幽门螺杆菌黏附素A的原核表达、纯化及其结晶条件搜索

Searching for conditions of prokaryotic expression,purification and crystallization of Helicobacter pylori adhesin A

目的原核表达、纯化幽门螺杆菌（Helicobacterpylori，11．pylori）黏附素A（麒pyloriadhesinA，HpaA），并初步搜索其结晶条件。方法采用TMHMM和SignalP．4．1软件预测HpaA蛋白中的跨膜序列和信号肽序列，根据GenBank中登录的编码HpaA的基因序列hp0797去掉预测出的信号肽序列后设计引物，以H．pylori标准株26695基因组为模板，PCR扩增编码HpaA的基因序列，插入原核表达载体pET-22b，构建重组表达质粒pET-22b-HpaA，转化大肠埃希菌（Ecoil）BL21（DE3），IPTG25℃诱导表达，表达的重组蛋白经亲和层析、阴离子交换层析和分子筛层析进行纯化。将纯化产物浓缩至10mg／ml，采用结晶搜索试剂盒，通过悬滴汽相扩散法搜索HpaA的结晶条件，并在此基础上配制条件进行优化。结果去掉序列中编码信号肽区域或跨膜结构域的密码子，选择HpaA的36-260位氨基酸进行克隆，构建的重组原核表达质粒pET．22b-HpaA经双酶切及测序鉴定，证明构建正确；表达的重组蛋白HpaA相对分子质量约27000，表达量约占菌体总蛋白的10％以上，主要以可溶形式表达，纯化后的纯度可达99％以上。在多种条件下，均有结晶生长，晶体多为薄片状、针状或簇状，在20％PEG3350，0．1mol／LHEPES（pH7．0）条件下，晶体形状类似长方体，外形有所改善。结论成功表达了HpaA蛋白，初步掌握其结晶条件，为下一步晶体结构学的研究及其在H．pylori感染与致病中的作用机制奠定了基础。

Objective To express Helicobacterpylori adhesion A （HpaA） in prokaryotic cells, purify the expressed product, and preliminarily search the condition for crystallization. Methods The transmembrane and signal peptide sequences in HpaA protein were predicted by Using TMHMM and SignaiP-4. 1 software, based on which a truncated form of HpaA was amplified by PCR using the genome ofH. pylori isolate 26695 as template and inserted into vector pET-22b. The constructed recombinant plasmid pET-22b-HpaA was transformed to E. coli BL21 （DE3） for expression of HpaA under induction of IPTG at 25 ~C. The expressed product was purified by affinity chromatography, anion exchange chromatography and gel filtration chromatography. The purified protein was concentrated to a concentration of 10 mg/ml, of which the condition for crystallization was searched by hanging-drop vapour diffusion method using Hampton Research Crystal Screen Kits. Results Restriction analysis and sequencing proved that recombinant plasmid pET-22b-HpaA was constructed correctly. The expressed recombinant HpaA, with a relative molecular mass of about 27 000, contained more than 10% of total somatic protein, mainly existed in a soluble form, and reached a purity of more than 99% after purification. Small crystals appeared under various conditions, which were in laminar, acicular or clustery forms. However, in presence of 20% PEG3350 and 0. 1 mol/L HEPES （pH 7. 0）, the crystals were in rectangle form, of which the appearance was improved. Conclusion HpaA was success-fully expressed, of which the condition for crystallization was preliminarily determined, which laid a foundation of further study on structure of crystals as well as the action mechanism of HpaA in infection and pathopoiesis of H. pylori.