Construction of the Eukariotic Expression Vector for s1 Gene Fragment of SARS-CoV and its Immunol ogical Effects

CAI Heng-ling; WU Yi-mou; WAN Yan-ping; XIAO Jian-hua; YANG Qiu-lin; ZENG Qiao; ZHAO Fei-jun; TANG Man-juan

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October 2005

CAI Heng-ling, WU Yi-mou, WAN Yan-ping, XIAO Jian-hua, YANG Qiu-lin, ZENG Qiao, ZHAO Fei-jun and TANG Man-juan. Construction of the Eukariotic Expression Vector for s1 Gene Fragment of SARS-CoV and its Immunol ogical Effects[J]. Virologica Sinica, 2005, 20(5): 464-467.

Citation: CAI Heng-ling, WU Yi-mou, WAN Yan-ping, XIAO Jian-hua, YANG Qiu-lin, ZENG Qiao, ZHAO Fei-jun, TANG Man-juan. Construction of the Eukariotic Expression Vector for s1 Gene Fragment of SARS-CoV and its Immunol ogical Effects .VIROLOGICA SINICA, 2005, 20(5) : 464-467.

SARS病毒s1基因片段真核表达载体的构建及免疫效果

蔡恒玲 ¹ ,
吴移谋 ^1* ,
万艳平 ¹ ,
肖建华 ² ,
杨秋林 ² ,
曾桥 ² ,
赵飞骏 ¹ ,
唐曼娟 ¹

1.
1.湖南衡阳南华大学病原微生物学教研室 2.南华大学寄生虫教研室

摘要

本研究根据GenBank登录的BJ01株SARS-CoV序列合成801bpS1基因片段,该片段被亚克隆至真核表达载体pcDNA3.1(+)得到重组质粒pcDNA3.1(+)/S1;转染Hela细胞,SDS-PAGE、Western-Blotting鉴定蛋白表达;肌注免疫BALB/c小鼠,利用ELISA法检测免疫后小鼠的抗SARS-CoVIgG及IFN-γ水平,MTT法检测T细胞增殖活性。结果显示,重组质粒pcDNA3.1(+)/S1可在Hela细胞内表达S1蛋白,免疫后小鼠的T细胞增殖活性增强,抗SARS-CoVIgG与IFN-γ水平升高。本实验说明pcDNA3.1(+)/S1可诱导小鼠产生一定的体液免疫和细胞免疫应答。
- SARS相关冠状病毒
- , S1基因
- , 免疫活性
- , 真核表达

Construction of the Eukariotic Expression Vector for s1 Gene Fragment of SARS-CoV and its Immunol ogical Effects

Abstract

The 801 base pairs of S1 gene fragment were synthesized based on the Sever acute respiratory syndrome associated coronavirus(SARS-CoV)BJ01 strain registered in GenBank.The synthetical DNA was subcloned into the appropriate site of pcDNA3.1(+) eukariotic expression vector to construct a recombinant plasmid pcDNA3.1(+)/S1;The recombinant plasmid pcDNA3.1(+)/S1 was transfected into Hela cells and the expressed protein was identified by SDS-PAGE and Western-blotting;BALB/c mice were immunized with pcDNA3.1(+)/S1 by i.m.The level of anti-SARS-CoV IgG and IFN-γ in BALB/c mice after immunization were detected by ELISA and T cell proliferation activity was tested by MTT.It was found that the recombinant plasmid pcDNA3.1(+)/S1 could express S1 protein in Hela cells,T cell proliferative activity,the level of anti-SARS-CoV IgG and IFN-γ increased after immunization.It revealed that pcDNA3.1(+)/S1 could induce moderate cellular and humoral immunological reaction in BALB/c mice.
- SARS-CoV
- , S1 gene
- , Immunological activity
- , Eukariotic expression

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

Construction of the Eukariotic Expression Vector for s1 Gene Fragment of SARS-CoV and its Immunol ogical Effects

Abstract: The 801 base pairs of S1 gene fragment were synthesized based on the Sever acute respiratory syndrome associated coronavirus(SARS-CoV)BJ01 strain registered in GenBank.The synthetical DNA was subcloned into the appropriate site of pcDNA3.1(+) eukariotic expression vector to construct a recombinant plasmid pcDNA3.1(+)/S1;The recombinant plasmid pcDNA3.1(+)/S1 was transfected into Hela cells and the expressed protein was identified by SDS-PAGE and Western-blotting;BALB/c mice were immunized with pcDNA3.1(+)/S1 by i.m.The level of anti-SARS-CoV IgG and IFN-γ in BALB/c mice after immunization were detected by ELISA and T cell proliferation activity was tested by MTT.It was found that the recombinant plasmid pcDNA3.1(+)/S1 could express S1 protein in Hela cells,T cell proliferative activity,the level of anti-SARS-CoV IgG and IFN-γ increased after immunization.It revealed that pcDNA3.1(+)/S1 could induce moderate cellular and humoral immunological reaction in BALB/c mice.