蛇毒cystatin基因合成及其在大肠杆菌的表达

Gene Synthesis of cystatin from Snake Venom and its Expression in E.coli

目的 研究蛇毒 cystatin基因的合成并在大肠杆菌系统内表达。 方法 根据中华眼镜蛇毒 cystatin蛋白的氨基酸序列合成 cystatin基因的 4个片段 ,通过缓慢退火 PCR方法将其拼接为完整的 cystatin基因 ,经Bam H 及 Sac 双酶切后定向克隆到原核表达载体 p ET- 4 2 a(+)中 ,PCR及测序鉴定 ,转化宿主菌 BL 2 1 (DE3) ,异丙基硫代 -β- D半乳糖苷 (IPTG)诱导 ,SDS- PAGE凝胶电泳分析表达产物 ,GST· Mag琼脂糖树脂纯化融合蛋白 ,Western- blot鉴定。 结果 成功合成蛇毒 cystatin基因 ,并构建原核表达质粒 p ET- 4 2 a(+) /GST- cystatin,在大肠杆菌 BL 2 1 (DE3)中表达融合蛋白 GST- cystatin,SDS- PAGE凝胶浓度扫描显示表达量占菌体总蛋白的 30 % ,Western- blot证实纯化蛋白为重组 cystatin蛋白。 结论 合成蛇毒 cystatin基因并在大肠杆菌中表达 。

Objective To investigate gene synthesis of the cystatin from snake venom and its expression in Escherichia coli. Methods The four fragments of cystatin gene of snake venom were artificially synthesized according to its amino acid sequence. Slowly annealing PCR methods were conducted to obtain the full length of cystatin gene. The artificial gene was cloned into prokaryotic expression vector pET 42a(+) at BamH Ⅰ/Sac Ⅰ site. The cloned insert was identified by PCR and sequence analysis. The recombinant expression plasmid was transformed into E.coli. BL21(DE3) and GST cystatin fusion protein expression was conducted with IPTG. The fusion protein was purified by GST·Mag TM agarose beads. The expressed protein was identified by SDS PAGE and Western blot. Results The cystatin gene from snake venom was successfully synthesized and inserted into the prokaryotic expression vector pET 42a(+). The recombinant expression plasmid pET 42a(+)/GST cystatin was constructed. GST cystatin fusion protein was expressed in E.coli. BL21(DE3) and its amounts were about 30% of total bacterial proteins. The recombinant GST cystatin fusion protein from snake venom was obtained after purification by GST·Mag TM agarose beads. The recombinant cystatin protein was identified by Western blot. Conclusion The full length of cystatin gene from snake venom and its recombinant protein were obtained.