不可分型流感嗜血杆菌外膜蛋白P6的重组表达及免疫保护作用研究

Recombination expression and immanoprotective effects of the P6 outer membrane protein of nontypeable Haemophilus influenzae

目的研究不可分型流感嗜血杆菌（NTHi）重组外膜蛋白P6的理化性质和对小鼠的免疫保护作用。方法利用PCR技术，以NTHi基因组为模板，扩增出编码外膜蛋白P6的基因片段；构建pET-32a—P6重组质粒；转化到大肠杆菌B121（DE3）中进行表达，并对表达条件进行了优化。通过阴离子交换和凝胶过滤层析对蛋白进行纯化。经SDS—PAGE、Western blot等对蛋白的理化性质进行初步分析，继而免疫BALB／c小鼠，用同源菌经腹腔攻击，比较免疫组和对照组小鼠死亡率。结果实现了该蛋白在大肠杆菌中的高效表达，产物表达形式为可溶性表达，纯化后蛋白纯度可达95％，收率可达5mg／g（蛋白／湿菌），相对分子质量（Mr）为14145．848，Western blot证实重组P6蛋白能与菌源提纯P6蛋白免疫小鼠后的抗血清发生特异性反应。动物实验结果表明重组P6蛋白在小鼠体内能诱生高滴度的抗体，显示免疫组小鼠存活率明显高于对照组（P〈0．01）。结论重组NTHi外膜蛋白P6的原核表达产物为可溶性蛋白，在小鼠体内能诱生高效价抗体，在小鼠感染模型中具有保护作用，为NTHi疫苗的研发提供了基础资料。

Objective To clone and express recombinant outer membrane protein P6, determine its optimal expression conditions and to investigate the immunoprotective effects of the P6 protein on mice. Methods The P6 gene of nontypeable Haemophilus influenzae （NTHi） was amplified by PCR from the NTHi genome and cloned into expression vector pET-32a （ ＋ ） to generate the pET-32a-P6 recombinants. They were confirmed by nuclease digestion and sequence analysis. The verified recombinant was transformed into E. coli BL21 （ DE3 ）. Its optimal expression conditions were determined such as engineering strains, the concentration of IPTG, inducing temperature, inducing time, different medium etc. The recombinant protein was purified by Q SepharoseTMXL ion exchange and gel filtration chromatography. The protein was analyzed by SDS-PAGE, Western blot and sequencing. BALB/c mice were immunized with recombinant protein before challenged by NTHi through intraperitoneal injection. Then the mortality rate of different group was compared. Results The recombinant P6 of NTHi was successfully constructed and expressed in E. coli at a relatively high level. The purity was up to 95% after purification. The relative molecular mass of the protein is 14 145. 848. The recombinant protein was confirmed to show specific reaction on the antiserum through Western blot. The animal experiments showed the mortality rates of immunization groups were significantly lower than that of the control group （ P 〈 0. 05 ）. Conclusion The successful expression of the recombinant P6 will be very helpful for the further study on development of vaccine, its purified, immunological activity and antibody preparation.