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目的:构建人Calcineurin B(CnB)基因表达载体,在大肠杆菌中高效表达并纯化Calcineurin B蛋白。方法:用RT-PCR方法从人胎脑总RNA中反转录扩增Calcineurin B,将其插入克隆载体pGEM-T-easy中,测序鉴定阳性克隆,并将鉴定正确的Calcineurin B亚克隆至表达载体pET-32b(+),构建重组表达载体pET-CnB。结果阳性重组子在大肠杆菌BL21(DE3)和BL21(DE3)pLysS中,经IPTG诱导可高效表达Calcineurin B,经15％SDS-PAGE分析可观察到分子量与理论值相符(39kD)的诱导表达条带,并发现Calcineurin B主要以可溶性形式表达。Western blot结果证实,39kD的表达条带可与抗硫氧还蛋白的单克隆抗体起特异反应。用His-bind亲和层析纯化得到了纯度较高的calcineurin B融合蛋白。结论:用基因工程技术在大肠杆菌中高效表达人calcineurin B、并得到了纯度较高的calcineurin B融合蛋白,为进一步研究其生物学功能及研制calcineurin B单克隆抗体奠定了基础。
OBJECTIVE: To construct human Calcineurin B (CnB) gene expression vector and express and purify Calcineurin B protein in Escherichia coli. Methods: Calcineurin B was reverse transcribed from human fetal brain RNA by RT-PCR and inserted into the cloning vector pGEM-T-easy. The positive clones were identified by sequencing and subcloned into the expression of the correctly identified Calcineurin B Vector pET-32b (+) to construct the recombinant expression vector pET-CnB. Results The recombinant plasmids were highly expressed in Escherichia coli BL21 (DE3) and BL21 (DE3) pLysS induced by IPTG. The 15% SDS-PAGE analysis showed that the induced recombinant plasmids with the molecular weight of 39kD Band, and found that Calcineurin B is mainly expressed in soluble form. Western blot results confirmed that 39kD expression of the band with the anti-thioredoxin monoclonal antibodies play a specific reaction. Purified by His-bind affinity chromatography to obtain high purity calcineurin B fusion protein. Conclusion: The high expression of human calcineurin B in Escherichia coli by genetic engineering technique and the high purity of calcineurin B fusion protein were obtained, which laid the foundation for further study on its biological function and development of monoclonal antibody against calcineurin B.