对氧磷酶的表达及活性研究.docVIP

  对氧磷酶的表达及活性研究# 李霞1，李晓瑞1，谭淑敏1，陈剑清1，舒特俊1，吴祥甫1，张耀洲1,2** （1. 浙江理工大学生命科学学院，杭州 310018； 5 10 15 20 25 30 35 40 2. 天津国际生物医药联合研究院，天津 300457） 摘要：对氧磷酶 1（paraoxonase1，PON1）是生物体内存在的一种广谱的非专一性酯酶。它 能通过催化磷酸酯键的水解，来降解有机磷酸、芳香羧酸酯和氨基甲酸酯，从而对水解有机 磷杀虫剂及降解神经毒剂发挥重要作用。本文构建了对氧磷酶 1 基因的原核表达载体 pET-32a-PON1，转化大肠杆菌 BL21（DE3）,经过 IPTG 诱导，成功表达目的蛋白。利用镍 柱亲和层析纯化后免疫新西兰雄兔制备抗体。同时,我们通过 bac-to-bac 系统构建了重组杆状 病毒 vBmPON1，将重组杆状病毒感染家蚕 BmN 细胞进行扩增得到具有一定感染复数的病 毒接种家蚕五龄幼虫及蚕蛹，经 western blotting 鉴定知目的蛋白 PON1 在家蚕幼虫及蚕蛹中 均得到表达，且在家蚕幼虫血淋巴中目的蛋白分布于上清中，有利于进一步分离纯化。将收 集的发病家蚕幼虫血淋巴经过滤、分级离心及超滤初步处理后，对目的蛋白进行了活性检测， 证实了家蚕生物反应器可以表达出有一定活性的目的蛋白，为进一步研究其与哺乳动物体内 天然存在的 PON1 区别奠定了基础。 关键词：分子生物学；对氧磷酶 1；重组杆状病毒；家蚕生物反应器；活性检测 中图分类号：Q71 The Expression and Activiy Research of PON1 Li Xia1, Li Xiaorui1, Tan Shumin1, Chen Jianqing1, Shu Tejun1, Wu Xiangfu1, Zhang Yaozhou1,2 (1. Institute of Biochemistry, Zhejiang Sci-tech University, HangZhou 310018; 2. Tianjin International Joint Academy of Biotechnology and Medicine, TianJin 300457) Abstract: PON1 is a broad-spectrum and non-specific esterase existing in vivo of the organisms. It can degrade the organophosphate ,the aromatic carboxylic acid ester and the carbamic acid ester through catalyzing the hydrolysis of phosphate bond.Thus，It may play an improtant role in catalyzing the hydrolysis of organophosphorus insecticide and the degradation of the nerve agent In this paper,The PON1 gene was inserted into the prokaryotic expression vector pET-32a by DNA recombination technology.Then the recombinant expression vector was transformed into E.coli BL21（DE3）.Induced with IPTG，the encoded protein was synthesized successfully.The targart protein PON1 was purified with Ni-NTA affinity chromatography column,and then,the purified product was used as an antigen for immunity to rabbit.to produce polyclonal antibody.At the same time,we contructed the recombinant baculovirus vBmPON1 through the bac-to-bac system and infected into BmN cells ， virus with definite multiplicity of infection was acquired.Inoculate the re