CAJ | 학술논문

目的：SARS冠状病毒（ SARS-CoV）核衣壳蛋白（ N蛋白）在杆状病毒-昆虫系统的表达，研究其抗原性。方法：利用PCR方法扩增SARS-NP全长基因，使用BamHⅠ和Sal Ⅰ限制性内切酶定向插入Bac-to-Bac系统的pFastBac HTC载体，构建重组质粒转化DH10 Bac细胞，获得重组杆粒DNA后转染Sf9细胞，在High Five细胞中表达SARS-NP；表达产物经Ni-NTA亲和层析纯化及免疫印迹和ELISA方法验证抗原性。结果：成功构建pFastBac HTC-SARS-NP重组质粒，并在High Five细胞高效表达，获得相对分子量约48 kD的重组蛋白质。经验证，重组蛋白能被SARS-CoV NP的特异性单抗（8E1A17）和兔免疫血清识别，能与SARS患者血清反应，与健康人血清、人冠状病毒229 E型和OC43型的杂交瘤培养上清、相对应的兔免疫血清、患者血清均无交叉反应，具良好抗原反应性。结论：SARS-NP成功表达于杆状病毒-昆虫系统，具较强抗原反应性，初步认为其与229 E型和OC43型感染血清无交叉反应性。
목적：SARS관상병독（ SARS-CoV）핵의각단백（ N단백）재간상병독-곤충계통적표체，연구기항원성。방법：이용PCR방법확증SARS-NP전장기인，사용BamHⅠ화Sal Ⅰ한제성내절매정향삽입Bac-to-Bac계통적pFastBac HTC재체，구건중조질립전화DH10 Bac세포，획득중조간립DNA후전염Sf9세포，재High Five세포중표체SARS-NP；표체산물경Ni-NTA친화층석순화급면역인적화ELISA방법험증항원성。결과：성공구건pFastBac HTC-SARS-NP중조질립，병재High Five세포고효표체，획득상대분자량약48 kD적중조단백질。경험증，중조단백능피SARS-CoV NP적특이성단항（8E1A17）화토면역혈청식별，능여SARS환자혈청반응，여건강인혈청、인관상병독229 E형화OC43형적잡교류배양상청、상대응적토면역혈청、환자혈청균무교차반응，구량호항원반응성。결론：SARS-NP성공표체우간상병독-곤충계통，구교강항원반응성，초보인위기여229 E형화OC43형감염혈청무교차반응성。
Objective:To express SARS-CoV nucleocapsid protein in Bac-to-Bac Baculovirus Expression System and analyze the antigenicity of the recombinant protein.Methods: The SARS-CoV nucleocapsid gene was amplified by PCR.The PCR product digested with BamHⅠand SalⅠrestriction endonucleases was cloned into vector pFastBac HTC of Bac-to-Bac Baculovirus expression system.Recombinant plasmid was transformed DH 10Bac cells to obtain the recombinant Bacmid DNA.Recombinant Bacmid DNA was transferred into Sf9 cells which were inducted to express the recombinant protein in High Five cells.After purified by Ni affinity chroma-tography ,the antigenicity of the recombinant protein was analyzed by Western blot and ELISA.Results:Recombinant plasmid was con-structed successfully.The recombinant protein with the relative molecular mass of 48 kD was efficiently expressed in High Five cells and purified successfully by Ni affinity chromatography.Western blot and ELISA analysis showed that the recombinant protein could be spe -cifically recognized by the monoclonal antibody to SARS-CoV N protein and immune serum from rabbits ,respectively.The recombinant protein can specifically reacted with serum from SARS patients ,not with serum from healthy persons and patients infected with hCoV-229 E and hCoV-OC43.Conclusion: SARS-CoV nucleocapsid protein has been expressed successfully in the Bac-to-Bac Baculovirus Expression System ,and obtained good antigenicity.It is preliminary deemed that it can't reacted with serum from patients infected with hCoV-229E and hCoV-OC43.