融合基因VH-mms13的构建及其蛋白表达鉴定  

Construction of fusion gene VH-mms13 and identification of its protein

作　　者：杨柳青[1] 孔登[1] 王雪耘[1] 王晓红[1] 孟丽[1] 王小柯[1] 

机构地区：[1]潍坊医学院生物化学与分子生物学教研室,山东潍坊261053

出　　处：《中国生化药物杂志》2015年第12期6-8,12,共4页Chinese Journal of Biochemical Pharmaceutics

基　　金：山东省科技攻关计划(2009GG10002079)

摘　　要：目的构建原核表达载体p ET30a(+)-VH-mms13,诱导表达后鉴定融合蛋白表达。方法分别扩增单克隆抗体基因的重链可变区VH基因和细菌磁小体膜蛋白基因mms13基因,采用重叠延伸PCR技术(splicing by overlap extension,SOE-PCR)构建融合基因VH-linker-mms13,并将融合基因插入pET30a(+)载体,酶切、测序验证;将重组质粒导入大肠杆菌DE3中,0.4 mmol/L异丙基疏代半乳糖苷(Isopropylβ-D-thiogalactoside,IPTG)诱导表达,产物经SDS-PAGE电泳和Western blot双重鉴定。结果 PCR鉴定构建的融合基因VH-mms13大小为738 bp,与理论值相符,测序结果表明序列无误;转入DE3经IPTG诱导,在包含体中检测到融合蛋白表达;Western blot结果显示该表达蛋白可与His-tag抗体特异性结合,蛋白大小符合融合蛋白理论预期。结论成功构建了融合基因VH-mms13的表达载体p ET30a(+)-VH-mms13,且融合蛋白反应原性良好,为生物磁靶向药物的研发奠定了基础。Objective To construct the prokaryotic expression vector pET30a（ ＋）-VH-mms13 and identification of its protein after induced with IPTG. Method Heavy chain variable region VH gene of typeⅣcollagenase monoclonal antibody and magnetosome membrane protein gene mms13 were amplified separately,the fusion gene VH-linker-mms13 were synthesized by SOE-PCR technique and inserted into p ET30a（ ＋） plasmid,which was confirmed by restriction enzyme digest and sequencing. Then the recombinant plasmid p ET30a（ ＋）-VH-mms13 was transform into E. coli DE3 and induced with 0. 4 mmol / L IPTG. The fused protein was identified by SDS-PAGE and Western blot. Results The length of fusion gene VH-mms13 was 738 bp,and the sequence was correct. After induced with IPTG,the fused protein was found in the inclusion body and Western blot results suggested that the fused protein can bind with His-tag antibody specifically. Conclusion Expression vector p ET30a（ ＋）-VH-mms13 is successfully constructed and the fusion protein has good immunogenicity,which lay the foundation for the development of biomagnetism-targeted drug.

关 键 词：VH mms13 重叠延伸PCR 融合蛋白 

分 类 号：Q78[生物学—分子生物学]