人源抗狂犬病毒糖蛋白稳定型抗体精氨酸密码子突变株的构建及其在巴斯德毕赤酵母中的分泌表达

目的：对人源抗狂犬病毒糖蛋白（GPRV）单链二硫键稳定抗体（ScdsFv）进行精氨酸密码子修饰，实现其在酵母中的分泌表达，并检测其生物学活性。方法：参照巴斯德毕赤酵母偏好密码子，对抗GPRV ScdsFv原核表达基因进行密码子修饰，并通过点基因融合技术构建ScdsFv重组酵母表达基因，连接pPIC9K构建重组表达质粒pPIC9K-ScdsFv，电转化毕赤酵母GS115，经筛选后进行甲醇诱导表达。结果：SDS-PAGE及Western blot检测到重组表达质粒在30℃经甲醇诱导表达的蛋白相对分子质量为30000；荧光抗体试验验证ScdsFv能靶向结合GPRV；MTT试验说明ScdsFv能中和狂犬病毒，具有一定的细胞保护作用。结论：重组ScdsFv在酵母系统中可以有效表达。具有较好的生物学活性。

Construction and Secreted Expression of Human Anti-GPRV Arg Mutant Strain in Pichia pastoris

Objective： To modify the Arg codons of human anti-GPRV（glycoprotein of rabies virus） ScdsFv, to express ScdsFv in Pichia pastoris and study its bioactivity. Methods： According to the partiality codon of P.pastoris, prokaryotic expression ScdsFv gene was modified, and secreted expression ScdsFv was reconstructed by SOE, synthesized and cloned into pPIC9K to construct the recombinant expression vector pPIC9K-ScdsFv. The pPIC9K-ScdsFv was transformed into yeast host strain GSll5 and induced by methanol. Results： SDS-PAGE and Western blot identified that ScdsFv was ex- pressed after induced by methanol at 30℃ and was observed as a band of 30 000, fluorescent antibody test verified that ScdsFv could target GPRV, and MTT demonstrated that ScdsFv could neutralize the rabies virus to protect cells. Conclusion： The human anti-GPRV ScdsFv was efficiently expressed in P.pastoris and showed natural biological activities.