粉尘螨变应原DerfMalf6的克隆,表达及生物信息学分析.docVIP

*【基金项目】 国家自然科学基金项目（NO. NS-F NO. NS-F；江苏省卫生厅招标项目（NO. Z200914）;江苏省卫生职业技术教育研究立项课题（NO. J200907）；江苏省“六大人才高峰”第六批项目资助。 **【通讯作者】 崔玉宝，Email： HYPERLINK mailto: （通讯作者不是指导导师） 【作者简介】 孙金霞 (1986-)，女，江苏省盐城人，助教，主要从事过敏性疾病及相关过敏原的研究。 Email： HYPERLINK mailto: 粉尘螨变应原Der f Mal f 6的克隆、表达及生物信息学分析* 孙金霞，周鹰，杨李，俞黎黎，王运刚，卞勇华，崔玉宝** (盐城卫生职业技术学院，江苏盐城224005) 【摘要】 目的 克隆粉尘螨变应原Der f Mal f 6(简称Mal f 6)编码基因并构建其原核表达体系。 方法 提取粉尘螨总RNA，根据GenBank (AY283280.1)设计并合成引物，RT-PCR扩增Der f Mal f 6全长基因，克隆至pCold TF DNA载体，转化至E.coli JM109，取阳性克隆测序；将pCold TF- Mal f 6质粒转化至BL21，IPTG诱导表达，SDS验证产物。生物信息学软件预测Mal f 6的理化特性、结构和功能。 结果 RT-PCR获得全长为495bp的Mal f 6基因。原核表达后经SDS电泳显示其全细胞、上清及沉淀物均有蛋白表达，上清表达量较高。生物信息学分析显示该蛋白分子由164个氨基酸组成，分子量为17.7KDa，二级结构由α螺旋(4.88%)，延伸主链(37.8%)，无规则卷曲(57.32%)组成，是亲水性细胞质蛋白，具有肽酰-脯氨酰反式异构酶活性。 结论 粉尘螨变应原Mal f 6原核表达获得成功，为该变应原进一步研究及规模生产并应用于临床奠定了基础。 【关键词】粉尘螨；变应原；原核表达；生物信息学 【中图文分类号】R384.4 Cloning, expression, and analysis of the Mal f 6 allergen from Dermatophagoides farinae in China Sun Jinxia, Zhou Ying, Yu Lili , Yang Li, Wang Yungang, Bian Yonghua, Cui Yubao (Yancheng Institute of Health Science, Yancheng, Jiangsu 224005, China) 【Abstract】 Objectives To acquire the cDNA of Der f Mal f 6 gene (Mal f 6) from Dermatophagoides farinae (D. farinae) in China, and construct a prokaryotic expression system. Methods Total RNA was extracted from D. farinae. The Mal f 6 gene was amplified by RT-PCR and cloned into pCold TF DNA vecter. The pCold TF-Mal f 6 was then transformed into E.coli BL21 and induced to express target protein by IPTG. It was then identified by SDS. Bioinformatic tools were used to analysis the physiochemical properties, secondary structure and function of Mal f 6. Results The Mal f 6 gene was obtained by RT-PCR and was 495bp in full length. The constrcuted plasmid pCold TF-Mal f 6 was then transformed into E.coli BL21, and induced to express protein. SDSdetected a specific band in whole cells, supernatant, and precipitate containing pCold TF- Mal f 6. Bioinformatics indicated that the allergen c