汉滩病毒核蛋白的分段表达及抗原表位分析
作者：薛小平 徐志凯 马文煜 尹文 张芳琳 闫岩 吴兴安 白文涛
单位：第四军医大学微生物教研室，西安 710032
关键词：汉滩病毒； 抗原表位； 核蛋白
中国病毒学000303摘要：在大肠杆菌中对汉滩病毒S基因4种不同长度片段的重组表达质粒进行诱导表达。结果表明表达的4种GST-NP融合蛋白均以不溶性包含体形式存在于菌体细胞内，表达量分别占菌体蛋白总量的29-36%，分子量分别约为72 kD、66 kD、54 kD和44 kD。Western blot显示54 kD和72 kD融合蛋白用酶标抗汉滩病毒NP McAb 1A8和抗GST McAb 3C11染色呈阳性反应。66 kD和44 kD融合蛋白仅与酶标3C11呈阳性反应。用凝血酶切去GST，获得4种汉滩病毒重组核蛋白(rNP)，分子量分别约为44 kD、40 kD、26 kD和16 kD。用19株McAb对表达产物做抗原位点分析，结果完整的rNP可与19株McAb中的13株反应，McAb反应谱与天然NP相同；S1.1 kb表达产物(N-端1-37和C端402-429位aa缺失)和S 0.5 kb表达产物(N-端1-274位aa缺失)与19株McAb均不发生反应；S0.7 kb表达产物(C-端275-429位aa缺失)可与5株组特异性McAb反应。表明汉坦病毒核蛋白上的抗原位点主要存在于N-端1-37位aa区段内。
, http://www.100md.com
中图分类号：Q786.R373 文献标识码：A
文章编号：1003-5125(2000)03-0220-06
Expression of Truncated HTNV Nucleoprotein and
Analysis of Antigenic epitope
XUE Xiao-ping，XU Zhi-kai，MA Wen-yu
(Dept. of Microbiology, Fourth Military Medical University, Xi'an 710032, China)
Abstract: The expressing vector carring various truncated fragments of S gene of HTNV strain 76-118 was constructed and the vector efficienly expressed in E.coli. The result demonstrated that the GST-NP fusion proteins exist in the form of inclusion bodies in E.coli, the expressing amount accounted for 29-36% of the total proteins, and the molecular weights were 72 kD, 66 kD, 54 kD and 44 kD, respectively. Western blot showed that all of the four fusion proteins were positive stained with HPR-labeled anti-GST McAb 3C11, but only the 72 kD and 54 kD fusion proteins were positive stained with HRP-labeled anti-NP McAb 1A8. Four recombinant NP (rNP), which molecular weights were 44 kD, 40 kD, 26 kD and 16 kD respectively, were obtained by removing GST from purified GST-NP fusion proteins with thrombin. Mapping of antigenic epitope was done by 19 strains McAb. The result showed that 72 kD fusion protein could react with 13 strains McAb, which was same as authentic NP of HTNV. The truncated fusion protein (deleting aa 1-37 at N-teminal and aa 402-429 at c-terminal) expressed by S1.1 kb fragment and S 0.5 kb fragment (deleting aa 1-274 at N-terminal) did not react with all 19 strains McAb. The truncated fusion protein (deleting aa 275-429 at c-terminal) expressed by S 0.7 kb fragment reacted with 5 strains of group-specific McAb, which suggested that antigenic epitopes on NP of HTNV were located in the N-terminal of NP and distribution of group-specific and type-specific antigenic epitope showed some regional.
, 百拇医药
Key words: Hantaan virus; Nucleocapsid protein; Mapping epitope
基金项目：国家自然科学基金资助项目(39570038)；军队杰出青年基金资助项目[JB)]
作者简介：薛小平(1951年-)，男，陕西人，教授，博士，主要从事出血热病毒结构与功能的研究
参考文献
[1] 徐志凯，汪美先，姜绍谆，等.用McAb对肾综合征出血热50 kD蛋白的分析[J].病毒学报，1988，4：113
[2] 徐志凯，王海涛，肖毅，等. 肾综合征出血热病毒50 kD结构蛋白的抗原位点分析[J].单克隆抗体通讯，1992，8(2)：30
, http://www.100md.com [3] Schmaljohhn CS, Jernings G, Hay et al. Coding strategy of the S genome segment of Hantann virus [J]. Virology, 1986,155:633
[4] 尹文，徐志凯，闫岩，等.汉坦病毒S基因的分段表达及其表达产物的鉴定[J].细胞与分子免疫学杂志，1998，1：23
[5] Mifang L, Dexin L, Shu yan X et al. Antigenic and molecular characterization of Hantavirus isolates from China virus [J]. Virus Research, 1994,31:219
[6] 陈伯权，倪大石，沈宏开，等.广谱肾综合征出血热病毒单克隆抗体A35的生物学性状[J].病毒学报，1987，3(3)：296
, 百拇医药
[7] 唐家琪，陈竟芳，张炳根，等.肾综合征出血热病毒单克隆抗体的制备及其特性鉴定[J].中华微生物和免疫学杂志，1987，7(4)：237
[8] 萨姆布鲁克 J，弗里奇 EF，曼尼阿蒂斯T著. 金冬雁等译.分子克隆实验指南[M]，第二版，北京：科学出版社，1992
[9] Sigimoto S, yokoo Y. Purification of recombinant salmon growth hormone expressed is Escherichia coli [J]. Biotechno. Lett, 1991,13:289
[10] Fack F, Hungle-Durr B, Sung D, et al. Epitope mapping by phage display random gene fragment libraries. Expression of various truncated fragments of S gene of HTNV strain 76-118 and analysis of antigenic epitope [J]. J. Immunol. Methe. 1997,206(102):43
