论文部分内容阅读
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。
OBJECTIVE: To express and purify human papillomavirus type 16 (HPV-16) E1 protein in prokaryotic cells and prepare HPV-16 E1 specific antibody. Methods: HPV-16 E1 gene was amplified by PCR and cloned into prokaryotic expression vector pMAL-p2x. The recombinant plasmid was transformed into E. coli BL-21 strain. The conditions for the soluble expression of HPV-16 El protein were optimized by changing the induction temperature and IPTG concentration, and the protein was purified by maltose affinity chromatography. The purified protein was used to immunize New Zealand white rabbits subcutaneously and footpad to prepare antiserum. The serum titer and specificity were determined by ELISA and Western blot. Results: The results of digestion and sequencing showed that HPV-16 E1 gene was cloned into pMAL-p2x. The optimized conditions for inducing the expression of HPV-16 E1 fusion protein were 28 ℃ and IPTG concentration was 0.5 mmol / L. HPV-16 E1 soluble protein was purified by maltose affinity chromatography, with a purity of 95.7%. West-ern blot showed that the prepared antiserum specifically binds to HPV-16 E1 protein. The ELISA results showed that the titer of the obtained antiserum reached more than 1: 64000. CONCLUSION: HPV-16 E1 protein was expressed and purified in E. coli. The protein was used to immunize New Zealand white rabbits to prepare high-titer anti-HPV-16 E1 protein serum and provide antigen and antibody for the study of HPV-16 E1 function .