重组G5P蛋白的原核表达纯化及与PrP^Sc结合能力的鉴定

Identification of Expression of Recombinant G5P and Its Binding Ability with PrP^Sc

目的利用基因克隆技术制备重组G5P蛋白,并鉴定其结合朊病毒(PrPSc)的能力。方法化学合成G5P全长基因核苷酸片段,将其插入原核表达载体pET-28a-c(+)质粒中,获得pET-28a-c(+)-G5P重组质粒。将其转化到E.coli BL21(DE3)中进行IPTG诱导表达,经Ni+亲和层析柱纯化,获得纯化重组G5P蛋白。制备重组G5P蛋白偶联磁珠,将其与含有或不含有PrP^Sc的人脑匀浆液混合,进行PrP^Sc的捕获实验。利用Western blot检测技术鉴定重组G5P蛋白与PrP^Sc结合的特异性与结合力。结果成功获得纯化的重组G5P蛋白,捕获实验显示重组G5P蛋白偶联磁珠仅从散发性克雅氏病(sCJD)患者的脑匀浆液中捕获到朊蛋白,而从非CJD患者的脑匀浆液中未捕获到朊蛋白。捕获的朊蛋白经蛋白酶K处理后仍能检出Western blot条带,证明重组G5P蛋白结合的朊蛋白为PrP^Sc而非正常朊蛋白。将sCJD患者的脑匀浆液稀释10000倍后重组G5P蛋白仍可捕获到PrP^Sc。结论该研究利用基因克隆技术成功获得了纯化的重组G5P蛋白。该蛋白具有特异性结合PrP^Sc的能力,并具有较高亲和力。这为下一步研发新型朊病毒检测技术和滤除技术打下基础。

Objective To prepare recombinant G5 P protein by gene cloning technology and to evaluate its binding ability to PrP^Sc.Methods The full-length nucleotide fragment of G5 P gene was chemically synthesized and inserted into the prokaryotic expression vector pET-28 a-c(+)plasmid.The recombinant pET-28 a-c(+)-G5 P plasmid was transformed into E.coli BL21(DE3)for IPTG inducible expression.The recombinant G5 P protein was purified by Ni+affinity column.The recombinant G5 P protein coupled magnetic beads were incubated with human brain homogenate with or without PrP^Sc,for the capture assay.The specificity and binding force of recombinant G5 P protein to PrP^Sc were identified by Western blotting.Results The purified recombinant G5 P protein was successfully prepared,and the capture assay showed that the recombinant G5 P protein coupled magnetic beads could only capture prions from the brain homogenate of sCJD patients.Western blot bands could still be detected after proteinase K treatment of the captured prions,which proves that the prions bound by the recombinant G5 P protein were PrP^Sc rather than abnormal prions.PrP^Sc could still be captured by recombinant G5 P protein after 10000 times dilution of brain homogenate in sCJD patients.Conclusion The recombinant G5 P protein was successfully obtained by gene cloning technology.The protein has the ability to specifically bind to PrP^Sc and has a high affinity,which lays a foundation for developing new prion detection and filtration technologies.