摘要
基于NCBI数据库中固氮菌nif D基因的核苷酸序列,设计并合成特异性引物,以甘蔗内生固氮菌Klebsiella variicola DX120E为模板,通过PCR方法获得了Klebsiella variicola DX120E nif D基因的ORF;以p ET30a(+)为原核表达载体,构建重组表达质粒p ET30a-nif D。将正确的重组质粒转入原核表达菌株BL21(DE3),在28℃培养条件下经1 mmol/L的异丙基硫代半乳糖苷(isopro-ply-β-D-thiogalactopyranoside,IPTG)诱导表达;通过SDS-PAGE电泳检测融合蛋白表达情况。结果表明,本研究克隆得到甘蔗内生固氮菌Klebsiella variicola DX120E nif D基因的ORF为1 452 bp,编码483个氨基酸;原核诱导表达后经提纯和质谱鉴定,该蛋白等电点为5.90,分子量为53.87 k D,与预期大小一致。该蛋白在原核表达系统中以包涵体的形式成功表达。利用Ni柱亲和层析方法进一步获得了纯度较高的重组蛋白,为今后单克隆抗体的制备、蛋白质印记杂交检测(Western Bloting)等研究奠定了基础。
According to the nif D nucleic acid sequence of other nitrogen-fixing bacteria strains registered on NCBI, specific primers were designed and an ORF of nif D gene was obtained by PCR using the template of the endogenous nitrogen-fixing bacteria strain Klebsiella variicola DX120 E from sugarcane. The recombinant plasmid p ET30a-nif D was constructed by using p ET30a(+) as prokaryotic expression vector. The correct recombinant plasmid was transformed into prokaryotic expression strain BL21(DE3). The fusion protein was induced by1 mmol/L IPTG at 28℃ and detected by SDS-PAGE. Sequence analysis showed that the ORF of nif D gene cloned in this study was 1 452 bp encoding 483 amino acids, the prokaryotically expressed protein was purified and detected with MOD-TOF, and the molecular weight was 53.87 k D with the isoelectric point of 5.90, which was consistent with the expected size. The p ET30a-nif D was effectively expressed in the form of inclusion bodies in a prokaryotic expression system. The highly purified recombinant protein was obtained by Ni affinity chromatography, which laid a foundation for further research on antibody preparation, enzyme activity in vitroand western blot analysis.
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2015年第3期512-520,共9页
Genomics and Applied Biology
基金
国家自然科学基金(31171504)
广西八桂学者岗位专项经费
广西特聘专家岗位专项经费
广西自然科学基金项目(2013NXNSFAA019082)
国家863计划项目(2013AA102604)共同资助
