HPV-16 E1蛋白的表达、纯化及其抗体制备

Prokaryotic expression,purification of human papillomavirus type 16 E1 protein and Preparation of it's Antiserum

目的:在原核细胞中表达、纯化人乳头状瘤病毒16型(HPV-16)E1蛋白并制备HPV-16 E1特异性抗体。方法:PCR扩增HPV-16 E1基因,将其构建至原核表达载体pMAL-p2x,转化大肠杆菌BL-21菌株。通过改变诱导温度与IPTG浓度优化HPV-16 E1蛋白可溶性表达的条件,并用麦芽糖亲和层析法纯化该蛋白。纯化蛋白经皮下及足垫免疫新西兰白兔制备抗血清,ELISA和Western blot检测血清效价和特异性。结果:酶切和测序结果表明HPV-16 E1基因已克隆入pMAL-p2x。经优化筛选出HPV-16 E1融合蛋白诱导表达的最佳条件为28℃,IPTG浓度0.5 mmol/L。经麦芽糖亲和层析法纯化获得了HPV-16 E1可溶蛋白,纯度达到95.7%。West-ern blot检测证明制备的抗血清能与HPV-16 E1蛋白特异性结合,ELISA检测表明获得的抗血清效价达到1∶640 000以上。结论:在大肠杆菌中表达并纯化获得了HPV-16 E1蛋白,该蛋白免疫新西兰白兔制备了高效价的HPV-16 E1蛋白抗血清,为HPV-16 E1功能研究提供了抗原和检测用抗体。

AIM： To express and purify the human papillomavirus type 16 E1 protein in E.coli and prepare the antibody against HPV-16 E1.METHODS： HPV-16 E1 gene was amplified by PCR and cloned into prokaryotic expression vector pMAL-p2x,and the recombinant plasmid was transformed into E.coil BL-21.We optimize the soluble expression condition of fusion protein by induction with different IPTG concentration and different temperature.The expressed fusion protein was purified by mahose affinity column Chromatography.To prepare the anti-serum,New Zealand white rabbits were immunized with purified HPV-16 E1 protein via hypodermic and volar.Western blot and ELISA analyzed the serum＇s specificity against HPV-16 E1 and serum titers.RESULTS： Restriction endonuclease analysis and DNA sequencing showed HPV-16 E1 was cloned into the plasmid pMAL-p2x.Based on the optimization experiments,it concluded that the best soluble expression conditions for the HPV-16 E1 fusion protein involved addition of IPTG to a final concentration of 0.5 mmol/L and then further incubation at 28℃.The purity of the HPV-16 E1 fusion protein was over 95.7% after purification.ELISA and Western blotting showed the titers of the anti-serum were above 1∶640 000,and the anti-serum can specifically bind with HPV-16 E1 protein.CONCLUSION： We have ingathered the HPV-16 E1 fusion protein by expressing in E.coli and purifying,and the antibody against HPV-16 E1 was prepared with the fusion protein immunizing New Zealand white rabbits.This work will provide an antigen and detection antibody for further study on the HPV-16 E1 function.