蜜蜂幼虫芽胞杆菌金属蛋白酶基因的原核表达及多克隆抗体的制备

Prokaryotic Expression of Paenibacillus larvae Metalloprotease Gene and Preparation of Polyclonal Antibodies

克隆蜜蜂幼虫芽胞杆菌金属蛋白酶基因PLMP,构建其原核表达载体后进行重组蛋白的表达及纯化,接种公兔制备多克隆抗体,以期为研究幼虫芽胞杆菌快速检测方法奠定基础。用PCR方法扩增幼虫芽胞杆菌PLMP基因,扩增产物克隆至表达载体pGEX-4T-1并转化至E.coliBL21(DE3)感受态中。经不同的诱导时间、IPTG浓度诱导表达PLMP重组蛋白,经SDS-PAGE电泳切胶回收纯化目的蛋白。用目的蛋白对公兔进行4次免疫后,采集血清制备多抗。PCR扩增得到1 242bp的PLMP基因片段;构建原核表达载体pGEX-4T-1-PLMP,经终浓度0.8mmol/L IPTG、37℃诱导表达5h得到分子质量为70ku目的蛋白;用目的蛋白免疫公兔得到的多抗经间接ELISA检测效价达到1∶12 800,Western blot检测出现条带,证明所制备的多抗能够用于PLMP抗原的检测。研究结果构建了表达幼虫芽胞杆菌金属蛋白酶基因PLMP的原核载体,获得了PLMP蛋白,制备了兔抗PLMP多克隆抗体,为建立幼虫芽胞杆菌快速检测方法奠定了基础。

The study cloned the PLMP gene from Paenibacillus larvae of honeybees,constructed the prokaryotic expression vector for expressing and purifying of recombinant protein,prepared polyclonal antibodies against recombinant proteins in male rabbits,so as to lay a foundation for the study of the rapid detection method of Paenibacillus larvae.The PLMP gene was amplified by PCR and cloned into the prokaryotic expression vector pGEX-4 T-1,and then the recombinant vector was transformed into E.coliBL21（DE3）.The PLMP recombinant protein was induced by different time and different IPTG concentrations,and purified by cutting SDS-PAGE gel extraction.After the four times immunization with purpose protein in male rabbits,sera were collected and polyclonal antibodies were prepared.The PLMP gene fragment of 1242 bp was successfully cloned,prokaryotic expression vector pGEX-4 T-1-PLMP was constructed,the recombinant protein about 70 ku was obtained after inducing the pGEX-4 T-1-PLMP with 0.8 mmol/L IPTG at 37℃for 5 hrs.The titer of the polyclonal antibodies against rabbit immunized with the recombinant protein was 1∶12 800 by ELISA and a band appeared on nitrocellulose by Western blot,this showed that the obtained antibodies could be applied to detect the PLMP antigen.The pGEX-4 T-1-PLMP was successfully constructed and PLMP recombinant protein was expressed.Simultaneously,the polyclonal antibodies were prepared,and it laid the foundation to establish the method for rapid detection of Paenibacillus larvae.