FSHR部分基因的真核表达质粒构建和生物信息学分析  

Cloning and Bioinformatics Analysis of FSHR Gene Fragment

作　　者：朱秀兰[1] 马文丽[1] 德伟[2] 彭翼飞[1] 阴常欣[1] 郑文岭[1,3] 

机构地区：[1]南方医科大学基因工程研究所,广州510515 [2]南京医科大学生物化学与分子生物学系,南京210029 [3]华南基因组中心,广州510800

出　　处：《热带医学杂志》2009年第3期238-240,252,共4页Journal of Tropical Medicine

基　　金：广东省重点实验室建设基金(No.2004B60144)。

摘　　要：目的克隆FSHR基因部分片段(145～330bp)(FSHRn),构建真核表达重组质粒,预测其编码蛋白作为候选避孕疫苗的可行性。方法提取小鼠睾丸组织的总RNA,利用RT-PCR技术反转录成cDNA,按照GenBank中小鼠FSHRN-端序列设计引物,扩增基因片段并插入pcDNA3.1/myc-His(-)B载体,重组质粒经PCR、双酶切和测序鉴定后用VectorNTI9.0软件作生物信息学分析。结果扩增片段长度为186bp,测序结果与已知序列吻合,重组真核表达质粒经PCR和双酶切鉴定获得正确重组子,生物信息学分析其编码蛋白抗原性肽段主要集中在15～20aa、22～27aa、32～36aa、42～48aa、58～67aa,与人的基因同源性为90%。结论成功克隆了FSHRn基因片段并构建了pcDNA3.1/FHSRn真核表达重组质粒;FSHRn具有良好的抗原性,FSHRn蛋白可作为良好的动物候选疫苗,为此段蛋白的深入研究和人类男性避孕疫苗的研制打下基础。Objective To clone the fragment （145-330 bp） of FSHR gene （FSHRn）, construct pcDNA3.1/ FHSRn recombinant eukaryotic expression plasmid and predict the feasibility of the encoding protein as a contraceptive vaccine candidate. Methods The FSHRn DNA fragment was amplified by RT-PCR using specific primers, and then cloned into pcDNA3.1/myc-His （-）B vector to yield the recombinant plasmid. Bioinformatics analysis was performed using the Vector NTI9.0 software. Results A 186 bp long fragment of the mouse FSHR gene was successfully amplified and cloned. The recombinant expression vector pET32a-FSHRn was identified successfully by PCR and incision enzyme digestion. The cloned fragment showed a 100% homology with the mouse gene （Genbank/NCBI：NM_013523.2）. Bioinformatics analysis indicated that the antigenic epitopes were mainly located at 15-20aa, 22-27aa, 32-36aa, 42- 48aa and 58-67aa. The cloned gene fragment had a 90% homology with the human gene. Conclusion The FSHRn gene was successfully cloned and inserted into the pcDNA3.1/myc-His（-）B vector. The encoding protein might have a favourable antigenicity and could be used as a contraceptive vaccine candidate. The cloned gene fragment may be used for the subsequent FSHR researches and the development of male contraceptive vaccine.

关 键 词：FSHR 疫苗 真核表达 生物信息学 

分 类 号：R382.5[医药卫生—医学寄生虫学] R714.51[医药卫生—基础医学]