功能未知基因LOC410296的表达载体构建及小鼠抗血清制备  

Construction of expression vector and preparation of antiserum for the undefined gene LOC401296

作　　者：沈丽萍[1,2] 陈虹[2] 张学清[2] 周建平[3] 林仲武[4] 王治东[2] 陈英[1,2] 

机构地区：[1]安徽医科大学,合肥230032 [2]军事医学科学院放射与辐射医学研究所,北京100850 [3]军事医学科学院毒物药物研究所,北京100850 [4]军事医学科学院科技部,北京100850

出　　处：《军事医学》2014年第3期212-215,共4页Military Medical Sciences

摘　　要：目的为深入研究功能未知基因LOC401296,首先获取其全长序列,实现LOC401296分子的真核和原核表达,并制备针对LOC401296蛋白的抗血清。方法采用PCR方法克隆LOC401296编码区全长序列,利用pET28B和pCMV-Myc质粒构建LOC401296原核表达和真核表达载体,用异丙基硫代半乳糖苷(IPTG)诱导目的蛋白原核表达,目的蛋白经纯化、复性后免疫小鼠制备抗血清。结果 PCR克隆得到LOC401296编码区全长序列,成功构建LOC401296原核及真核表达载体,IPTG诱导目的蛋白在大肠杆菌体内表达,用纯化蛋白免疫小鼠获得抗人LOC401296蛋白血清,ELISA检测抗血清效价达1∶64 000,Western印迹证实LOC401296真核表达载体成功表达且抗血清特异性良好。结论获得LOC401296基因编码区全长序列,成功构建了LOC401296基因表达载体,分别在大肠杆菌和真核细胞内实现LOC401296蛋白原核表达和真核表达,获得小鼠抗人LOC401296蛋白抗血清,为LOC401296基因功能研究奠定了基础。Objective To clone the full-length CDS sequence of LOC 401296 , an undefined gene that we found recently, and to obtain both its expression in eukaryotic cells or E.coli and antiserum to LOC401296 protein.Methods The full-length CDS sequence of LOC401296 was amplified by polymerase chain reaction （PCR）.Then we established the expression vectors pET28B-LOC401296 and pCMV-Myc-LOC401296 by cloning the full-length CDS sequence into vector PET28B and vector pCMV-Myc respectively.Isopropyl Thiogalactoside （IPTG） was used to induce LOC401296 expression in E.coli.Furthermore,the protein purified and refolded was used to immunize BALB /c mice.The titer of the antiserum collected from immunized mice was identified by ELISA assay and Western blot .Results We cloned the full-length CDS sequence of LOC401296 was successfully .Protein LOC401296 was expressed as was expected and the mouse anti-human LOC401296 antiserum was obtained .The antiserum titer reaching 1∶64 000 was identified by ELISA .Besides, Western blot analysis showed that the antiserum could be used to detect protein LOC 401296 .Conclusion The full-length sequence of LOC401296 is obtained and the mouse anti-human LOC401296 antiserum becomes available .This study can contribut to further research on the undefined gene LOC 401296 .

关 键 词：LOC401296 功能未知基因 原核表达 真核表达 蛋白纯化 抗血清 

分 类 号：Q78[生物学—分子生物学]