Construction of Human calcineurin B Gene Expression Vector
Zhang Huili Huang Zanchun Zhang Yu Xu Pin Cheng Yu Hui RutaiSino-German Laborary, Cardiovascular Institute and Fu Wai Hospital, CAMS and PUMC, Beijing (100037)
Objective To construct the human calcineurin B expression vector and express and purify Calcineurin B protein in E.coli BL21 (DE3) and BL21(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 se-quence 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 of calcineurihB 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.
【CateGory Index】： R346