关键作用。

Objective To clone and express three VAR2CSA DBL domains（DBL4/5/6） encoded by the var2csa of a Hainan（2）isolate of Plasmodium falciparum,and analyze the difference of CSA-binding activity as well as immunological recognition among different domains.Methods The DBL domains were amplified by PCR and cloned into the vector pMD18-T,which were further verified by enzyme digestion and sequencing.The inserts with correct sequences were subcloned into the prokaryotic expression vector pET-22b,transformed into E.coli BL21（DE3） and induced by IPTG.The recombinant proteins were purified by affinity chromatograph and further identified by SDS-PAGE and Western blot.Activities of CSA-binding of the three recombinant DBL domains were assayed by ELISA method.The immunological recognition of different recombinant DBL domains was measured by ELISA,and the data were statistically analyzed using SPSS17.0 software.Results Enzyme digestion and DNA sequencing confirmed that the recombinant expression plasmids were constructed.The recombinant proteins were successfully purified and confirmed by Western blot.DBL5 had a higher affinity to the CSA receptor.The levels of specific IgG to the DBL4,DBL5 and DBL6 domains were significantly different and the DBL5 domain was dominantly recognized.Conclusion DBL5 may play the major role in binding and immune response during PAM.