Dutta Noton Kumar, Mazumdar Kaushiki, Lee Byoung-Hee, Baek Min-Won, Kim Dong-Jae, Na Yi-Rang, Park Sung-Hoon, Lee Hyun-Kyoung, Kariwa Hiroaki, Mai Le Quynh, Park Jae-Hak
Laboratory Animal Medicine, College of Veterinary Medicine and KRF Zoonotic Disease Priority Research Institute, Seoul National University, Gwanak-Gu, Seoul, Republic of Korea.
Immunol Lett. 2008 Jun 15;118(1):65-71. doi: 10.1016/j.imlet.2008.03.003. Epub 2008 Apr 8.
It is believed today that nucleocapsid protein (N) of severe acute respiratory syndrome (SARS)-CoV is one of the most promising antigen candidates for vaccine design. In this study, three fragments [N1 (residues: 1-422); N2 (residues: 1-109); N3 (residues: 110-422)] of N protein of SARS-CoV were expressed in Escherichia coli and analyzed by pooled sera of convalescence phase of SARS patients. Three gene fragments [N1 (1-1269 nt), N2 (1-327 nt) and N3 (328-1269 nt)-expressing the same proteins of N1, N2 and N3, respectively] of SARS-N were cloned into pVAX-1 and used to immunize BALB/c mice by electroporation. Humoral (by enzyme-linked immunosorbent assay, ELISA) and cellular (by cell proliferation and CD4(+):CD8(+) assay) immunity was detected by using recombinant N1 and N3 specific antigen. Results showed that N1 and N3 fragments of N protein expressed by E. coli were able to react with sera of SARS patients but N2 could not. Specific humoral and cellular immunity in mice could be induced significantly by inoculating SARS-CoV N1 and N3 DNA vaccine. In addition, the immune response levels in N3 were significantly higher for antibody responses (IgG and IgG1 but not IgG2a) and cell proliferation but not in CD4(+):CD8(+) assay compared to N1 vaccine. The identification of antigenic N protein fragments has implications to provide basic information for the design of DNA vaccine against SARS-CoV. The present results not only suggest that DNA immunization with pVax-N3 could be used as potential DNA vaccination approaches to induce antibody in BALB/c mice, but also illustrates that gene immunization with these SARS DNA vaccines can generate different immune responses.
如今人们认为，严重急性呼吸综合征（SARS）冠状病毒的核衣壳蛋白（N）是疫苗设计中最有前景的抗原候选物之一。在本研究中，SARS冠状病毒N蛋白的三个片段[N1（残基：1 - 422）；N2（残基：1 - 109）；N3（残基：110 - 422）]在大肠杆菌中表达，并用SARS患者恢复期的混合血清进行分析。SARS - N的三个基因片段[N1（1 - 1269 nt）、N2（1 - 327 nt）和N3（328 - 1269 nt），分别表达与N1、N2和N3相同的蛋白质]被克隆到pVAX - 1中，并通过电穿孔法用于免疫BALB/c小鼠。使用重组N1和N3特异性抗原检测体液免疫（通过酶联免疫吸附测定，ELISA）和细胞免疫（通过细胞增殖和CD4(+)：CD8(+)测定）。结果表明，大肠杆菌表达的N蛋白的N1和N3片段能够与SARS患者的血清发生反应，但N2不能。接种SARS冠状病毒N1和N3 DNA疫苗可显著诱导小鼠产生特异性体液免疫和细胞免疫。此外，与N1疫苗相比，N3疫苗在抗体反应（IgG和IgG1，但不包括IgG2a）和细胞增殖方面的免疫反应水平显著更高，但在CD4(+)：CD8(+)测定中并非如此。抗原性N蛋白片段的鉴定为抗SARS冠状病毒DNA疫苗的设计提供了基础信息。目前的结果不仅表明用pVax - N3进行DNA免疫可作为在BALB/c小鼠中诱导抗体的潜在DNA疫苗接种方法，还表明用这些SARS DNA疫苗进行基因免疫可产生不同的免疫反应。
