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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 the var2csa gene DBL4, DBL5 and DBL6 of HN (2) of Plasmodium falciparum in Hainan strain and compare their affinity with chondroitin sulfate A (CSA) by ELISA. (1), (2) VAR2CSA different DBL region immune response differences. Methods Three pairs of primers were designed according to the DBL region sequence. The target fragment was amplified by PCR and ligated with pMD18-T cloning vector. The recombinant plasmid pET-22b was identified by PCR, restriction enzyme digestion and sequencing. The recombinant protein was expressed by transformation, induction, purification and purified by SDS-PAGE and Western blot. The adhesion experiments of recombinant proteins with CSA and the immune recognition experiments with patient’s sera were performed by ELISA. Results The results of enzyme digestion and DNA sequencing showed that the expression vector was successfully constructed, expressed and purified three DBL recombinant proteins with the same molecular weight as the expected size. Western blott assay showed that the target protein was immunoreactive. ELISA showed that all the DBL5 regions Significantly higher OD405 values than the other two DBL regions, and the DBL5 region was significantly recognized by the serum of pregnant-related malaria patients. Conclusion The expression of recombinant proteins in three DBL regions of var2csa gene was successful, and the adhesive ability of DBL5 region to CSA was stronger. The immune response of DBL5 region in human body may play a key role in disease resistance and immunity.