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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 a fragment of FSHR gene (145 ~ 330bp) (FSHRn) and construct a eukaryotic recombinant plasmid to predict its feasibility as a candidate contraceptive vaccine. Methods Total RNA of mouse testis was extracted and reverse transcribed into cDNA by RT-PCR. Primers were designed according to FSHRN-terminal sequence of mouse and mouse in GenBank. The gene fragment was amplified and inserted into pcDNA3.1 / myc-His (-) B vector The recombinant plasmids were identified by PCR, double enzyme digestion and sequencing. VectorNTI9.0 software was used for bioinformatics analysis. Results The length of amplified fragment was 186bp. The sequencing result was consistent with the known sequence. The recombinant plasmid was identified by PCR and double enzyme digestion. The bioinformatics analysis showed that the antigenic peptides mainly concentrated in 15 ~ 20aa , 22 ~ 27aa, 32 ~ 36aa, 42 ~ 48aa, 58 ~ 67aa, and 90% homology with human. Conclusion FSHRn gene fragment was successfully cloned and pcDNA3.1 / FHSRn eukaryotic expression recombinant plasmid was constructed. FSHRn has good antigenicity, FSHRn protein can be used as a good animal candidate vaccine, in-depth study of this protein and human male contraception vaccine Lay the foundation for the development.