德国小蠊主要变应原Bla g 2在毕赤酵母菌中的表达

目的 在毕赤酵母菌（Pichia pastoris）中表达德国小蠊主要变应原 Bla g 2，以获得其可溶性蛋白。 方法 根据已知的Bla g 2基因序列设计带有酶切位点的引物，PCR扩增德国小蠊变应原Bla g 2基因，经酶切后将该基因连接到毕赤酵母真核表达载体pGAPZaA，获重组质粒pGAPZaA-Bla g 2，该重组质粒线性化后，经电转化法转化毕赤酵母GS115，用PCR筛选出重组子并在YPD培养基中表达。用Ni²⁺亲和层析法纯化重组蛋白Bla g2，用蛋白质印迹（Western blotting）分析其免疫学活性。 结果 重组质粒pGAPZaA-Bla g 2转化毕赤酵母GS115后进行表达，十二烷基磺酸钠?鄄聚丙烯酰胺凝胶电泳（SDS-PAGE）分析显示，重组蛋白Bla g 2表达产物以可溶性分子形式存在，相对分子质量（Mr）为45 000。培养3 d的表达量占上清总蛋白的50 %。表达产物重组蛋白Bla g 2经Ni²⁺亲和层析纯化后，Western blotting分析，在约Mr 45 000处有1条明显的特异性条带，该蛋白具有免疫学活性。 结论 获得了真核表达的德国小蠊主要变应原重组蛋白Bla g 2，该蛋白以可溶性蛋白的形式分泌到培养液中，避免了原核表达的变复性过程。

Expression of Allergen Bla g 2 from Blattella germanica in Pichia pastoris

Objective To express the major allergen of Blattella germanica (Bla g 2) in Pichia pastoris and obtain the soluble protein. Methods The known Bla g 2 gene was used to design the primers which had the restriction enzyme sites. PCR method was applied to obtain the Bla g 2 gene. The gene fragment was then cut and ligated with the Pichia expression vector pGAPZaA, resulting in a recombinant plasmid pGAPZaA-Bla g 2. The linearized pGAPZaA-Bla g 2 was transformed into Pichia pastoris GS115 through electroporation, then screened to positive transformants, and the protein was expressed in YPD medium. Purification of the recombinant protein was achieved by metal (Ni2 ) chelating affinity chromatography and Western-blotting assay indicated its IgE binding capacity. Results With the expressed reeombinanl protein, SDS-PAGE showed the presence of the product in the supernatant of the culture with Mr 45 000. After 3 days culture, the recombinant protein occupied 50% of the total proteins in the supernatant. The recombinant protein was purified and Western-blot demonstrated an adequate IgE binding capacity of the product. Conclusion A recombinant protein of Bla g 2 has been obtained, which is soluble in the supernatant and therefore can avoid a process of denaturalization and renaturation of the recombinant.