柑橘溃疡病致病基因pthA核定位信号肽抗血清的制备及其抑菌研究

Preparation of Antiserum of the Recombinant pthA-NLS and Its Inhibition Effect on Citrus Canker Disease

用PCR法扩增位于柑橘溃疡病菌致病基因pthAC-末端的3个核定位信号序列,并将其克隆到原核表达载体PET32a(+)上,经双酶切及核酸序列测定重组质粒(pthA-NLS),其序列与GenBank中pthA的相关序列有99.9%的同一性。重组质粒转化大肠杆菌BL21(DE3)后诱导了重组多肽的表达,并用Ni2+-NTA纯化柱得到了48kD的纯化重组多肽。把重组多肽注入免疫Balb/c小白鼠,制备了相应的抗血清,Western blotting和ELISA分析结果表明,抗血清可特异地结合重组多肽,亦可识别溃疡病菌PthA天然蛋白,获得的抗血清可以用于柑橘溃疡病的检测。利用抗血清与溃疡病菌混合接种离体冰糖橙叶片,发现抗血清能推迟溃疡病菌的致病过程,且病斑比对照小,但未能达到抗病程度。pthA基因末端核定位信号序列的克隆、原核表达及抗血清的制备为进一步研究pthA的致病机理和研发溃疡病快速分子检测技术奠定了基础。

The sequence encoding three nuclear localizing signals（NLSs）at the C-terminal of pthA,was amplified by PCR from the plasmid of Xanthomonas axonopodis pv.citri.and cloned into PET32a（＋）vector.The recombinant plasmid named pthA-NLS was identified by restriction digestion and sequence analysis.The sequence of the cloned NLSs had 99.9% of similarity with that of pthA in GenBank.The recombinant fusion protein was expressed in E.coli BL21（DE3）and analyzed by 12% SDS-PAGE.A 48 kD of recombinant fusion protein was purified with Ni^2＋-NTA resin.The antiserum was obtained by immunizing Balb/c mouse with the purified recombinant protein and then identified by Western blotting and ELISA analysis.The result demonstrated that the antiserum could specifically bind to the recombinant protein and the PthA from X.axonopodis pv.citri,as well as to partially inhibit the disease development.The successful cloning,expression and the preparation of pthA-NLS specific mouse antiserum offered the ways for better understanding the pathogenesis of pthA and the development of a rapid method for molecular detection of citrus canker.