天蚕素 B-黄体生成素释放激素结合部位重组基因的构建及其抑癌功能的研究

目的构建天蚕素B-黄体生成素释放激素结合部位（CB-LHRH’）重组基因,探讨CB-LHRH’蛋白成为新型抗肿瘤靶向药物的可能性。方法设计并人工合成CB-LHRH’重组基因序列,应用昆虫细胞．杆状病毒表达系统表达目的蛋白并通过蛋白斑点印迹法鉴定,采用二甲氧唑黄（XTT）比色法检测不同剂量的目的蛋白对卵巢上皮性癌细胞株SKOV3（LHRH受体阴性）和子宫内膜癌细胞株HEC-1B（LHRH受体阳性）细胞的生长抑制率,并进行镜下形态观察。结果蛋白斑点印迹法检测结果显示,重组杆状病毒感染的Si9细胞裂解液呈现棕色,证实CB-LHRH’蛋白已表达。重组杆状病毒感染的昆虫细胞系Sf9细胞裂解液对SKOV3及HEC-1B细胞的生长抑制率,随作用时间的延长和剂量的增加而增加,SKOV3细胞的生长抑制率从（5.03±0.08）％增加至（53.24±1.22）％,HEC-1B细胞的生长抑制率从（5.13±0.37）％增加至（56.16±1.08）％。相同剂量的重组杆状病毒感染的St9细胞裂解液,作用相同时间,其对HEC-1B细胞的生长抑制率高于SKOV3细胞,差异有统计学意义（P〈0.01）。经重组杆状病毒感染的St9细胞裂解液作用后,镜下可见癌细胞出现明显改变甚至成片坏死崩解为无定形的颗粒状物质。结论CB-LHRH’蛋白可有效杀伤LHRH受体阳性的HEC-1B细胞,有望成为治疗表达LHRH受体的肿瘤的抗肿瘤靶向药物。

Construction and expression of recombinant cecropin B-binding site of luteinizing hormone releasing hormone gene and its anticancer function

OBJECTIVE: To construct and express recombinant cecropin B-binding site of luteinizing hormone releasing hormone (CB-LHRH') gene, and to evaluate the anticancer function of CB-LHRH' on human ovarian cancer cell line SKOV3 and human endometrial cancer cell line HEC-1B. METHODS: The sequence of the cDNA encoding CB-LHRH' was designed, artificially synthesized, verified by DNA sequence analysis and expressed by Bac-to-Bac baculovirus expression system. The expression of CB-LHRH' proteins were identified by western dot blot using rabbit polyclonal antibody against LHRH as the primary antibody. To determine the anticancer effects of the CB-LHRH' protein, ovarian cancer cell line SKOV3 and endometrial adenocarcinoma cell line HEC-1B were treated by different doses of the CB-LHRH' protein. Cell growth inhibition assay was performed using the 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl) 5 (phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) kit at different times, and cell morphologic changes were observed under the inverted microscope. RESULTS: The inhibitory rate of proliferation by CB-LHRH' increased with the increase of dose and time respectively: SKOV3 cell, from (5.03 +/- 0.08)% to (53.24 +/- 1.22)%; HEC-1B cell, from (5.13 +/- 0.37)% to (56.16 +/- 1.08)%. The inhibitory effect on HEC-1B cell was stronger than that on SKOV3 cell (P < 0.01). There were obvious cell morphologic changes. CONCLUSION: The CB-LHRH' protein has anticancer activity, probably through the LHRH receptor, and it may be expected to be a potential candidate as peptide drugs for targeted cancer therapy.