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目的克隆小鼠无纤毛柱状上皮细胞分泌蛋白(CCSP)基因并构建其真核表达重组体,鉴定重组CCSP的生物学活性。方法采用TRIzol法提取小鼠肺组织总RNA,反转录-聚合酶链反应(RT-PCR)对CCSP基因进行扩增,Bam H I和XhoⅠ双酶切扩增产物与真核表达载体p CDNA3.1(+),酶切产物连接并转化入感受态细胞Trans 5α,阳性重组质粒p CDNA3.1(+)-m CCSP经PCR、酶切和测序鉴定正确后,通过DNAfectin 2100 DNA转染试剂将重组质粒转染人胚肾(HEK293T)细胞,采用Western blot法检测表达产物。脂多糖分别刺激转染有p CDNA3.1(+)-m CCSP和p CDNA3.1(+)质粒DNA的HEK 293T细胞,利用磷脂酶A2的水解作用,以掺入大肠杆菌膜的[3H]标记的油酸为酶解底物,检测HEK 293T细胞内PLA2的活力。结果聚合酶链反应结果显示,从小鼠肺组织中扩增出约290 bp的片段。菌落PCR、双酶切及测序结果显示,重组质粒p CDNA3.1(+)-m CCSP构建成功且序列正确。Western blot结果显示,在转染p CDNA3.1(+)-m CCSP的HEK293T细胞中有CCSP蛋白的表达,蛋白质分子量约为10 k Da,而转染空质粒组未见表达。CCSP蛋白可以显著降低脂多糖诱导的HEK 293T细胞内PLA2的活力。结论成功构建了真核表达重组体p CDNA3.1(+)-m CCSP,且重组蛋白CCSP具有生物学活性,为进一步研究其功能奠定了基础。
Objective To clone mouse ciliated epithelial cell secreting protein (CCSP) gene and construct its recombinant eukaryotic expression vector to identify the biological activity of recombinant CCSP. Methods Total RNA was extracted from the lungs of mice by TRIzol method. The CCSP gene was amplified by reverse transcription - polymerase chain reaction (RT - PCR). The amplified product of Bam HI and Xho Ⅰ was digested with restriction endonuclease pCDNA3. The recombinant plasmid pCDNA3.1 (+) - m CCSP was identified by restriction endonuclease digestion and DNA sequencing. The DNAfectin 2100 DNA transfection reagent Recombinant plasmids were transfected into human embryonic kidney (HEK293T) cells and the expression products were detected by Western blot. Lipopolysaccharide stimulated HEK 293T cells transfected with pCDNA3.1 (+) - m CCSP and pCDNA3.1 (+) plasmid DNA, respectively, and hydrolyzed with [3H] The labeled oleic acid was used as substrate for enzymatic hydrolysis to detect the activity of PLA2 in HEK 293T cells. Results The results of polymerase chain reaction showed that a fragment of about 290 bp was amplified from mouse lung tissue. The results of colony PCR, double enzyme digestion and sequencing showed that the recombinant plasmid pCDNA3.1 (+) - m CCSP was successfully constructed and its sequence was correct. Western blot results showed that CCSP protein was expressed in HEK293T cells transfected with pCDNA3.1 (+) - m CCSP, with a molecular weight of about 10 kDa, but no expression was found in transfected empty plasmid. CCSP protein can significantly reduce LPS-induced PLA2 activity in HEK 293T cells. Conclusion The recombinant eukaryotic expression vector pCDNA3.1 (+) - m CCSP was successfully constructed and the recombinant protein CCSP has the biological activity, which laid the foundation for further study of its function.