2种家蚕蛋白酶抑制剂的重组表达及产物对胰岛素口服吸收的影响  

Recombinant Expression of Two Silkworm Protease Inhibitors and Their Effect on Absorption of Orally-taken Insulin

作　　者：赵梦茹 王丹[1,2] 夏良存 李司 张文平[1,2] 

机构地区：[1]浙江理工大学生命科学学院,杭州310018 [2]浙江省家蚕生物反应器和生物医药重点实验室,杭州310018

出　　处：《蚕业科学》2017年第6期929-936,共8页ACTA SERICOLOGICA SINICA

基　　金：浙江省科技厅公益技术研究社会发展项目(No.2016C330-80);浙江理工大学科研启动基金项目(No.16042187-Y);浙江省科技厅公益技术研究农业项目(No.2016C32012)

摘　　要：口服胰岛素(INS)治疗糖尿病存在因胃肠道的蛋白酶而发生药物降解的问题。从家蚕蛹中分离了Bm Kazal、Bm CP82种蛋白酶抑制剂,设计其编码基因引物序列克隆2个目的基因,构建重组表达载体p ET28a-Bm Kazal和p ET32a-Bm CP8在大肠埃希菌(Escherichia coli)中表达并纯化了重组Bm Kazal和Bm CP8蛋白。将这2种蛋白酶抑制剂与INS混匀制成INS口服悬液给小鼠灌胃,研究其对INS口服吸收的影响。试验结果表明:按1.5 IU/kg剂量皮下注射INS的对照组小鼠,血药达峰浓度C_(max)为(7.56±1.40)m IU/L,达峰时间T_(max)为1 h;口服0.5μg/μL Bm CP8+15 IU/kg INS组、0.5μg/μL Bm Kazal+15 IU/kg INS组、0.5μg/μL Bm CP8+0.5μg/μL Bm Kazal+15 IU/kg INS组小鼠的血药达峰浓度C_(max)分别为(6.07±0.51)、(7.12±0.89)、(9.20±2.00)m IU/L,达峰时间T_(max)均为3 h;而按15 IU/kg剂量单纯口服INS对照组小鼠的血药浓度一直维持在4.32 m IU/L左右。以上结果说明Bm CP8、Bm Kazal能够协同INS经小鼠胃肠道口服吸收入血液,而且二者联合使用效果更好,使小鼠体内对INS的口服生物利用度由1.51%提高至4.17%,有促进小鼠对胰岛素口服吸收的作用。Orally-taken insulin for treating diabetes has the problem of drug degradation due to existence of proteases in stomach and intestine. In this study,two protease inhibitors named Bm Kazal and Bm CP8 were isolated from silkworm（ Bombyx mori） pupa. Subsequently,primer sequences were designed according to their coding genes for cloning the two target genes. After the two recombinant plasmids,p ET28 a-Bm Kazal and p ET32 a-Bm CP8,were constructed and expressed in E. coli,purified Bm Kazal and Bm CP8 proteins were obtained. These two protease inhibitors were added into insulin as pharmaceutical excipient to create oral suspensions which were then used to treat mice intragastrically for evaluating their effect on absorption of orally-taken insulin. The results indicated that,in control mice receiving insulin（ 1. 5 IU/kg） via subcutaneous injection,the peak concentration of insulin in blood（ C_（max）） was（ 7. 56±1. 40） m IU/L and the time of peak appearance（ T_（max）） was 1 h. In experimental groups of 0. 5 μg/μL Bm CP8＋15 IU/kg INS,0. 5 μg/μL BmKazal＋15 IU/kg INS and 0. 5 μg/μL Bm CP8 ＋ 0. 5 μg/μL Bm Kazal＋15 IU/kg INS,C_（max） was（ 6. 07±0. 51）,（ 7. 12±0. 89） and（ 9. 20 ± 2. 00） m IU/L respectively and T_（max） was 3 h in all groups. However,in control mice receiving insulin（ 15 IU/kg） via oral intake,insulin concentration in blood was maintained at about 4. 32 m IU/L all the time.The above results indicate that protease inhibitors Bm CP8 and Bm Kazal could facilitate permeation of orally-taken insulin from gastrointestinal tract into blood and combined use of them had better effect. These two protease inhibitors could increase bioavailability of orally-taken insulin from 1. 51% to 4. 17%,showing good effect in promoting absorption of orally-taken insulin in mice.

关 键 词：胰岛素 重组家蚕蛋白酶抑制剂 口服吸收 小鼠 

分 类 号：Q78[生物学—分子生物学] R965[医药卫生—药理学]