人LDHB的原核表达及体外抑制剂筛选模型的建立

Expression of human LDHB in prokaryote and establishment of its inhibitor screening model

目的建立人乳酸脱氢酶B(LDHB)体外抑制剂筛选模型。方法用一对5'端分别带NdeⅠ、XhoⅠ位点的引物扩增LDHB cDNA。LDHB cDNA经NdeⅠ+XhoⅠ双酶切后与同样酶切的pET-28a质粒连接,连接产物转化大肠杆菌DH5α。将筛选的重组质粒转化大肠杆菌BL21(DE3),用0.2 mmol/L IPTG诱导LDHB表达,镍离子亲和层析柱纯化LDHB。以丙酮酸钠和NADH为底物,通过检测340 nm波长处光密度值的变化,确定LDHB及其抑制剂棉酚的最适浓度,并计算模型的Z因子。结果 LDHB的表达量为25 mg/L,LDHB、棉酚的最适浓度分别为22.5μg/L、10μmol/L,筛选模型的Z因子为0.91。结论建立了成本低、可靠性强、适合高通量的LDHB体外抑制剂筛选模型。

Objective To establish an in vitro inhibitor screening model of human lactate dehydrogenase B（ LDHB）. Methods LDHB cDNA was amplified by a pair of primers,flanked with Nde I and Xho I restriction enzymes sites at the 5＇ end,respectively.LDHB cDNA and p ET-28a plasmids were codigested by Nde I and Xho I,and then ligated. The ligation mixture was transformed into E. coli DH5α. Recombinant plasmid pET-28a-LDHB was screened and transformed into E. coli BL21（ DE3）,0. 2 mmol/L IPTG was used to induce the expression of LDHB,and fusion protein LDHB was purified by Ni^（2＋） affinity column. NADH and pyruvate sodium were used as substrates of LDHB. LDHB enzymatic activity was determined by the change of OD（340 nm） of NADH. The model was optimized to apply the best working condition of LDHB and gossypol（ LDHB inhibitor）. Results LDHB was expressed in BL21（DE3）with the yield of 25 mg/L. The optimal LDHB concentration was 22. 5 μg/L. The Z score was 0. 91 using 10 μmol/L gossypol as positive control. Conclusion This assay is reliable,cost-effective and suitable for high-throughput screening of LDHB inhibitor.