重组人唾液淀粉酶A的制备及活性鉴定  

Production and Activity Identification of Recombinant Human Salivary Amylase A

作　　者：刘洁 金科华 LIU Jie;JIN Ke-hua(School of Nursing,Hubei University of Science and Technology,Xianning Hubei 437100,China)

机构地区：[1]湖北科技学院护理学院,湖北咸宁437100 [2]湖北科技学院基础医学院

出　　处：《湖北科技学院学报（医学版）》2021年第4期292-294,F0003,共4页Journal of Hubei University of Science and Technology(Medical Sciences)

基　　金：湖北省大学生课外创新项目(S201910927036);湖北科技学院教学改革项目(2017XB010)。

摘　　要：目的利用基因工程制备重组人唾液淀粉酶A(rSAA),用其替代唾液中的SAA水解淀粉。方法设计一对带酶切位点的引物,PCR扩增SAA cDNA全长,将pET-28a质粒和PCR产物双酶切、连接。连接产物热激法转化大肠杆菌DH5α,于含卡那霉素的LB平板筛选抗性菌落,培养抗性菌落,抽提质粒,进行酶切和测序鉴定。将重组质粒(记为pS1)转化大肠杆菌BL21(DE3),用含卡那霉素的LB平板筛选抗性菌落(记为BL-pS1),培养BL-pS1,ITPG诱导rSAA表达。收集菌体,重悬于pH6.8的磷酸缓冲液,超声裂解,离心取上清,测试其水解淀粉的活性。结果构建了SAA cDNA的原核表达载体pS1,rSAA在宿主菌BL21(DE3)中获得可溶性表达,rSAA能高效水解淀粉。结论用rSAA取代唾液中SAA水解淀粉操作安全、取用方便、利于环保。Objective To produce recombinant salivary amylase A(rSAA)by genetic engineering and use rSAA to hydrolyse starch instead of SAA collected from saliva.Methods A pair of primers with restriction sites were designed to amplify the full length of SAA cDNA by PCR.The pET-28a plasmid and PCR product were double-digested and ligated.Ligation products were used to transform E.coli DH5αcompetent cells by heat shock.Resistant colonies were screened in LB plate containing kanamycin.The resistant colonies were cultured,plasmids were extracted.The construct was identified by restriction enzyme digestion.The recombinant plasmid(pS1)transformed to E.coli BL21(DE3),resistant colonies(BL-PS1)were screened with LB plate containing kanamycin.Resistant colonies BL-PS1 were cultured,rSAA was expressed by ITPG induction.The bacteria were collected and resuspended in phosphoric acid buffer at pH6.8,and the supernatant was obtained by centrifuge and used to test the activity of hydrolyzing starch.Results Prokaryotic expression vector inserted with SAA cDNA was constructed,soluble rSAA was expressed in BL21(DE3),and could hydrolyze starch efficiently.Conclusion The use of rSAA to replace SAA produced in saliva to hydrolyse starch has the advantages of safe operation,easy access,environmental protection.

关 键 词：唾液淀粉酶A pET-28a质粒 淀粉 

分 类 号：Q787[生物学—分子生物学]