人Calcineurin B基因克隆、表达和纯化

Construction of Human calcineurin B Gene Expression Vector

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 the human calcineurin B expression vector and express and purify Calcineurin B protein in E.coli BL21 （DE3） and BL2 1（DE3）pLysS. Methods Total RNA isolated from human fetal brain tissue was used as a template. The entire coding region of the calcineurin B was amplified, purified and inserted into pGEM-T-easy vector. The sequence of the inserted fragment was verified by restriction enzyme digestion, PCR identification and DNA sequencing. The DNA fragment containing the entire coding sequence was excised from pGEM-T-easy vector by digestion with Nco Ⅰ and BamH Ⅰ and subcloned into the expression vector pET32b（＋） under the control of the T7 RNA polymerase promoter. Correct pET-CnB plasmids were transformed into E.coli BL21 （DE3） and BL21 （DE3）pLysS. Results After IPTG induction, a high level expression ofcalcineurinB was obtained. SDS-PAGE showed that the E.coli with recombinant expression vector could express a 39kD soluble protein. Western blot analysis show that anti-Trx McAb specifically bound to the 39kD band. Pure recombinant calcineurin protein was obtained by high-affinity His-bind purification system. Conclusion We cloned calcineurin B to express the protein in E. coli in order to study the biological function and make the monoclonal antibody of calcineurin.