赖型钩体重组质粒pDJH_2亚克隆及其在大肠杆菌中的高效表达

Subclones of Fragment DNA Recombinant rpDJH 2 of L. interrogans serovar lai Strain 017 and It's Expression of High Level in E.coli

为了探讨ｐＤＪＨ２的１．９ｋｂ０１７株钩体ＤＮＡ片段可否作为钩体重组疫苗广谱保护性抗原的候选，采用重组ＤＮＡ技术，将ｐＤＪＨ２的１．９ｋｂＤＮＡ片段分别与ｐＴ７－７，ｐＲＳＥＴ系列重组，转化入大肠杆菌进行亚克隆，ＩＰＴＧ诱导表达。结果显示：阳性亚克隆ｐＤＪｔ．ｐＤＪｒＢ１能在大肠杆菌中高效表达；对其进行ＳＤＳ－ＰＡＧＥ分析，可见６８ｋｄ和２３ｋｄ处出现新带，与钩体０１７株外膜同分子量蛋白带位置一致，免疫印迹（特异性抗０１７株外膜抗血清）在６８ｋｄ和２３ｋｄ处分别有印迹；用ｐＤＪｔ亚克隆重组质粒主动免疫豚鼠，可抵抗强毒力株攻击，表现出免疫保护作用。本实验结果提示：（１）ｐＤＪＨ２１．９ｋｂ重组ＤＮＡ片段能以不同质粒为载体，在不同大肠杆菌株中高效表达；（２）该重组ＤＮＡ片段表达产物——６８ｋｄ和２３ｋｄ蛋白，可能是赖型钩体０１７株外膜的保护性抗原。

Fragment of 1.9kb recombinant DNA of pDJH 2 was linked with vectors pT7 7 and pRSETs. Then they were transformed into E.coli JM109 (DE3) respectively. Expression of subclones was achieved in E.coli JM109 (DE3) with IPTG inducement. SDS PAGE showed that the molecular weights of products were 68kd and 23kd respectively. The amount of production seemed to be higher than that of the outer membrane proteins of L.interrogans serovar strain 017 in nature. Immunoblotting of pDJt and pDJrB 2(both are subclones) with the specific antiserum of anti OMP of L.interrogans serovar lai strain 017 and the experiment of initiative immuno protection in guinea pigs showed both proteins 68kd and 23kd might be the antigens of immuno protection on the outer membrane of L.interrogans serovar lai strain 017.