淋巴细胞脉络丛脑膜炎病毒核蛋白合成基因的表达及应用

Expression and Application of Synthetic Gene of Lymphocytic Choriomenin-gitis Virus Nucleoprotein

目的 用合成基因表达产物取代淋巴细胞脉络丛脑膜炎病毒（ＬＣＭＶ）或其他方法重组蛋白抗原， 用于ＬＣＭＶ感染的诊断。方法 采用Ｐｒｏｓｉｓ软Ｓ件对ＬＣＭＶ核蛋白（Ｎｐ）全长氨基酸序列进行亲疏水性分析，合成ＬＣ－ ＭＶ ｓＲＮＡ第１８１６－２１７８位核苷酸序列编码的Ｎｐ第３８０～５００位氨基酸基因，克隆在Ｈｉｓ－ｔａｇ ｐＥＴ－２８ａ融合表达，表 达产物经固定化金属配体亲和层析纯化，建立ＥＬＩＳＡ检测试剂。结果 表达产物主要以包涵体形式存在，表达量 达２０％以上。表达产物经Ｎｉ－ＮＴＡ－Ａｇａｒｏｓｅ纯化后纯度达到９０％以上。经检测９３份人血清．有６份病毒抗体阳 性，阳性率为６．５％（６／９３），与全病毒抗原检测结果７．５％（７／９３）相近。结论 用合成基因表达产物取代ＬＣＭＶ抗原 检测病毒抗体，不仅可以消除病毒传播可能，而且特异性强。为ＬＣＭＶ感染诊断及生物材料质量控制提供有效方 法。

Objective To express the synthetic gene of lymphocytic choriomeningitis virus(LCMV) and use the expressed product for the diagnosis of LCMV infection. Methods The hydrophilicity and hydro-phobicity of the whole length of amino acid sequence of nucleoprotein(Np) of LCMV were analyzed by Prosis software.Then the nucleotide sequence at the sites 1816-2178 of LCMV sRNA encoding the 380th-500th ami-no acids of Np was synthesized and cloned to His-tag pET-28a vector for expression. The expressed product was purified by Ni-NTA-Agarose chromatography and used for detecting LCMV by ELISA kit. Results Most of the expressed product existed in the form of inclusion body and contained more than 20% of somatic protein. After being purified, the purity of it reached above 90% .The expressed product was used for detecting 93 human serum specimens. No significant difference was observed in the positive rates detected by the expressed product(6.5%, 6/93) and the whole LCMV antigen(7.5%, 7/93). Conclusion The exp ressed product of synthetic gene of LCMV can be used for detecting LMCV infection as an alternative of LCMV antigen. It showed strong specificty and could block the possible transmission of LCMV. The study provide an effective method for the diagnosis of LCMV infection and the quality control of biological materials.