文档介绍：mFSHR基因N端片段的序列分析、原核表达载体构建及其表达
(作者:___________单位: ___________邮编: ___________)
作者:黄海雁,文梦灵,张惠玲
【摘要】目的:克隆小鼠卵泡刺激素受体(FSHR) 基因N端部分片段(2890aa)(mFSHRn);预测其编码蛋白作为候选避孕疫苗的可行性;构建原核表达重组质粒,并在大肠杆菌中表达。方法: 提取小鼠睾丸组织总RNA,利用逆转录聚合酶链反应(RTPCR)技术反转录成cDNA,按照GeneBank中小鼠FSHR N端序列设计引物,扩增基因片段并插入pET32a载体,测序鉴定后做生物信息学分析;质粒转化E. coli BL21(DE3)感受态菌株诱导表达蛋白。结果:扩增片段长度为186 bp, 测序结果与预期结果完全一致,生物信息学分析其编码蛋白具有良好的抗原性;重组原核表达质粒经鉴定获得正确重组子并诱导表达。结论: mFSHRn蛋白可作为良好的候选避孕疫苗,成功构建了pET32amFHSRn原核表达重组质粒,获得可表达mFHSRn的大肠杆菌菌株,为后续的相关研究奠定了基础。
【关键词】卵泡刺激素受体;原核; 表达;生物信息学分析
Objective: To clone follicle stimulating hormone receptor gene N terminal fragment (28
90aa) (mFSHRn), and to forecast the possibility of its encoding protein being pregnancy ine, and to construct prokaryotic expression binant plasmid and express it in colibacillus. Methods: Mouse testis RNA was extracted and inversely transcribed to cDNA by means of RTPCR. The primer was designed according to mouse FSHR N terminal sequence of Genebank, and then gene fragments were amplified and pET32a vector was inserted. After sequencing, bioinformatics was analyzed. Expression protein was induced by plasmid transformation E. coli BL21 (DE3) competence strain. Results: The amplification length was 186bp, sequencing and expectation pletely concord. Bioinformatics analysis showed the encoding protein had satisfactory antigenicity. The restructuring prokaryotic expression plasmid obtained recon and induced : mFSHRn protein can be a reliable candidate of pregnancy ine. We essfully construct pET32amFHSRn prokaryotic expression binant plasmid and gain mFHSRn expressing colibacillus strain, and lay a foundation for further study.
[KEY WORDS] Follicle stimulating hormone receptor;Protocaryon; Expres
sion; Bioinformatics analysis
卵泡刺激素(folliclestimulating hormone ,FSH)是一种糖蛋白激素,通过特定的G蛋白偶联受体在细胞膜表面发挥作用。卵泡刺激素受体(folliclestimulating hormone receptor, FSHR)是一种寡聚糖蛋白构成的膜受体, 4个单体由二硫键连接,属于G蛋白偶联受体家族。人FSHR由695个氨基酸组成:氨基端17个氨基酸为疏水性信号肽;紧接信号肽的是349个氨基酸组成的亲水性结构