CAJ | 학술논문

刺突蛋白(S)和核心蛋白(N)是SARS冠状病毒的主要结构蛋白.在病毒细胞受体结合和病毒包装过程起重要作用.重组融合表达这2种蛋白具有较高的诊断学价值.对SARS病毒N蛋白和S蛋白氨基酸序列进行计算机分析,选择含有优势抗原表位的N蛋白1～227位氨基酸片段和S蛋白450～650位氨基酸片段,采用序列重叠延伸策略(sequence overlapping extension,SOE)构建编码N1-227-Linker-S450-650新型融合蛋白的基因片段,导入原核表达载体,实现融合蛋白在大肠杆菌的高效表达.利用组氨酸标签亲和层析的方法纯化,获得高纯度的融合蛋白.对该融合蛋白的结构特征模拟分析的结果显示,其免疫化学性质均无显著改变.采用ELISA和Western印迹方法对其识别SARS冠状病毒特异性抗体的能力进行初步鉴定,显示该融合蛋白具有较好的抗原性和特异性,可有效特异性地检测恢复期SARS病人血清中抗SARS冠状病毒结构蛋白的抗体,可以作为SARS冠状病毒感染的辅助诊断手段.
자돌단백(S)화핵심단백(N)시SARS관상병독적주요결구단백.재병독세포수체결합화병독포장과정기중요작용.중조융합표체저2충단백구유교고적진단학개치.대SARS병독N단백화S단백안기산서렬진행계산궤분석,선택함유우세항원표위적N단백1～227위안기산편단화S단백450～650위안기산편단,채용서렬중첩연신책략(sequence overlapping extension,SOE)구건편마N1-227-Linker-S450-650신형융합단백적기인편단,도입원핵표체재체,실현융합단백재대장간균적고효표체.이용조안산표첨친화층석적방법순화,획득고순도적융합단백.대해융합단백적결구특정모의분석적결과현시,기면역화학성질균무현저개변.채용ELISA화Western인적방법대기식별SARS관상병독특이성항체적능력진행초보감정,현시해융합단백구유교호적항원성화특이성,가유효특이성지검측회복기SARS병인혈청중항SARS관상병독결구단백적항체,가이작위SARS관상병독감염적보조진단수단.
The spike (S) and nucleocapsid (N) proteins, which are responsible for viral binding to cell surface receptors and the formation of ribonucleoprotein complexes during virion assembling, are major structure proteins of severe acute respiratory syndrome coronavirus (SARS-CoV). The expression of recombinant protein may give more accurate result for detecting SARS-CoV infection. A novel fusion protein,comprising of two fragments of N and S proteins from SARS-CoV, was prepared. Our computer-assisted analysis suggested that the immunodominant domains were located in the amino acid residues 1-227 of N protein and 450-650 of S protein, further the fusion of the two fragments did not change the immunochemical characteristics. The complementary DNA(cDNA) encoding N1-227 fused with S450-650 was obtained by sequence overlapping extension (SOE), and named NLS. It was cloned into pET-28a ( + ), an expression vector for His-tag fusion protein. This new constructed fusion protein was prokaryotic expressed in E. coli,and purified by metal chelate affinity chromatography with the purity over 95 %. The purified fusion protein was identified by anti-His monoclonal antibody and convalescence SARS patients serum. The NLS protein based ELISA showed that NLS maintained appropriate antigenicity and specificity to react with the sera of convalescent SARS patients. The functional NLS protein were successfully expressed and purified. And the fusion protein based ELISA can be used for detection of antibodies (Abs) against the S and N proteins of SARS-CoV. It may provided a novel diagnostic tool and have the potential application in developing of anti-SARS vaccine.