慢病毒敲减质粒pLKO.1-hSRF的构建及鉴定

目的:构建敲减人血清反应因子SRF基因的慢病毒质粒,为进一步研究SRF在口腔鳞状细胞癌中的作用提供工具。方法:利用Thermo Fisher公司RNAi网站设计出针对SRF基因特异性敲减的片段,然后通过对pLKO.1载体进行双酶切,胶回收纯化后,经T4连接酶将双酶切线性化载体与设计的目的片段进行连接,转化和质粒提取,采用双酶切以及测序的方法对重组质粒进行序列鉴定。利用293T细胞对构建正确的pLKO.1-hSRF质粒进行病毒包装后感染SAS口腔鳞癌细胞,经嘌呤霉素筛选获得稳定株,并通过Western blot和实时荧光定量PCR对稳定转染细胞株的敲减效率进行验证。结果:构建出的慢病毒敲减质粒pLKO.1-hSRF经测序和酶切鉴定均正确,感染该慢病毒质粒的SAS细胞后,其SRF蛋白表达量和mRNA水平与对照组相比均显著下降(P<0.01)。结论:慢病毒敲减质粒pLKO.1-hSRF构建成功,获得SRF低表达的SAS细胞株。国家自然科学基金(81671001,81771079,207201);;福建省中青年骨干人才项目(2015-ZQN-ZD-35);;厦门医学院科研基金(K2015-06);;厦门市重大疾病联合攻关项目(3502Z20179051

Prokaryotic expression of SIRT5 and preparation of its polyclonal antibody

目的克隆小鼠的SIrT5基因,及制备SIrT5多克隆抗体。方法提取小鼠肝细胞总rnA进行rT-PCr,PCr法扩增SIrT5基因,将此基因克隆至PET-28A载体,转化E.COlI bl21(dE3),经异丙-β-d-硫代吡喃半乳糖苷(IPTg)诱导表达后,利用nI 2+-nTA亲和层析柱纯化;纯化的目的蛋白免疫bAl/C小鼠后,收获血清并鉴定。结果成功构建了SIrT5原核表达载体,并能在宿主菌E.COlI bl21(dE3)中高效表达;利用纯化的蛋白制备了理想的多克隆抗体。结论利用分子克隆技术,获得了高纯度的SIrT5蛋白并制备了多克隆抗体,为进一步研究奠定了基础。Objective To construct prokaryotic expression vector of SIRT5and prepare its polyclonal antibody.Methods Total RNA isolated from mice liver cell was RT-PCR.Sirt5was amplified by RT-PCR and cloned into pET-28aplasmid,and the recombinant plasmid was transformed into E.coli BL21(DE3).After induction by IPTG,the fusion protein of SIRT5was produced,the protein was purified by Ni 2+-NTA afinity chromatography.Bal/c mice were immuned with the purified protein of SIRT5,and mice serum was collected for identification.Results SIRT5was successfully constructed and highly expressed in E.coli BL21(DE3).The purified protein was used to prepare polyclonal antibody.Conclusion Using moleccular cloning techonlogy,purified SIRT5protein can be obtained and prepared its polyclonal antibody,which may provide an foundation for further investigation