恶性疟原虫海南株var2csa基因DBL区蛋白的原核表达及功能分析

Cloning and Expression of var2csa DBL Domains from Plasmodium falciparum Hainan Isolate and Functional Analysis of the Recombinant Protein

目的克隆、表达恶性疟原虫海南株变异抗原基因(var)家族之一var2csa基因的血型抗原结合基团(duffy anti-gen-binding ligand,DBL)4、DBL5、DBL6区,比较其与硫酸软骨素A(chondroitin sulfate A,CSA)黏附能力的差异。方法 PCR扩增恶性疟原虫海南株基因组DNA中3个目的片段,将扩增产物与pMD18-T克隆载体连接,经酶切、测序鉴定正确后克隆至表达载体pET-22b上,转化至大肠埃希菌BL21(DE3)中,以异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达重组蛋白,并用组氨酸标签融合蛋白纯化柱(His GraciTrap)纯化,经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、蛋白质印迹(Western blotting)检测目的蛋白。ELISA检测不同DBL区重组蛋白与CSA的结合情况。结果 PCR扩增获得目的片段分别为996、859和894bp,酶切及DNA测序结果均显示重组质粒构建成功,在大肠埃希菌BL21(DE3)中成功表达并纯化3个DBL区重组蛋白,DBL4区、DBL5区和DBL6区的相对分子质量分别为Mr 439800,Mr 34500,Mr 36000。West-ern blotting检测结果显示,目的蛋白具有反应原性。ELISA结果显示,不同蛋白浓度条件下DBL5区均明显比其他两个DBL区的吸光度(A405值)高(P<0.05),表明DBL5区与CSA的黏附能力较强。结论 var2csa基因3个DBL区重组蛋白表达成功,DBL5区与CSA的黏附能力较强。

Objective To clone and express three VAR2CSA duffy antigen-binding ligand（DBL） domains（DBL4/5/6） encoded by var2csa gene of a Hainan isolate of Plasmdium falciparum,and study the difference of chondroitin sulfate A（CSA）-binding activity among them.Methods Three DBL domains was amplified by PCR and cloned into the vector pMD18-T.The recombinant plasmids were identified by enzyme digestion and sequencing,and then subcloned into the prokaryotic expression vector pET-22b.The recombinant plasmid was transformed into E.coli BL21（DE3） and followed by expression of the protein induced by IPTG.The recombinant protein was purified with His GraciTrap kit and identified by SDS-PAGE and Western blotting.CSA-binding activity of the three recombinant DBL domains was assayed by ELISA.Results The target genes were amplified with the length of 996 bp,859 bp and 894 bp.The constructed recombinant plasmids were identified by enzyme digestion and DNA sequencing.The recombinant proteins（DBL4/5/6） were purified,the relative molecular mass of DBL4,DBL5 and DBL6 was Mr 439 800,Mr 34 500 and Mr 36 000,respectively.The purified protein has been confirmed with immunogenicity by Western blotting.The results of adhesion experiment indicated that A405 values of DBL5 domain with different concentration were significantly higher than that of DBL4 and DBL6.Conclusion The three recombinant proteins（DBL4/5/6） of VAR2CSA DBL domains were expressed,and DBL5 domain has high binding affinity with CSA.