乙脑病毒prME蛋白与鼠GM-CSF编码基因的融合构建与表达  

Construction and Expression of Recombinant Plasmid Encoding prME Protein Derived from Japanese Encephalitis Virus and Granulocyte-macrophage Colony-stimulating Factor of BALB/c Murine

作　　者：翟永贞[1] 周言[1] 马力[1] 冯国和[1] 

机构地区：[1]中国医科大学附属盛京医院感染科,沈阳110004

出　　处：《中国医科大学学报》2010年第9期706-709,共4页Journal of China Medical University

基　　金：国家自然科学基金资助项目(30471547);辽宁省自然科学基金资助项目(20042072);辽宁省教育厅高校科研基金资助项目(05L498);辽宁省教育厅高校科研基金资助项目(L2010589)

摘　　要：目的构建乙脑病毒prME蛋白与鼠GM-CSF融合基因重组子并建立稳定细胞表达株。方法套式-RT-PCR法从BALB/c鼠脾组织获取GM-CSF编码基因,用限制性内切酶从含乙脑病毒prME蛋白基因重组子获取prME蛋白基因,定向克隆至同一真核表达载体pcDNA3.1(+)不同酶切位点,构建重组子pJME/GM-CSF并经酶切及DNA测序分析。脂质体法将pJME/GM-CSF转染中华仓鼠卵巢(CHO)细胞。Western blot法检测转染的CHO细胞中融合蛋白表达。结果 pJME/GM-CSF经BamH I/EcoRI和BamH I/Not I酶切释出的插入子片段(2001 bp、2472bp)分别与预期结果相符合。所编码的融合蛋白相对分子量(Mr)为85×103。结论 pJME/GM-CSF成功构建,转染的CHO细胞可稳定表达融合蛋白。Objective Construction and expression of recombinant plasmid fusing encoding prME protein derived from Japanese encephalitis virus （JEV） and GM-CSF of BALB/c mouse. Methods GM-CSF gene was ampli/ied by nested-RT-PCR from BALB/c spleen cells. JEV prME protein gene was eluted by the digestion with restrict/on endonucleases BamH I/EcoR I from pJME piasmid. Genetic fusion of prME protein and GM-CSF are subcloned info pcDNA3.1 （＋） eukaryotic vector, and named as pJME/GM-CSF.The recombinant was confirmed by restriction enzymes digestion and DNA sequencing, and then transfected into China hamster ovary （CHO） cells by Lipofectamine2000. pJME/ GM-CSF expression in CHO cells was examined by Western blot. Results We observed 2001 bp and 2472 bp DNA fragments when pJME/GM-CSF was digested with BamHI/EcoRI and BamHI/NotI respectively as expected. The estimated molecular weight of the fusion protein was 85kD. Conclusion The recombinant pJME/GM-CSF was constructed and transfected into CHO cells successfully with pJME/GMCSF stably expressed.

关 键 词：脑炎病毒 日本 病毒蛋白质类 粒细胞-巨噬细胞集落刺激因子 CHO细胞 

分 类 号：R512.32[医药卫生—内科学]