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目的从人附睾组织中克隆OCTN2基因,构建PGEX-2T-OCTN2载体,原核表达人OCTN2重组蛋白。方法应用RT-PCR方法从人附睾组织中克隆OCTN2,利用ECoRⅠ和BamHⅠ酶切位点把OCTN2克隆到PGEX-2T载体,酶切和测序鉴定后,大量诱导重组蛋白并回收。结果从人附睾组织中克隆到人OCTN2的基因片段,构建了PGEX-2T-OCTN2载体,表达了预期相对分子质量的融合蛋白并大量回收。结论成功克隆OCTN2,表达并大量回收其融合蛋白,为制备人OCTN2特异性抗体、进一步研究附睾肉碱转运机制奠定了前期基础。
Objective To clone the OCTN2 gene from human epididymis, construct PGEX-2T-OCTN2 vector and express prokaryotic recombinant human OCTN2 protein. Methods OCTN2 was cloned from human epididymis by RT-PCR. OCTN2 was cloned into PGEX-2T vector using EcoRⅠand BamHⅠ restriction sites. After digestion and sequencing, the recombinant protein was induced and recovered. Results The gene fragment of human OCTN2 was cloned from human epididymis and PGEX-2T-OCTN2 vector was constructed. The fusion protein with the expected relative molecular mass was expressed and recovered in large quantities. Conclusion OCTN2 was cloned successfully, and its fusion protein was expressed and recovered in large quantities. This study laid the foundation for the further study of carnitine transport mechanism in order to prepare human OCTN2 specific antibodies.