收稿日期：1999-04-19，修回日期：1999-06-14, http://www.100md.com
单位：第四军医大学微生物教研室，西安 710032
关键词：汉滩病毒； 抗原表位； 核蛋白
中国病毒学000303摘要：在大肠杆菌中对汉滩病毒S基因4种不同长度片段的重组表达质粒进行诱导表达。结果表明表达的4种GST-NP融合蛋白均以不溶性包含体形式存在于菌体细胞内，表达量分别占菌体蛋白总量的29-36%，分子量分别约为72 kD、66 kD、54 kD和44 kD。Western blot显示54 kD和72 kD融合蛋白用酶标抗汉滩病毒NP McAb 1A8和抗GST McAb 3C11染色呈阳性反应。66 kD和44 kD融合蛋白仅与酶标3C11呈阳性反应。用凝血酶切去GST，获得4种汉滩病毒重组核蛋白(rNP)，分子量分别约为44 kD、40 kD、26 kD和16 kD。用19株McAb对表达产物做抗原位点分析，结果完整的rNP可与19株McAb中的13株反应，McAb反应谱与天然NP相同；S1.1 kb表达产物(N-端1-37和C端402-429位aa缺失)和S 0.5 kb表达产物(N-端1-274位aa缺失)与19株McAb均不发生反应；S0.7 kb表达产物(C-端275-429位aa缺失)可与5株组特异性McAb反应。表明汉坦病毒核蛋白上的抗原位点主要存在于N-端1-37位aa区段内。
, http://www.100md.com
中图分类号：Q786.R373 文献标识码：A
文章编号：1003-5125(2000)03-0220-06
Expression of Truncated HTNV Nucleoprotein and
Analysis of Antigenic epitope
XUE Xiao-ping，XU Zhi-kai，MA Wen-yu
(Dept. of Microbiology, Fourth Military Medical University, Xi'an 710032, China)
Abstract: The expressing vector carring various truncated fragments of S gene of HTNV strain 76-118 was constructed and the vector efficienly expressed in E.coli. The result demonstrated that the GST-NP fusion proteins exist in the form of inclusion bodies in E.coli, the expressing amount accounted for 29-36% of the total proteins, and the molecular weights were 72 kD, 66 kD, 54 kD and 44 kD, respectively. Western blot showed that all of the four fusion proteins were positive stained with HPR-labeled anti-GST McAb 3C11, but only the 72 kD and 54 kD fusion proteins were positive stained with HRP-labeled anti-NP McAb 1A8. Four recombinant NP (rNP), which molecular weights were 44 kD, 40 kD, 26 kD and 16 kD respectively, were obtained by removing GST from purified GST-NP fusion proteins with thrombin. Mapping of antigenic epitope was done by 19 strains McAb. The result showed that 72 kD fusion protein could react with 13 strains McAb, which was same as authentic NP of HTNV. The truncated fusion protein (deleting aa 1-37 at N-teminal and aa 402-429 at c-terminal) expressed by S1.1 kb fragment and S 0.5 kb fragment (deleting aa 1-274 at N-terminal) did not react with all 19 strains McAb. The truncated fusion protein (deleting aa 275-429 at c-terminal) expressed by S 0.7 kb fragment reacted with 5 strains of group-specific McAb, which suggested that antigenic epitopes on NP of HTNV were located in the N-terminal of NP and distribution of group-specific and type-specific antigenic epitope showed some regional.
, 百拇医药
Key words: Hantaan virus; Nucleocapsid protein; Mapping epitope
基金项目：国家自然科学基金资助项目(39570038)；军队杰出青年基金资助项目[JB)]
作者简介：薛小平(1951年-)，男，陕西人，教授，博士，主要从事出血热病毒结构与功能的研究
参考文献
[1] 徐志凯，汪美先，姜绍谆，等.用McAb对肾综合征出血热50 kD蛋白的分析[J].病毒学报，1988，4：113
[2] 徐志凯，王海涛，肖毅，等. 肾综合征出血热病毒50 kD结构蛋白的抗原位点分析[J].单克隆抗体通讯，1992，8(2)：30
, http://www.100md.com [3] Schmaljohhn CS, Jernings G, Hay et al. Coding strategy of the S genome segment of Hantann virus [J]. Virology, 1986,155:633
[4] 尹文，徐志凯，闫岩，等.汉坦病毒S基因的分段表达及其表达产物的鉴定[J].细胞与分子免疫学杂志，1998，1：23
[5] Mifang L, Dexin L, Shu yan X et al. Antigenic and molecular characterization of Hantavirus isolates from China virus [J]. Virus Research, 1994,31:219
[6] 陈伯权，倪大石，沈宏开，等.广谱肾综合征出血热病毒单克隆抗体A35的生物学性状[J].病毒学报，1987，3(3)：296
, 百拇医药
[7] 唐家琪，陈竟芳，张炳根，等.肾综合征出血热病毒单克隆抗体的制备及其特性鉴定[J].中华微生物和免疫学杂志，1987，7(4)：237
[8] 萨姆布鲁克 J，弗里奇 EF，曼尼阿蒂斯T著. 金冬雁等译.分子克隆实验指南[M]，第二版，北京：科学出版社，1992
[9] Sigimoto S, yokoo Y. Purification of recombinant salmon growth hormone expressed is Escherichia coli [J]. Biotechno. Lett, 1991,13:289
[10] Fack F, Hungle-Durr B, Sung D, et al. Epitope mapping by phage display random gene fragment libraries. Expression of various truncated fragments of S gene of HTNV strain 76-118 and analysis of antigenic epitope [J]. J. Immunol. Methe. 1997,206(102):43
收稿日期：1999-04-19，修回日期：1999-06-14, http://www.100md.com