家蚕核型多角体病毒酪氨酸蛋白磷酸酯酶基因的克隆及在大肠杆菌中表达

Cloning of a Protein Tyrosine Phosphatase Gene of Bombyx mori Nuclear Polyhedrosis Virus and Its Expression in Escherichia coli

采用PCR方法克隆了家蚕核型多角体病毒中国镇江株 (BmNPV ZJstrain)的酪氨酸蛋白磷酸酯酶基因(ptp) ,测定了该基因的核苷酸序列 ,比较了与相关病毒相应基因的同源性 ,并将该基因插入到原核表达质粒在大肠杆菌中进行了表达。该基因的编码部分由 5 0 7个核苷酸组成 ,编码 16 8个氨基酸残基的蛋白多肽 ,其中含有酪氨酸蛋白磷酸酶酯催化部位的“HC”基序。该基因与苜蓿银纹夜蛾核型多角体病毒 (AcMNPV) ptp 基因和BmN PV -T3 株 (日本 )拟为的 ptp基因核苷酸序列的同源性分别为 96 8%和 98 2 % ,蛋白质氨基酸序列的同源性分别为 97 0 %和 97 6 %。将该基因插入到温度诱导型表达质粒pBV2 2 1的温控启动子PRPL 之后 ,在大肠杆菌JM10 9中表达了具有催化活性的蛋白质 ,分子量约为 19kD ,表达产物能催化对硝基酚磷酸钠 (PNPP)的脱磷酸反应 ,这种催化作用可被钒酸钠和ZnCl2

A protein tyrosine phosphatase (PTP) gene was cloned from Bombyx mori nuclear polyhedrosis virus,Zhenjiang strain(BmNPV-ZJ),sequenced and then expressed in Escherichia coli. The gene consists of 507 nucleotides and is predicted to encode a protein of 168 amino acids containing the 'HC' motif of protein tyrosine phosphatase catalytic site.An identity of 96.8% in nucleotide sequence is shared with its homologue in Autographa californica multiple nuclear polyhedrosis virus(AcMNPV) and 98.2% with its postulated homologue in BmNPV T3 strain (Japan).The similarity of the cloned PTP in amino acid sequence is 97% to AcMNPV PTP and 97.6% to the postulated PTP of BmNPV T3 strain.The ptp gene was inserted into the expression vector pBV221 and expressed in E.coli JM109 under the control of the bacteriophage λ P RP L promoter regulated by the temperature sensitive repressor.The expressed 19 kD protein has the catalytic activity dephosphorylating p-nitrophenyl phosphate(PNPP).The catalytic activity toward PNPP was inhibited by sodium vanadate and ZnCl 2,both are effective inhibitors of PTPs.